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Sample records for induces p53-dependent apoptosis

  1. Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice

    PubMed Central

    Gentil Dit Maurin, A; Lemercier, C; Collin-Faure, V; Marche, P N; Jouvin-Marche, E; Candéias, S M

    2015-01-01

    The production of T cell receptor αβ+ (TCRαβ+) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-β) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4+CD8+ double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4−CD8− (double-negative, DN) cKit+CD25+ thymocytes, where TCR-β gene rearrangement is initiated. However, TCR-αβ−CD8+ immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αβ+ thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αβ− progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis. PMID:24635132

  2. Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells

    PubMed Central

    Hirsch, Matthew L.; Fagan, B. Matthew; Dumitru, Raluca; Bower, Jacquelyn J.; Yadav, Swati; Porteus, Matthew H.; Pevny, Larysa H.; Samulski, R. Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication. PMID:22114676

  3. Radiation-Induced Salivary Gland Dysfunction Results From p53-Dependent Apoptosis

    SciTech Connect

    Avila, Jennifer L.; Grundmann, Oliver; Burd, Randy; Limesand, Kirsten H.

    2009-02-01

    Purpose: Radiotherapy for head-and-neck cancer causes adverse secondary side effects in the salivary glands and results in diminished quality of life for the patient. A previous in vivo study in parotid salivary glands demonstrated that targeted head-and-neck irradiation resulted in marked increases in phosphorylated p53 (serine{sup 18}) and apoptosis, which was suppressed in transgenic mice expressing a constitutively active mutant of Akt1 (myr-Akt1). Methods and Materials: Transgenic and knockout mouse models were exposed to irradiation, and p53-mediated transcription, apoptosis, and salivary gland dysfunction were analyzed. Results: The proapoptotic p53 target genes PUMA and Bax were induced in parotid salivary glands of mice at early time points after therapeutic radiation. This dose-dependent induction requires expression of p53 because no radiation-induced expression of PUMA and Bax was observed in p53-/- mice. Radiation also induced apoptosis in the parotid gland in a dose-dependent manner, which was p53 dependent. Furthermore, expression of p53 was required for the acute and chronic loss of salivary function after irradiation. In contrast, apoptosis was not induced in p53-/- mice, and their salivary function was preserved after radiation exposure. Conclusions: Apoptosis in the salivary glands after therapeutic head-and-neck irradiation is mediated by p53 and corresponds to salivary gland dysfunction in vivo.

  4. P53 dependent and independent apoptosis induced by lidamycin in human colorectal cancer cells.

    PubMed

    Chen, Lihui; Jiang, Jianming; Cheng, Chunlei; Yang, Ajing; He, Qiyang; Li, Diandong; Wang, Zhen

    2007-06-01

    Enediyne compound is one class of antibiotics with very potent anti-cancer activity. However, the role of p53 in enediyne antibiotic-induced cell killing remains elusive. Here we reported the involvement of p53 signaling pathway in apoptosis induction by lidamycin (LDM), a member of the enediyne antibiotic family. We found that LDM at low drug concentration of 10 nmol/L induces apoptotic cell death much more effectively in human colorectal cancer cells with wild type p53 than those with mutant or deleted p53. p53 is functionally activated as an early event in response to low dose LDM that precedes the significant apoptosis induction. The primarily activation of mitochondria as well as the activation of p53 transcriptional targets such as Puma, Bad and Bax in HCT116 p53 wild type cells further demonstrates the key role of p53 in mediating the compound-induced apoptosis. This is further supported by the observation that the absence of Bax or Puma decreases apoptosis dramatically while Bcl-2 overexpression confers partially resistance after drug treatment. Activation of p53 signaling pathway leads to activation of caspases and caspases inhibitor VAD-fmk completely blocks low dose LDM induced apoptosis through the inhibition of mitochondria pathway. In contrast, LDM at higher concentration causes rapid apoptosis through more direct DNA damaging mechanism that is independent of activation of p53 and caspases and cannot be blocked by caspase inhibitor. Taken together, LDM induces apoptosis in a p53-dependent manner when given at low doses, but in a p53-independent manner when given at high doses. This dosage-dependent regimen can be applied to cancer clinic based upon the p53 status of cancer patients. PMID:17534142

  5. p14(ARF) Prevents Proliferation of Aneuploid Cells by Inducing p53-Dependent Apoptosis.

    PubMed

    Veneziano, Lorena; Barra, Viviana; Lentini, Laura; Spatafora, Sergio; Di Leonardo, Aldo

    2016-02-01

    Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14(ARF) is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14(ARF) is involved in the maintenance of aneuploid cells in primary human cells, suggesting that it could be part of a pathway controlling their proliferation. To investigate this aspect further, p14(ARF) was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14(ARF) Re-expression reduced the number of aneuploid cells in MAD2 post-transcriptionally silenced cells. Also aberrant mitoses, frequently displayed in MAD2-depleted cells, were decreased when p14(ARF) was expressed at the same time. In addition, p14(ARF) ectopic expression in MAD2-depleted cells induced apoptosis associated with increased p53 protein levels. Conversely, p14(ARF) ectopic expression did not induce apoptosis in HCT116 p53KO cells. Collectively, our results suggest that the tumor suppressor p14(ARF) may have an important role in counteracting proliferation of aneuploid cells by activating p53-dependent apoptosis. PMID:25752701

  6. Cadmium Induces p53-Dependent Apoptosis in Human Prostate Epithelial Cells

    PubMed Central

    Aimola, Pierpaolo; Carmignani, Marco; Volpe, Anna Rita; Di Benedetto, Altomare; Claudio, Luigi; Waalkes, Michael P.; van Bokhoven, Adrie; Tokar, Erik J.; Claudio, Pier Paolo

    2012-01-01

    Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl2 and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl2 concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis. PMID:22448262

  7. p53-dependent apoptosis contributes to di-(2-ethylhexyl) phthalate-induced hepatotoxicity.

    PubMed

    Ha, Mei; Wei, Li; Guan, Xie; Li, Lianbing; Liu, Changjiang

    2016-01-01

    Di-(2-ethylhexyl) phthalate (DEHP) is used extensively in many personal care and consumer products, resulting in widespread non-occupational human exposure through multiple routes and media. DEHP has various deleterious effects including hepatotoxicity. p53 protein is a central sensor in cell apoptosis. In order to clarify the roles of p53 in DEHP-induced hepatotoxicity, Sprague-Dawley (SD) rats were dosed daily with DEHP by gavage for 30 days; BRL cells (rat liver cell line) were treated with DEHP for 24 h after pretreatment with NAC or small interfering RNA (siRNA). Results indicated that after exposure to DEHP, hepatic histological changes such as hepatocyte edema, vacuolation and hepatic sinusoidal dilation, and increased apoptosis index were observed. In the liver, DEHP induced oxidative stress and DNA damage, which activated p53 in vivo and in vitro. Pretreatment with NAC significantly reduced ROS level and p53 expression in BRL cells. The suppressed Mdm2 also contributed to p53 accumulation. Activated p53 mediated hepatocyte apoptosis via the intrinsic mitochondrial pathway, inhibiting anti-apoptotic Bcl-2 and Bcl-xL and inducing pro-apoptotic Bax, cytochrome c and caspases. In p53-silenced BRL cells, hepatocyte apoptosis mediated by p53 was attenuated. PCNA protein level was upregulated after p53 gene silencing. However, the Fas/FasL apoptotic pathway did not exhibit activated signs in DEHP-caused hepatotoxicity. Taken together, DEHP-caused oxidative stress and Mdm2 downregulation contribute to p53 activation. The p53-dependent apoptotic pathway plays critical and indispensable roles in DEHP-induced hepatotoxicity, while the Fas/FasL pathway does not involve in this molecular event. PMID:26549752

  8. Novel small molecule induces p53-dependent apoptosis in human colon cancer cells

    SciTech Connect

    Park, Sang Eun; Min, Yong Ki; Ha, Jae Du; Kim, Bum Tae; Lee, Woo Ghil . E-mail: bigguy@krict.re.kr

    2007-07-06

    Using high-throughput screening with small-molecule libraries, we identified a compound, KCG165 [(2-(3-(2-(pyrrolidin-1-yl)ethoxy)-1,10b-dihydro-[1,2,4]triazolo[1,5-c] quinazolin-5(6H)-one)], which strongly activated p53-mediated transcriptional activity. KCG165-induced phosphorylations of p53 at Ser{sup 6}, Ser{sup 15}, and Ser{sup 20}, which are all key residues involved in the activation and stabilization of p53. Consistent with these findings, KCG165 increased level of p53 protein and led to the accumulation of transcriptionally active p53 in the nucleus with the increased occupancy of p53 in the endogenous promoter region of its downstream target gene, p21{sup WAF1/CIP}. Notably, KCG165-induced p53-dependent apoptosis in cancer cells. Furthermore, we suggested topoisomerase II as the molecular target of KCG165. Together, these results indicate that KCG165 may have potential applications as an antitumor agent.

  9. 2-Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway.

    PubMed

    Schweikl, Helmut; Petzel, Christine; Bolay, Carola; Hiller, Karl-Anton; Buchalla, Wolfgang; Krifka, Stephanie

    2014-03-01

    Resin monomers of dental composites like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation of dental pulp-derived cells, or induce cell death via apoptosis. The induction of apoptosis is related to the availability of the antioxidant glutathione, although a detailed understanding of the signaling pathways is still unknown. The present study provides insight into the causal relationship between oxidative stress, oxidative DNA damage, and the specific signaling pathway leading to HEMA-induced apoptosis in RAW264.7 mouse macrophages. The differential expression of the antioxidative enzymes superoxide dismutase, glutathione peroxidase, and catalase in HEMA-exposed cells indicated oxidative stress, which was associated with the cleavage of pro-caspase 3 as a critical apoptosis executioner. A 2-fold increase in the amount of mitochondrial superoxide anions after a 24 h exposure to HEMA (6-8 mM) was paralleled by a considerable decrease in the mitochondrial membrane potential (MMP). Additionally, expression of proteins critical for the signaling of apoptosis through the intrinsic mitochondrial pathway was detected. Transcription-dependent and transcription-independent mechanisms of p53-regulated apoptosis were activated, and p53 was translocated from the cytosol to mitochondria. HEMA-induced transcriptional activity of p53 was indicated by increased levels of PUMA localized to mitochondria as a potent inducer of apoptosis. The expression of Bcl-xL and Bax suggested that cells responded to stress caused by HEMA via the activation of a complicated and antagonistic machinery of pro- and anti-apoptotic Bcl-2 family members. A HEMA-induced and oxidative stress-sensitive delay of the cell cycle, indicating a DNA damage response, occurred independent of the influence of KU55399, a potent inhibitor of ATM (ataxia-telangiectasia mutated) activity. However, ATM, a protein kinase which

  10. Dysfunctional telomeres induce p53-dependent and independent apoptosis to compromise cellular proliferation and inhibit tumor formation.

    PubMed

    Wang, Yang; Wang, Xinwei; Flores, Elsa R; Yu, Jian; Chang, Sandy

    2016-08-01

    Aging is associated with progressive telomere shortening, resulting in the formation of dysfunctional telomeres that compromise tissue proliferation. However, dysfunctional telomeres can limit tumorigenesis by activating p53-dependent cellular senescence and apoptosis. While activation of both senescence and apoptosis is required for repress tumor formation, it is not clear which pathway is the major tumor suppressive pathway in vivo. In this study, we generated Eμ-myc; Pot1b(∆/∆) mouse to directly compare tumor formation under conditions in which either p53-dependent apoptosis or senescence is activated by telomeres devoid of the shelterin component Pot1b. We found that activation of p53-dependent apoptosis plays a more critical role in suppressing lymphoma formation than p53-dependent senescence. In addition, we found that telomeres in Pot1b(∆/∆) ; p53(-/-) mice activate an ATR-Chk1-dependent DNA damage response to initiate a robust p53-independent, p73-dependent apoptotic pathway that limited stem cell proliferation but suppressed B-cell lymphomagenesis. Our results demonstrate that in mouse models, both p53-dependent and p53-independent apoptosis are important to suppressing tumor formation. PMID:27113195

  11. Neuropeptide Y protects kidney against cisplatin-induced nephrotoxicity by regulating p53-dependent apoptosis pathway.

    PubMed

    Kim, Namoh; Min, Woo-Kie; Park, Min Hee; Lee, Jong Kil; Jin, Hee Kyung; Bae, Jae-Sung

    2016-05-01

    Cisplatin is a platinum-based chemotherapeutic drug for treating various types of cancers. However, the use of cisplatin is limited by its negative effect on normal tissues, particularly nephrotoxicity. Various mechanisms such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and apoptosis are involved in the adverse effect induced by cisplatin treatment. Several studies have suggested that neuropeptide Y (NPY) is involved in neuroprotection as well as restoration of bone marrow dysfunction from chemotherapy induced nerve injury. However, the role of NPY in chemotherapy- induced nephrotoxicity has not been studied. Here, we show that NPY rescues renal dysfunction by reducing the expression of pro-apoptotic proteins in cisplatin induced nephrotoxicity through Y1 receptor, suggesting that NPY can protect kidney against cisplatin nephrotoxicity as a possible useful agent to prevent and treat cisplatin-induced nephrotoxicity. [BMB Reports 2016; 49(5): 288-292]. PMID:26728272

  12. TEL/ETV6 induces apoptosis in 32D cells through p53-dependent pathways

    SciTech Connect

    Yamagata, Tetsuya; Maki, Kazuhiro; Waga, Kazuo; Mitani, Kinuko . E-mail: kinukom-tky@umin.ac.jp

    2006-08-25

    TEL is an ETS family transcription factor that is critical for maintaining hematopoietic stem cells in adult bone marrow. To investigate the roles of TEL in myeloid proliferation and differentiation, we introduced TEL cDNA into mouse myeloid 32Dcl3 cells. Overexpression of TEL repressed interleukin-3-dependent proliferation through blocking cell cycle progression. Also, the presence of TEL triggered apoptosis through the mitochondrial intrinsic pathway on exposure to granulocyte colony-stimulating factor. We found an increase in p53 protein and its DNA binding in the TEL-overexpressing cells. Forced expression of TEL stimulated transcription via the p53-responsive element and increased the expression of cellular target genes for p53 such as cell cycle regulator p21 and apoptosis inducer Puma. Consistently, induction of apoptosis was delayed by pifithrin-{alpha} treatment and completely blocked by increased expression of Bcl-2 in the TEL-overexpressing cells. These data collectively suggest that TEL exerts a tumor suppressive function through augmenting the p53 pathway and facilitates normal development of myelopoiesis.

  13. Chromium oxide nanoparticle-induced genotoxicity and p53-dependent apoptosis in human lung alveolar cells.

    PubMed

    Senapati, Violet Aileen; Jain, Abhishek Kumar; Gupta, Govind Sharan; Pandey, Alok Kumar; Dhawan, Alok

    2015-10-01

    Chromium oxide (Cr2 O3 ) nanoparticles (NPs) are being increasingly used as a catalyst for aromatic compound manufacture, abrading agents and as pigments (e.g., Viridian). Owing to increased applications, it is important to study the biological effects of Cr2 O3 NPs on human health. The lung is one of the main exposure routes to nanomaterials; therefore, the present study was designed to determine the genotoxic and apoptotic effect of Cr2 O3 NPs in human lung epithelial cells (A549). The study also elucidated the molecular mechanism of its toxicity. Cr2 O3 NPs led to DNA damage, which was deduced by comet assay and cytokinesis block micronucleus assay. The damage could be mediated by the increased levels of reactive oxygen species. Further, the oxygen species led to a decrease in mitochondrial membrane potential and an increase in the ratio of BAX/Bcl-2 leading to mitochondria-mediated apoptosis induced by Cr2 O3 NPs, which ultimately leads to cell death. Hence, there is a need of regulations to be imposed in NP usage. The study provided insight into the caspase-dependent mechanistic pathway of apoptosis. PMID:26086747

  14. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway

    PubMed Central

    Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

    2016-01-01

    Aim: Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Methods: Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Results: Lidocaine (0.005%−0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50−800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Conclusion: Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway. PMID:27041463

  15. Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit

    PubMed Central

    2013-01-01

    Background Complementary medicines, including homeopathy, are used by many patients with cancer, usually alongside with conventional treatment. However, the molecular mechanisms underneath the anti-cancer effect, if any, of these medicines have still remained unexplored. To this end we attempted to evaluate the efficacy of calcarea carbonica, a homeopathic medicine, as an anti-cancer agent and to delineate the detail molecular mechanism(s) underlying calcerea carbonica-induced tumor regression. Methods To investigate and delineate the underlying mechanisms of calcarea carbonica-induced tumor regression, Trypan blue dye-exclusion test, flow cytometric, Western blot and reverse transcriptase-PCR techniques were employed. Further, siRNA transfections and inhibitor studies were used to validate the involvement of p53 pathway in calcarea carbonica-induced apoptosis in cancer cells. Results Interestingly, although calcarea carbonica administration to Ehrlich’s ascites carcinoma (EAC)- and Sarcoma-180 (S-180)-bearing Swiss albino mice resulted in 30-35% tumor cell apoptosis, it failed to induce any significant cell death in ex vivo conditions. These results prompted us to examine whether calcarea carbonica employs the immuno-modulatory circuit in asserting its anti-tumor effects. Calcarea carbonica prevented tumor-induced loss of effector T cell repertoire, reversed type-2 cytokine bias and attenuated tumor-induced inhibition of T cell proliferation in tumor-bearing host. To confirm the role of immune system in calcarea carbonica-induced cancer cell death, a battery of cancer cells were co-cultured with calcarea carbonica-primed T cells. Our results indicated a "two-step" mechanism of the induction of apoptosis in tumor cells by calcarea carbonica i.e., (1) activation of the immune system of the host; and (2) induction of cancer cell apoptosis via immuno-modulatory circuit in p53-dependent manner by down-regulating Bcl-2:Bax ratio. Bax up-regulation resulted in

  16. p53-Dependent apoptosis induced in human bronchial epithelial (16-HBE) cells by PM(2.5) sampled from air in Guangzhou, China.

    PubMed

    Zhou, Bo; Liang, Guiqiang; Qin, Huiyan; Peng, Xiaowu; Huang, Jiongli; Li, Qin; Qing, Li; Zhang, Li'e; Chen, Li; Ye, Li; Niu, Piye; Zou, Yunfeng

    2014-12-01

    Epidemiological studies have shown that air pollution particulate matter (PM) is associated with increased respiratory morbidity and mortality. However, the mechanisms are not fully understood. Oxidative stress-mediated apoptosis plays an important role in the occurrence of respiratory diseases. In this study, human bronchial epithelial (16-HBE) cells were exposed to different concentrations (16-128 µg/ml) of PM(2.5) for 24 h to investigate the apoptosis induced by PM(2.5). The results showed that PM(2.5) exposure significantly induced apoptosis, DNA strand breaks, and oxidative damage in a dose-dependent manner in 16-HBE cells. The expression of p53 and p73 increased significantly along with the dose of PM(2.5) in 16-HBE cells, whereas the expression of p21(Cip1/WAF1) decreased; the expression of mdm2 increased and then decreased, but not significantly. Taken together, these observations indicate that PM(2.5) may lead to oxidative damage and induce apoptosis through the p53-dependent pathway in 16-HBE cells. p53-Dependent apoptosis mediated by DNA strand breaks may be an important mechanism of PM(2.5)-induced apoptosis in 16-HBE cells. PMID:25133668

  17. Leukocyte Elastase Induces Lung Epithelial Apoptosis via a PAR-1–, NF-κB–, and p53-Dependent Pathway

    PubMed Central

    Suzuki, Tomoko; Yamashita, Cory; Zemans, Rachel L.; Briones, Natalie; Van Linden, Annemie; Downey, Gregory P.

    2009-01-01

    Leukocyte elastase induces apoptosis of lung epithelial cells via alterations in mitochondrial permeability, but the signaling pathways regulating this response remain uncertain. Here we investigated the involvement of proteinase-activated receptor-1 (PAR-1), the transcription factor NF-κB, and the protooncogene p53 in this pathway. Elastase-induced apoptosis of lung epithelial cells correlated temporally with activation of NF-κB, phosphorylation, and nuclear translocation of p53, increased p53 up-regulated modulator of apoptosis (PUMA) expression, and mitochondrial translocation of Bax resulting in enhanced permeability. Elastase-induced apoptosis was also prevented by pharmacologic inhibitors of NF-κB and p53 and by short interfering RNA knockdown of PAR-1, p53, or PUMA. These inhibitors prevented elastase-induced PUMA expression, mitochondrial translocation of Bax, increased mitochondrial permeability, and attenuated apoptosis. NF-κB inhibitors also reduced p53 phosphorylation. We conclude that elastase-induced apoptosis of lung epithelial cells is mediated by a PAR-1–triggered pathway involving activation of NF-κB and p53, and a PUMA- and Bax-dependent increase in mitochondrial permeability leading to activation of distal caspases. Further, p53 contributes to elastase-induced apoptosis by both transcriptional and post-transcriptional mechanisms. PMID:19307610

  18. Astemizole-Histamine induces Beclin-1-independent autophagy by targeting p53-dependent crosstalk between autophagy and apoptosis.

    PubMed

    Jakhar, Rekha; Paul, Souren; Bhardwaj, Monika; Kang, Sun Chul

    2016-03-01

    Apoptosis and autophagy are genetically regulated, evolutionarily conserved processes that can jointly seal cancer cell fates, and numerous death stimuli are capable of activating either pathway. Although crosstalk between apoptosis and autophagy is quite complex and sometimes contradictory, it remains a key factor determining the outcomes of death-related pathologies such as cancer. In the present study, exposure of MCF-7 breast cancer cells to HIS and the H1 receptor antagonist AST both alone and together with HIS (AST-HIS) led to generation of intracellular ROS, which induced massive cellular vacuolization through dilation of the ER and mitochondria. Consequently, apoptosis by Bax translocation, cytochrome c release, and caspase activation were triggered. In addition, AST-HIS caused ER stress-induced autophagy in MCF-7 cells, as evidenced by an increased LC3-II/LC3-I ratio, with surprisingly no changes in Beclin-1 expression. Non-canonical autophagy was induced via p53 phosphorylation, which increased p53-p62 interactions to enhance Beclin-1-independent autophagy as evidenced by immunocytochemistry and immunoprecipitation. In the absence of Beclin-1, enhanced autophagy further activated apoptosis through caspase induction. In conclusion, these findings indicate that AST-HIS-induced apoptosis and autophagy can be regulated by ROS-mediated signaling pathways. PMID:26739061

  19. Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

    PubMed Central

    Lai, Chin-Yu; Tsai, An-Chi; Chen, Mei-Chuan; Chang, Li-Hsun; Sun, Hui-Lung; Chang, Ya-Ling; Chen, Chien-Chih

    2012-01-01

    Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (−/−) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity. PMID:22912688

  20. A new semisynthetic 1-O-acetyl-6-O-lauroylbritannilactone induces apoptosis of human laryngocarcinoma cells through p53-dependent pathway.

    PubMed

    Han, Yang-Yang; Tang, Jiang-Jiang; Gao, Rong-Fang; Guo, Xin; Lei, Ming; Gao, Jin-Ming

    2016-09-01

    Initiation of apoptosis is an important event for chemoprevention and chemotherapy of cancer. Naturally derived products had drawn growing attention as lead compounds for anticancer drug discovery. ABL-L, a semisynthetic analogue of natural sesquiterpenoid 1-O-acetylbritannilactone (ABL) isolated from Inula britannica, showed stronger suppression against three solid tumor cell lines with 4-10 fold improvement than ABL. However, its molecular mechanism of cell death induction has still not been determined. The present study evaluated the anticancer efficacy of ABL-L and its biological activities mechanism on human laryngocarcinoma cells HEp-2 in vitro. We found that ABL-L-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest. Typical apoptotic morphological and biochemical features were also observed in treated cells. Furthermore, the levels of the anti-apoptotic Bcl-2, pro-caspase 3/8/9 and poly(ADP-ribose) polymerase PARP decreased, and the level of pro-apoptotic Bax increased. Involvement of the caspase-mediated apoptosis was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. In addition, ABL-L induced a tumor suppressor p53 and its target genes expression p21, fas, noxa and puma. The results of p53 knockdown suggest that caspase-mediated apoptosis induced by ABL-L was in p53-dependent pathway on HEp-2 cells. Our data indicate that the cytotoxicity of the novel semisynthetic analogue ABL-L involved G1 cell cycle arrest and apoptosis via a p53-dependent, caspase-mediated pathway on human laryngocarcinoma cells. PMID:27262408

  1. Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model

    PubMed Central

    Martínez-Morentin, Leticia; Martínez, Lidia; Piloto, Sarah; Yang, Hua; Schon, Eric A.; Garesse, Rafael; Bodmer, Rolf; Ocorr, Karen; Cervera, Margarita; Arredondo, Juan J.

    2015-01-01

    The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox expression, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans. PMID:25792727

  2. Epothilones Suppress Neointimal Thickening in the Rat Carotid Balloon-Injury Model by Inducing Vascular Smooth Muscle Cell Apoptosis through p53-Dependent Signaling Pathway

    PubMed Central

    Son, Dong Ju; Jung, Jae Chul; Hong, Jin Tae

    2016-01-01

    Microtubule stabilizing agents (MTSA) are known to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and effectively reduce neointimal hyperplasia and restenosis. Epothilones (EPOs), non-taxane MTSA, have been found to be effective in the inhibition of VSMC proliferation and neointimal formation by cell cycle arrest. However, effect of EPOs on apoptosis in hyper-proliferated VSMCs as a possible way to reduce neointimal formation and its action mechanism related to VSMC viability has not been suited yet. Thus, the purposes of the present study was to investigate whether EPOs are able to inhibit neointimal formation by inducing apoptosis within the region of neointimal hyperplasia in balloon-injured rat carotid artery, as well as underlying action mechanism. Treatment of EPO-B and EPO-D significantly induced apoptotic cell death and mitotic catastrophe in hyper-proliferated VSMCs, resulting in cell growth inhibition. Further, EPOs significantly suppressed VSMC proliferation and induced apoptosis by activation of p53-dependent apoptotic signaling pathway, Bax/cytochrome c/caspase-3. We further demonstrated that the local treatment of carotid arteries with EPOs potently inhibited neointimal lesion formation by induction of apoptosis in rat carotid injury model. Our findings demonstrate a potent anti-neointimal hyperplasia property of EPOs by inducing p53-depedent apoptosis in hyper-proliferated VSMCs. PMID:27218463

  3. Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways

    SciTech Connect

    Li, Lin; Yue, Grace G.L.; Lau, Clara B.S.; Sun, Handong; Fung, Kwok Pui; Leung, Ping Chung; Han, Quanbin; Leung, Po Sing

    2012-07-01

    Pancreatic cancer is difficult to detect early and responds poorly to chemotherapy. A breakthrough in the development of new therapeutic agents is urgently needed. Eriocalyxin B (EriB), isolated from the Isodon eriocalyx plant, is an ent-kaurane diterpenoid with promise as a broad-spectrum anti-cancer agent. The anti-leukemic activity of EriB, including the underlying mechanisms involved, has been particularly well documented. In this study, we demonstrated for the first time EriB's potent cytotoxicity against four pancreatic adenocarcinoma cell lines, namely PANC-1, SW1990, CAPAN-1, and CAPAN-2. The effects were comparable to that of the chemotherapeutic camptothecin (CAM), but with much lower toxicity against normal human liver WRL68 cells. EriB's cytoxicity against CAPAN-2 cells was found to involve caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Moreover, the p53 pathway was found to be activated by EriB in these cells. Furthermore, in vivo studies showed that EriB inhibited the growth of human pancreatic tumor xenografts in BALB/c nude mice without significant secondary adverse effects. These results suggest that EriB should be considered a candidate for pancreatic cancer treatment. -- Highlights: ► We study Eriocalyxin B (EriB)'s cytotoxic effects on pancreatic cancer cell lines. ► EriB inhibits cell proliferation via mediation of apoptosis and cell cycle arrest. ► The effects are involved in caspase-dependent apoptosis and p53 pathway. ► In vivo study also shows EriB inhibits the growth of human pancreatic tumor. ► EriB can be a good candidate for chemotherapy in pancreatic cancer.

  4. An anthraquinone derivative from Luffa acutangula induces apoptosis in human lung cancer cell line NCI-H460 through p53-dependent pathway.

    PubMed

    Vanajothi, Ramar; Srinivasan, Pappu

    2016-06-01

    The current study was designed to evaluate the in vitro antiproliferative activity of 1,8-dihydroxy-4-methylanthracene-9,10-dione (DHMA) isolated from the Luffa acutangula against human non-small cell lung cancer cell line (NCI-H460). Induction of apoptosis and reactive oxygen species (ROS) generation was determined through fluorescence microscopic technique. Quantitative real-time PCR and western blotting analysis was carried out to detect the expression of pro-apoptotic (p53, p21, caspase-3, Bax, GADD45A, and ATM) and anti-apoptotic (NF-κB) proteins in NCI-H460 cell line. In silico studies also performed to predict the binding mechanism of DHMA with MDM2-p53 protein. The DHMA inhibited the cell viability of NCI-H460 cells in a dose-dependent manner with an IC50 of about 50 µg/ml. It significantly reduced cell viability correlated with induction of apoptosis, which was associated with ROS generation. The apoptotic cell death was further confirmed through dual staining and DNA fragmentation assay. DHMA significantly increased the expression of anti-apoptotic protein such as p53, p21, Bax, and caspase-3 but downregulated the expression of NF-κB in NCI-H460 cell line. In silico studies demonstrate that DHMA formed hydrogen bond interaction with key residues Trp26, Phe55 and Lys24 by which it disrupt the binding of p53 with MDM2 receptor. These findings suggested that DHMA induces apoptosis in NCI-H460 via a p53-dependent pathway. This the first study on cytotoxic and apoptosis inducing activity of DHMA from L. acutangula against NCI-H460 cell line. Therefore, DHMA has therapeutic potential for lung cancer treatment. PMID:26585176

  5. Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

    PubMed

    Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

    2011-11-01

    Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment. PMID:22011578

  6. Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells.

    PubMed

    Bañuelos, Adriana; Reyes, Elba; Ocadiz, Rodolfo; Alvarez, Elizabeth; Moreno, Martha; Monroy, Alberto; Gariglio, Patricio

    2003-08-01

    Human papillomavirus (HPV) E6 viral oncoprotein plays an important role during cervical carcinogenesis. This oncoprotein binds the tumor suppressor protein p53, leading to its degradation via the ubiquitin-proteasome pathway. Therefore, it is generally assumed that in HPV-positive cancer cells p53 function is completely abolished. Nevertheless, recent findings suggest that p53 activity can be recovered in cells expressing endogenous E6 protein. To investigate whether p53-dependent functions controlling genome integrity, cell proliferation, and apoptosis can be reactivated in cervical cancer cells, we examined the capacity of HeLa, INBL, CaSki, C33A, and ViBo cell lines to respond to neocarzinostatin (NCS), a natural product which induces single- and double-strand breaks in DNA. We found that NCS treatment inhibits cellular proliferation through G2 cell cycle arrest and apoptosis induction. This effect was preceded by nuclear accumulation of p53 protein and by an increase of p21 transcripts. Although apoptosis was blocked in ViBo cells (HPV-negative), nuclear accumulation of transcriptionally active p53 and inhibition of cell proliferation are observed after NCS treatment. These results suggest that HPV-positive cervical cancer cells are capable of responding efficiently to DNA damage provoked by NCS treatment through a p53-dependent pathway in spite of the presence of E6 protein. PMID:12750435

  7. A novel dithiocarbamate derivative induces cell apoptosis through p53-dependent intrinsic pathway and suppresses the expression of the E6 oncogene of human papillomavirus 18 in HeLa cells.

    PubMed

    Li, Yanhong; Qi, Hongxue; Li, Xiaobo; Hou, Xueling; Lu, Xueying; Xiao, Xiangwen

    2015-06-01

    Dithiocarbamates (DTCs) exhibit a broad spectrum of antitumor activities, however, their molecular mechanisms of antitumor have not yet been elucidated. Previously, we have synthesized a series of novel dithiocarbamate derivatives. These DTCs were examined for cytotoxic activities against five human cancer cell lines. In this study, one of dithiocarbamate (DTC1) with higher potential for HeLa cells was chosen to investigate molecular mechanisms for its anti-tumor activities. DTC1 could inhibit proliferation, and highly induce apoptosis in HeLa cells by activating caspase-3, -6 and -9; moreover, activities of caspase-3, -6 and -9 were inhibited by pan-caspase inhibitor, Z-VAD-FMK. Furthermore, DTC1 decreased the levels of Bcl-2 and Bcl-xL, and increased expression of cytosol cytochrome c, Bak, Bax and p53 in a time-dependent manner but had no effect on the level of Rb. It was shown that DTC1 induced HeLa cells apoptosis through a p53-dependent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Additionally, the relative expression of E6 and E7 were evaluated in HPV18-positive (HeLa cells) by real-time PCR and western blotting. The results firstly demonstrated that DTC1 suppressed both expression of E6 mRNA and E6 oncoprotein, but had no effect on the expression of E7 mRNA and protein in HPV18. Our results suggested that DTC1 may serve as novel chemotherapeutic agents in the treatment of cervical cancer and potential anti-HPV virus candidates that merit further studies. PMID:25772545

  8. 2.45 GHz Microwave Radiation Impairs Learning and Spatial Memory via Oxidative/Nitrosative Stress Induced p53-Dependent/Independent Hippocampal Apoptosis: Molecular Basis and Underlying Mechanism.

    PubMed

    Shahin, Saba; Banerjee, Somanshu; Singh, Surya Pal; Chaturvedi, Chandra Mohini

    2015-12-01

    A close association between microwave (MW) radiation exposure and neurobehavioral disorders has been postulated but the direct effects of MW radiation on central nervous system still remains contradictory. This study was performed to understand the effect of short (15 days) and long-term (30 and 60 days) low-level MW radiation exposure on hippocampus with special reference to spatial learning and memory and its underlying mechanism in Swiss strain male mice, Mus musculus. Twelve-weeks old mice were exposed to 2.45 GHz MW radiation (continuous-wave [CW] with overall average power density of 0.0248 mW/cm(2) and overall average whole body specific absorption rate value of 0.0146 W/Kg) for 2 h/day over a period of 15, 30, and 60 days). Spatial learning and memory was monitored by Morris Water Maze. We have checked the alterations in hippocampal oxidative/nitrosative stress, neuronal morphology, and expression of pro-apoptotic proteins (p53 and Bax), inactive executioner Caspase- (pro-Caspase-3), and uncleaved Poly (ADP-ribose) polymerase-1 in the hippocampal subfield neuronal and nonneuronal cells (DG, CA1, CA2, and CA3). We observed that, short-term as well as long-term 2.45 GHz MW radiation exposure increases the oxidative/nitrosative stress leading to enhanced apoptosis in hippocampal subfield neuronal and nonneuronal cells. Present findings also suggest that learning and spatial memory deficit which increases with the increased duration of MW exposure (15 < 30 < 60 days) is correlated with a decrease in hippocampal subfield neuronal arborization and dendritic spines. These findings led us to conclude that exposure to CW MW radiation leads to oxidative/nitrosative stress induced p53-dependent/independent activation of hippocampal neuronal and nonneuronal apoptosis associated with spatial memory loss. PMID:26396154

  9. The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

    PubMed Central

    Ho, Alan L; Schwartz, Gary K

    2012-01-01

    Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53−/− cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53-dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A. PMID:22293494

  10. SMC3 knockdown triggers genomic instability and p53-dependent apoptosis in human and zebrafish cells

    PubMed Central

    Ghiselli, Giancarlo

    2006-01-01

    Background The structural maintenance of chromosome 3 (SMC3) protein is a constituent of a number of nuclear multimeric protein complexes that are involved in DNA recombination and repair in addition to chromosomal segregation. Overexpression of SMC3 activates a tumorigenic cascade through which mammalian cells acquire a transformed phenotype. This has led us to examine in depth how SMC3 level affects cell growth and genomic stability. In this paper the effect of SMC3 knockdown has been investigated. Results Mammalian cells that are SMC3 deficient fail to expand in a clonal population. In order to shed light on the underlying mechanism, experiments were conducted in zebrafish embryos in which cell competence to undergo apoptosis is acquired at specific stages of development and affects tissue morphogenesis. Zebrafish Smc3 is 95% identical to the human protein, is maternally contributed, and is expressed ubiquitously at all developmental stages. Antisense-mediated loss of Smc3 function leads to increased apoptosis in Smc3 expressing cells of the developing tail and notocord causing morphological malformations. The apoptosis and the ensuing phenotype can be suppressed by injection of a p53-specific MO that blocks the generation of endogenous p53 protein. Results in human cells constitutively lacking p53 or BAX, confirmed that a p53-dependent pathway mediates apoptosis in SMC3-deficient cells. A population of aneuploid cells accumulated in zebrafish embryos following Smc3-knockdown whereas in human cells the transient downregulation of SMC3 level lead to the generation of cells with amplified centrosome number. Conclusion Smc3 is required for normal embryonic development. Its deficiency affects the morphogenesis of tissues with high mitotic index by triggering an apoptotic cascade involving p53 and the downstream p53 target gene bax. Cells with low SMC3 level display centrosome abnormalities that can lead to or are the consequence of dysfunctional mitosis and

  11. p53-dependent SIRT6 expression protects Aβ42-induced DNA damage

    PubMed Central

    Jung, Eun Sun; Choi, Hyunjung; Song, Hyundong; Hwang, Yu Jin; Kim, Ahbin; Ryu, Hoon; Mook-Jung, Inhee

    2016-01-01

    Alzheimer’s disease (AD) is the most common type of dementia and age-related neurodegenerative disease. Elucidating the cellular changes that occur during ageing is an important step towards understanding the pathogenesis and progression of neurodegenerative disorders. SIRT6 is a member of the mammalian sirtuin family of anti-aging genes. However, the relationship between SIRT6 and AD has not yet been elucidated. Here, we report that SIRT6 protein expression levels are reduced in the brains of both the 5XFAD AD mouse model and AD patients. Aβ42, a major component of senile plaques, decreases SIRT6 expression, and Aβ42-induced DNA damage is prevented by the overexpression of SIRT6 in HT22 mouse hippocampal neurons. Also, there is a strong negative correlation between Aβ42-induced DNA damage and p53 levels, a protein involved in DNA repair and apoptosis. In addition, upregulation of p53 protein by Nutlin-3 prevents SIRT6 reduction and DNA damage induced by Aβ42. Taken together, this study reveals that p53-dependent SIRT6 expression protects cells from Aβ42-induced DNA damage, making SIRT6 a promising new therapeutic target for the treatment of AD. PMID:27156849

  12. Identification of novel radiation-induced p53-dependent transcripts extensively regulated during mouse brain development.

    PubMed

    Quintens, Roel; Verreet, Tine; Janssen, Ann; Neefs, Mieke; Leysen, Liselotte; Michaux, Arlette; Verslegers, Mieke; Samari, Nada; Pani, Giuseppe; Verheyde, Joris; Baatout, Sarah; Benotmane, Mohammed A

    2015-01-01

    Ionizing radiation is a potent activator of the tumor suppressor gene p53, which itself regulates the transcription of genes involved in canonical pathways such as the cell cycle, DNA repair and apoptosis as well as other biological processes like metabolism, autophagy, differentiation and development. In this study, we performed a meta-analysis on gene expression data from different in vivo and in vitro experiments to identify a signature of early radiation-responsive genes which were predicted to be predominantly regulated by p53. Moreover, we found that several genes expressed different transcript isoforms after irradiation in a p53-dependent manner. Among this gene signature, we identified novel p53 targets, some of which have not yet been functionally characterized. Surprisingly, in contrast to genes from the canonical p53-regulated pathways, our gene signature was found to be highly enriched during embryonic and post-natal brain development and during in vitro neuronal differentiation. Furthermore, we could show that for a number of genes, radiation-responsive transcript variants were upregulated during development and differentiation, while radiation non-responsive variants were not. This suggests that radiation exposure of the developing brain and immature cortical neurons results in the p53-mediated activation of a neuronal differentiation program. Overall, our results further increase the knowledge of the radiation-induced p53 network of the embryonic brain and provide more evidence concerning the importance of p53 and its transcriptional targets during mouse brain development. PMID:25681390

  13. Low Dose Radiation Hypersensitivity is Caused by p53-dependent Apoptosis

    SciTech Connect

    Enns, L; Bogen, K; Wizniak, J; Murtha, A; Weinfeld, M

    2004-04-08

    Exposure to environmental radiation and the application of new clinical modalities, such as radioimmunotherapy, have heightened the need to understand cellular responses to low dose and low-dose rate ionizing radiation. Many tumor cell lines have been observed to exhibit a hypersensitivity to radiation doses below 50 cGy, which manifests as a significant deviation from the clonogenic survival response predicted by a linear-quadratic fit to higher doses. However, the underlying processes for this phenomenon remain unclear. Using a gel microdrop/flow cytometry assay to monitor single cell proliferation at early times post irradiation, we examined the response of human A549 lung carcinoma, T98G glioma and MCF7 breast carcinoma cell lines exposed to gamma radiation doses from 0 to 200 cGy delivered at 0.18 and 22 cGy/min. The A549 and T98G cells, but not MCF7 cells, showed the marked hypersensitivity at doses <50 cGy. To further characterize the low-dose hypersensitivity, we examined the influence of low-dose radiation on cell cycle status and apoptosis by assays for active caspase-3 and phosphatidylserine translocation (annexin-V binding). We observed that caspase-3 activation and annexin-V binding mirrored the proliferation curves for the cell lines. Furthermore, the low-dose hypersensitivity and annexin-V binding to irradiated A549 and T98G cells were eliminated by treating the cells with pifithrin, an inhibitor of p53. When p53-inactive cell lines (2800T skin fibroblasts and HCT116 colorectal carcinoma cells) were examined for similar patterns, we found that there was no HRS and apoptosis was not detectable by annexin-V or caspase-3 assays. Our data therefore suggest that low-dose hypersensitivity is associated with p53-dependent apoptosis.

  14. Novel p53-dependent anticancer strategy by targeting iron signaling and BNIP3L-induced mitophagy

    PubMed Central

    Wilfinger, Nastasia; Austin, Shane; Scheiber-Mojdehkar, Barbara; Berger, Walter; Reipert, Siegfried; Praschberger, Monika; Paur, Jakob; Trondl, Robert; Keppler, Bernhard K.; Zielinski, Christoph C.; Nowikovsky, Karin

    2016-01-01

    This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46. KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics. Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme. Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy. Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization. Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L. Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway. PMID:26517689

  15. RNF12 promotes p53-dependent cell growth suppression and apoptosis by targeting MDM2 for destruction.

    PubMed

    Gao, Kun; Wang, Chenji; Jin, Xiaofeng; Xiao, Jiantao; Zhang, Enceng; Yang, Xianmei; Wang, Dejie; Huang, Haojie; Yu, Long; Zhang, Pingzhao

    2016-05-28

    The oncoprotein MDM2 is an E3 ubiquitin ligase that targets tumor suppressor p53 for ubiquitination and proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Dysregulation of MDM2-p53 axis was frequently observed in human cancers. Originally, it is proposed that MDM2 degradation was mainly achieved by destructive self-ubiquitination. However, recent study suggests that MDM2 may be targeted for degradation by an external E3 ubiquitin ligase(s) under physiological levels. Here, we identified E3 ubiquitin ligase RNF12 as an MDM2-interacting protein through yeast two hybrid methods. We demonstrated that RNF12 targets MDM2 for ubiquitination and proteasomal-dependent degradation, which is independent of MDM2's self-ubiquitination activity. Accordingly, RNF12 elevates p53 protein level by abrogating MDM2-mediated p53 degradation and ubiquitination. Finally, we showed that RNF12 regulates cell growth suppression and DNA damage-induced apoptosis in a p53-dependent manner. Taken together, we establish RNF12 as a novel positive regulator of p53 pathway and an external E3 ubiquitin ligase for MDM2 destruction. These data shed light on the potential roles of RNF12 in MDM2-p53 axis and tumor suppression. PMID:26926424

  16. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling

    PubMed Central

    Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-01

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca2+). In the present study, we established conditions that allow the in vivo detection of Ca2+ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca2+ concentrations and, consequently, an increase in cell death in a p53-dependent pathway. PMID:25544762

  17. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation.

    PubMed

    Wang, Xinwei; Wei, Liang; Cramer, Julie M; Leibowitz, Brian J; Judge, Colleen; Epperly, Michael; Greenberger, Joel; Wang, Fengchao; Li, Linheng; Stelzner, Matthias G; Dunn, James C Y; Martin, Martin G; Lagasse, Eric; Zhang, Lin; Yu, Jian

    2015-01-01

    Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically. PMID:25858503

  18. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation

    PubMed Central

    Wang, Xinwei; Wei, Liang; Cramer, Julie M.; Leibowitz, Brian J.; Judge, Colleen; Epperly, Michael; Greenberger, Joel; Wang, Fengchao; Li, Linheng; Stelzner, Matthias G.; Dunn, James C. Y.; Martin, Martin G.; Lagasse, Eric; Zhang, Lin; Yu, Jian

    2015-01-01

    Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically. PMID:25858503

  19. Cytoplasmic CUL9/PARC ubiquitin ligase is a tumor suppressor and promotes p53-dependent apoptosis

    PubMed Central

    Pei, Xin-Hai; Bai, Feng; Li, Zhijun; Smith, Matthew D.; Whitewolf, Gabrielle; Jin, Ran; Xiong, Yue

    2011-01-01

    A wide range of cell stresses, including DNA damage, signal to p53 through post-translational modification of p53. The cytoplasmic functions of p53 are emerging as an important constituent of p53’s role in tumor suppression. Here we report that deletion of the Cul9 (formerly Parc) gene, which encodes an E3 ubiquitin ligase that binds to p53 and localizes in the cytoplasm, resulted in spontaneous tumor development, accelerated Eμ-Myc-induced lymphomagenesis and rendered mice susceptible to carcinogenesis. Cul9-p53 double mutant mice exhibited indistinguishable tumor phenotypes as p53 single mutant mice, indicating that the function of Cul9 in tumor suppression is largely mediated by p53. Deletion of Cul9 had no significant effect on cell cycle progression, but attenuated DNA damage-induced apoptosis. Ectopic expression of wild-type CUL9, but not a point mutant CUL9 deficient in p53 binding, promotes apoptosis. These results demonstrate CUL9 as a potential p53 activating E3 ligase in the cytoplasm. PMID:21487039

  20. Amphipathic silica nanoparticles induce cytotoxicity through oxidative stress mediated and p53 dependent apoptosis pathway in human liver cell line HL-7702 and rat liver cell line BRL-3A.

    PubMed

    Zuo, Daiying; Duan, Zhenfang; Jia, Yuanyuan; Chu, Tianxue; He, Qiong; Yuan, Juan; Dai, Wei; Li, Zengqiang; Xing, Liguo; Wu, Yingliang

    2016-09-01

    The aim of this study was to evaluate the potential cytotoxicity and the underlying mechanism of amphipathic silica nanoparticles (SiO2 NPs) exposure to human normal liver HL-7702 cells and rat normal liver BRL-3A cells. Prior to the cellular studies, transmission electron microscopy (TEM), dynamic light scattering (DLS), and X ray diffraction (XRD) were used to characterize SiO2 NPs, which proved the amorphous nature of SiO2 NPs with TEM diameter of 19.8±2.7nm. Further studies proved that exposure to SiO2 NPs dose-dependently induced cytotoxicity as revealed by cell counting kit (CCK-8) and lactate dehydrogenase (LDH) assays, with more severe cytotoxicity in HL-7702 cells than BRL-3A cells. Reactive oxygen species (ROS) and glutathione (GSH) assays showed elevated oxidative stress in both cells. Morphological studies by microscopic observation, Hochest 33258 and AO/EB staining indicated significant apoptotic changes after the cells being exposed to SiO2 NPs. Further studies by western blot indicated that SiO2 NPs exposure to both cells up-regulated p53, Bax and cleaved caspase-3 expression and down-regulated Bcl-2 and caspase-3 levels. Activated caspase-3 activity detected by colorimetric assay kit and caspase-3/7 activity detected by fluorescent real-time detection kit were significantly increased by SiO2 NPs exposure. In addition, antioxidant vitamin C significantly attenuated SiO2 NPs-induced caspase-3 activation, which indicated that SiO2 NPs-induced oxidative stress was involved in the process of HL-7702 and BRL-3A cell apoptosis. Taken together, these results suggested that SiO2 NPs-induced cytotoxicity in HL-7702 and BRL-3A cells was through oxidative stress mediated and p53, caspase-3 and Bax/Bcl-2 dependent pathway and HL-7702 cells were more sensitive to SiO2 NPs-induced cytotoxicity than BRL-3A cells. PMID:27187187

  1. Regulation of p53-dependent apoptosis, transcriptional repression, and cell transformation by phosphorylation of the 55-kilodalton E1B protein of human adenovirus type 5.

    PubMed Central

    Teodoro, J G; Branton, P E

    1997-01-01

    The adenovirus type 5 55-kDa E1B protein (E1B-55kDa) cooperates with E1A gene products to induce cell transformation. E1A proteins stimulate DNA synthesis and cell proliferation; however, they also cause rapid cell death by p53-dependent and p53-independent apoptosis. It is believed that the role of the E1B-55kDa protein in transformation is to protect against p53-dependent apoptosis by binding to and inactivating p53. It has been shown previously that the 55-kDa polypeptide abrogates p53-mediated transactivation and that mutants defective in p53 binding are unable to cooperate with E1A in transformation. We have previously mapped phosphorylation sites near the carboxy terminus of the E1B-55kDa protein at Ser-490 and Ser-491, which lie within casein kinase II consensus sequences. Conversion of these sites to alanine residues greatly reduced transforming activity, and although the mutant 55-kDa protein was found to interact with p53 at normal levels, it was somewhat defective for suppression of p53 transactivation activity. We now report that a nearby residue, Thr-495, also appears to be phosphorylated. We demonstrate directly that the wild-type 55-kDa protein is able to block E1A-induced p53-dependent apoptosis, whereas cells infected by mutant pm490/1/5A, which contains alanine residues at all three phosphorylation sites, exhibited extensive DNA fragmentation and classic apoptotic cell death. The E1B-55kDa product has been shown to exhibit intrinsic transcriptional repression activity when localized to promoters, such as by fusion with the GAL4 DNA-binding domain, even in the absence of p53. Such repression activity was totally absent with mutant pm490/1/5A. These data suggested that inhibition of p53-dependent apoptosis may depend on the transcriptional repression function of the 55-kDa protein, which appears to be regulated be phosphorylation at the carboxy terminus. PMID:9094635

  2. The absence of Prep1 causes p53-dependent apoptosis of mouse pluripotent epiblast cells.

    PubMed

    Fernandez-Diaz, Luis C; Laurent, Audrey; Girasoli, Sara; Turco, Margherita; Longobardi, Elena; Iotti, Giorgio; Jenkins, Nancy A; Fiorenza, Maria Teresa; Copeland, Neal G; Blasi, Francesco

    2010-10-01

    Disruption of mouse Prep1, which codes for a homeodomain transcription factor, leads to embryonic lethality during post-implantation stages. Prep1(-/-) embryos stop developing after implantation and before anterior visceral endoderm (AVE) formation. In Prep1(-/-) embryos at E6.5 (onset of gastrulation), the AVE is absent and the proliferating extra-embryonic ectoderm and epiblast, marked by Bmp4 and Oct4, respectively, are reduced in size. At E.7.5, Prep1(-/-) embryos are small and very delayed, showing no evidence of primitive streak or of differentiated embryonic lineages. Bmp4 is expressed residually, while the reduced number of Oct4-positive cells is constant up to E8.5. At E6.5, Prep1(-/-) embryos retain a normal mitotic index but show a major increase in cleaved caspase 3 and TUNEL staining, indicating apoptosis. Therefore, the mouse embryo requires Prep1 when undergoing maximal expansion in cell number. Indeed, the phenotype is partially rescued in a p53(-/-), but not in a p16(-/-), background. Apoptosis is probably due to DNA damage as Atm downregulation exacerbates the phenotype. Despite this early lethal phenotype, Prep1 is not essential for ES cell establishment. A differential embryonic expression pattern underscores the unique function of Prep1 within the Meis-Prep family. PMID:20826531

  3. Bcl6a function is required during optic cup formation to prevent p53-dependent apoptosis and colobomata

    PubMed Central

    Lee, Jiwoon; Lee, Bum-Kyu; Gross, Jeffrey M.

    2013-01-01

    Mutations in BCOR (Bcl6 corepressor) are found in patients with oculo-facio-cardio-dental (OFCD) syndrome, a congenital disorder affecting visual system development, and loss-of-function studies in zebrafish and Xenopus demonstrate a role for Bcor during normal optic cup development in preventing colobomata. The mechanism whereby BCOR functions during eye development to prevent colobomata is not known, but in other contexts it serves as a transcriptional corepressor that potentiates transcriptional repression by B cell leukemia/lymphoma 6 (BCL6). Here, we have explored the function of the zebrafish ortholog of Bcl6, Bcl6a, during eye development, and our results demonstrate that Bcl6a, like Bcor, is required to prevent colobomata during optic cup formation. Our data demonstrate that Bcl6a acts downstream of Vax1 and Vax2, known regulators of ventral optic cup formation and choroid fissure closure, and that bcl6a is a direct target of Vax2. Together, this regulatory network functions to repress p53 expression and thereby suppress apoptosis in the developing optic cup. Furthermore, our data demonstrate that Bcl6a functions cooperatively with Bcor, Rnf2 and Hdac1 in a common gene regulatory network that acts to repress p53 and prevent colobomata. Together, these data support a model in which p53-dependent apoptosis needs to be tightly regulated for normal optic cup formation and that Bcl6a, Bcor, Rnf2 and Hdac1 activities mediate this regulation. PMID:23669349

  4. Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

    PubMed

    Hare, Ian; Evans, Rebecca; Fortney, James; Moses, Blake; Piktel, Debbie; Slone, William; Gibson, Laura F

    2016-10-01

    Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted. PMID:27586146

  5. P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest

    PubMed Central

    Shamseddine, A A; Clarke, C J; Carroll, B; Airola, M V; Mohammed, S; Rella, A; Obeid, L M; Hannun, Y A

    2015-01-01

    Neutral sphingomyelinase-2 (nSMase2) is a ceramide-generating enzyme that has been implicated in growth arrest, apoptosis and exosome secretion. Although previous studies have reported transcriptional upregulation of nSMase2 in response to daunorubicin, through Sp1 and Sp3 transcription factors, the role of the DNA damage pathway in regulating nSMase2 remains unclear. In this study, we show that doxorubicin induces a dose-dependent induction of nSMase2 mRNA and protein with concomitant increases in nSMase activity and ceramide levels. Upregulation of nSMase2 was dependent on ATR, Chk1 and p53, thus placing it downstream of the DNA damage pathway. Moreover, overexpression of p53 was sufficient to transcriptionally induce nSMase2, without the need for DNA damage. DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest. Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest. Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3. These results identify nSMase2 as a novel p53 target gene, regulated by the DNA damage pathway to induce cell growth arrest. PMID:26512957

  6. p53 Dependent Apoptotic Cell Death Induces Embryonic Malformation in Carassius auratus under Chronic Hypoxia

    PubMed Central

    Dasgupta, Subrata; Sawant, Bhawesh T.; Chadha, Narinder K.; Pal, Asim K.

    2014-01-01

    Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos. PMID:25068954

  7. HZE particle radiation induces tissue-specific and p53-dependent mutagenesis in transgenic animals

    NASA Technical Reports Server (NTRS)

    Chang, P. Y.; Kanazawa, N.; Lutze-Mann, L.; Winegar, R.

    2001-01-01

    Transgenic animals, with the integrated target gene, provide a unique approach for measuring and characterizing mutations in any tissue of the animal. We are using the plasmid-based lacZ transgenic mice with different p53 genetic background to examine radiation-induced genetic damage resulting from exposure to heavy particle radiation. We measured lacZ mutation frequencies (MF) in the brain and spleen tissues at various times after exposing animals to an acute dose of 1 Gy of 1GeV/amu iron particles. MF in the spleen of p53+/+ animals increased up to 2.6-fold above spontaneous levels at 8 weeks post irradiation. In contrast, brain MF from the same animals increased 1.7-fold above controls in the same period. In the p53-/- animals, brain MF increased to 2.2-fold above spontaneous levels at 1 week after treatment, but returned to control levels thereafter. Radiation also induced alterations in the spectrum of mutants in both tissues, accompanied by changes in the frequency of mutants with deletions extending past the transgene into mouse genomic DNA. Our results indicate that the accumulation of transgene MF after radiation exposure is dependant on the tissue examined as well as the p53 genetic background of the animals.

  8. HZE particle radiation induces tissue-specific and p53-dependent mutagenesis in transgenic animals.

    PubMed

    Chang, P Y; Kanazawa, N; Lutze-Mann, L; Winegar, R

    2001-01-01

    Transgenic animals, with the integrated target gene, provide a unique approach for measuring and characterizing mutations in any tissue of the animal. We are using the plasmid-based lacZ transgenic mice with different p53 genetic background to examine radiation-induced genetic damage resulting from exposure to heavy particle radiation. We measured lacZ mutation frequencies (MF) in the brain and spleen tissues at various times after exposing animals to an acute dose of 1 Gy of 1GeV/amu iron particles. MF in the spleen of p53+/+ animals increased up to 2.6-fold above spontaneous levels at 8 weeks post irradiation. In contrast, brain MF from the same animals increased 1.7-fold above controls in the same period. In the p53-/- animals, brain MF increased to 2.2-fold above spontaneous levels at 1 week after treatment, but returned to control levels thereafter. Radiation also induced alterations in the spectrum of mutants in both tissues, accompanied by changes in the frequency of mutants with deletions extending past the transgene into mouse genomic DNA. Our results indicate that the accumulation of transgene MF after radiation exposure is dependant on the tissue examined as well as the p53 genetic background of the animals. PMID:11776257

  9. Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway.

    PubMed

    Bian, Tao; Gibbs, John D; Örvell, Claes; Imani, Farhad

    2012-01-01

    Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication. PMID:22662266

  10. Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression in HEK293 human cells and in murine neurons.

    PubMed

    Alves da Costa, Cristine; Paitel, Erwan; Mattson, Mark P; Amson, Robert; Telerman, Adam; Ancolio, Karine; Checler, Frédéric; Mattson, Marc P

    2002-03-19

    Presenilins 1 and 2 are two homologous proteins that, when mutated, account for most early onset Alzheimer's disease. Several lines of evidence suggest that, among various functions, presenilins could modulate cell apoptotic responses. Here we establish that the overexpression of presenilin 2 (PS2) and its mutated form Asn-141-Ile-PS2 alters the viability of human embryonic kidney (HEK)293 cells as established by combined trypan blue exclusion, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay, and propidium iodide incorporation FACS analyses. The two parent proteins increase the acetyl-DEVD-al-sensitive caspase-3-like activity in both HEK293 cells and Telencephalon specific murine neurons, modulate Bax and bcl-2 expressions, and enhance cytochrome C translocation into the cytosol. We show that overexpression of both wild-type and mutated PS2 increases p53-like immunoreactivity and transcriptional activity. We also establish that wild-type- and mutated PS2-induced caspase activation is reduced by p53 antisense approach and by pifithrin-alpha, a chemical inhibitor of p53. Furthermore, mouse fibroblasts in which the PS2 gene has been knocked out exhibited strongly reduced p53-transcriptional activity. Finally, we establish that the overexpression of both wild-type and mutated PS2 is accompanied by a drastic reduction of endogenous presenilin 1 (PS1) expression. Interestingly, pifithrin-alpha diminished endogenous PS2 immunoreactivity, whereas the inhibitor increases PS1 expression. Altogether, our data demonstrate that wild-type and familial Alzheimer's disease-linked PS2 trigger apoptosis and down-regulate PS1 expression through p53-dependent mechanisms. PMID:11904448

  11. Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

    PubMed Central

    Bernhart, Eva; Damm, Sabine; Heffeter, Petra; Wintersperger, Andrea; Asslaber, Martin; Frank, Saša; Hammer, Astrid; Strohmaier, Heimo; DeVaney, Trevor; Mrfka, Manuel; Eder, Hans; Windpassinger, Christian; Ireson, Christopher R.; Mischel, Paul S.; Berger, Walter; Sattler, Wolfgang

    2014-01-01

    Background Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo. Methods The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling. Results RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53wt (U87MG, A172, and primary GBM2), and p53mut (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53wt and p53mut cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53wt and p53mut GBM cells. PRKD2 knockdown in p53wt cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53mut GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation. Conclusions PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways. PMID:24463355

  12. The p53-dependent radioadaptive response

    NASA Astrophysics Data System (ADS)

    Ohnishi, Takeo

    We already reported that conditioning exposures at low doses, or at low dose-rates, lowered radiation-induced p53-dependent apoptosis in cultured cells in vitro and in the spleens of mice in vivo. In this study, the aim was to characterize the p53-dependent radioadaptive response at the molecular level. We used wild-type (wt) p53 and mutated (m) p53 containing cells derived from the human lung cancer H1299 cell line, which is p53-null. Cellular radiation sensitivities were determined with a colony-forming assay. The accumulation of p53, Hdm2, and iNOS was analyzed with Western blotting. The quantification of chromosomal aberrations was estimated by scoring dicentrics per cell. In wtp53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wtp53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were seen to increase about 2-4 fold after challenging irradiation following a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wtp53 cells, induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of Pifithrin-α (a p53 inhibitor), RITA or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an iNOS inhibitor) and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover, radioresistance developed when wtp53 cells were treated with ISDN (an NO generating agent) alone. These findings suggest that NO radicals are an initiator of the radioadaptive response acting through the activation of Hdm2 and the depression of p53 accumulations.

  13. Polychlorinated Biphenyl Quinone Metabolite Promotes p53-Dependent DNA Damage Checkpoint Activation, S-Phase Cycle Arrest and Extrinsic Apoptosis in Human Liver Hepatocellular Carcinoma HepG2 Cells.

    PubMed

    Song, Xiufang; Li, Lingrui; Shi, Qiong; Lehmler, Hans-Joachim; Fu, Juanli; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-11-16

    Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants. The toxic behavior and mechanism of PCBs individuals and congeners have been extensively investigated. However, there is only limited information on their metabolites. Our previous studies have shown that a synthetic PCB metabolite, PCB29-pQ, causes oxidative damage with the evidence of cytotoxicity, genotoxicity, and mitochondrial-derived intrinsic apoptosis. Here, we investigate the effects of PCB29-pQ on DNA damage checkpoint activation, cell cycle arrest, and death receptor-related extrinsic apoptosis in human liver hepatocellular carcinoma HepG2 cells. Our results illustrate that PCB29-pQ increases the S-phase cell population by down-regulating cyclins A/D1/E, cyclin-dependent kinases (CDK 2/4/6), and cell division cycle 25A (CDC25A) and up-regulating p21/p27 protein expressions. PCB29-pQ also induces apoptosis via the up-regulation of Fas/FasL and the activation of caspase 8/3. Moreover, p53 plays a pivotal role in PCB29-pQ-induced cell cycle arrest and apoptosis via the activation of ATM/Chk2 and ATR/Chk1 checkpoints. Cell cycle arrest and apoptotic cell death were attenuated by the pretreatment with antioxidant N-acetyl-cysteine (NAC). Taken together, these results demonstrate that PCB29-pQ induces oxidative stress and promotes p53-dependent DNA damage checkpoint activation, S-phase cycle arrest, and extrinsic apoptosis in HepG2 cells. PMID:26451628

  14. Trifluridine Induces p53-Dependent Sustained G2 Phase Arrest with Its Massive Misincorporation into DNA and Few DNA Strand Breaks.

    PubMed

    Matsuoka, Kazuaki; Iimori, Makoto; Niimi, Shinichiro; Tsukihara, Hiroshi; Watanabe, Sugiko; Kiyonari, Shinichi; Kiniwa, Mamoru; Ando, Koji; Tokunaga, Eriko; Saeki, Hiroshi; Oki, Eiji; Maehara, Yoshihiko; Kitao, Hiroyuki

    2015-04-01

    Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102, which consists of FTD and a thymidine phosphorylase inhibitor. Like 5-fluoro-2'-deoxyuridine (FdUrd), a deoxynucleoside form of 5-fluorouracil metabolite, FTD is sequentially phosphorylated and not only inhibits thymidylate synthase activity, but is also incorporated into DNA. Although TAS-102 was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, the mechanism of FTD-induced cytotoxicity is not completely understood. Here, we show that FTD as well as FdUrd induce transient phosphorylation of Chk1 at Ser345, and that this is followed by accumulation of p53 and p21 proteins in p53-proficient human cancer cell lines. In particular, FTD induced p53-dependent sustained arrest at G2 phase, which was associated with a proteasome-dependent decrease in the Cyclin B1 protein level and the suppression of CCNB1 and CDK1 gene expression. In addition, a p53-dependent increase in p21 protein was associated with an FTD-induced decrease in Cyclin B1 protein. Although numerous ssDNA and dsDNA breaks were induced by FdUrd, few DNA strand breaks were detected in FTD-treated HCT-116 cells despite massive FTD misincorporation into genomic DNA, suggesting that the antiproliferative effect of FTD is not due to the induction of DNA strand breaks. These distinctive effects of FTD provide insights into the cellular mechanism underlying its antitumor effect and may explain the clinical efficacy of TAS-102. PMID:25700705

  15. p53-dependent global nucleotide excision repair of cisplatin-induced intrastrand cross links in human cells.

    PubMed

    Bhana, Sara; Hewer, Alan; Phillips, David H; Lloyd, Daniel R

    2008-03-01

    Cisplatin is an extremely effective chemotherapeutic agent used for the treatment of testicular and other solid tumours. It induces a variety of structural modifications in DNA, the most abundant being the GpG- and ApG-1,2-intrastrand cross links formed between adjacent purine bases. These cross links account for approximately 90% of cisplatin-induced DNA damage and are thought to be responsible for the cytotoxic activity of the drug. In human cells, the nucleotide excision repair (NER) process removes the intrastrand cross links from the genome, the efficiency of which is likely to be an important determinant of cisplatin cytotoxicity. We have investigated whether the p53 tumour suppressor status affects global NER of cisplatin-induced intrastrand cross links in human cells. We have used a (32)P-postlabelling method to monitor the removal of GpG- and ApG-intrastrand cross links from two human cell models (the 041TR system, in which p53 is regulated by a tetracycline-inducible promoter, together with WI38 fibroblasts and the SV40-transformed derivative VA13) that each differ in p53 status. We demonstrate that the absence of functional p53 leads to persistence of both cisplatin-induced intrastrand cross links in the genome, suggesting that p53 regulates NER of these DNA lesions. This observation extends the role of p53 in NER beyond enhancing the removal of environmentally induced DNA lesions to include those of clinical origin. Given the frequency of p53 mutations in human tumours, these results may have implications for the use of cisplatin in cancer chemotherapy. PMID:18267949

  16. Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

    SciTech Connect

    Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C. . E-mail: mary-horne@uiowa.edu

    2006-12-10

    Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G{sub 1}/S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles

  17. Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer

    PubMed Central

    Thorenoor, Nithyananda; Faltejskova-Vychytilova, Petra; Hombach, Sonja; Mlcochova, Jitka; Kretz, Markus; Svoboda, Marek; Slaby, Ondrej

    2016-01-01

    We determined expression of 83 long non-coding RNAs (lncRNAs) and identified ZFAS1 to be significantly up-regulated in colorectal cancer (CRC) tissue. In cohort of 119 CRC patients we observed that 111 cases displayed at least two-times higher expression of ZFAS1 in CRC compared to paired normal colorectal tissue (P < 0.0001). By use of CRC cell lines (HCT116+/+, HCT116−/− and DLD-1) we showed, that ZFAS1 silencing decreases proliferation through G1-arrest of cell cycle, and also tumorigenicity of CRC cells. We identified Cyclin-dependent kinase 1 (CDK1) as interacting partner of ZFAS1 by pull-down experiment and RNA immunoprecipitation. Further, we have predicted by bioinformatics approach ZFAS1 to sponge miR-590-3p, which was proved to target CDK1. Levels of CDK1 were not affected by ZFAS1 silencing, but cyclin B1 was decreased in both cell lines. We observed significant increase in p53 levels and PARP cleavage in CRC cell lines after ZFAS1 silencing indicating increase in apoptosis. Our data suggest that ZFAS1 may function as oncogene in CRC by two main actions: (i) via destabilization of p53 and through (ii) interaction with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. However, molecular mechanisms behind these interactions have to be further clarified. PMID:26506418

  18. AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation

    SciTech Connect

    Morita, Akinori; Ariyasu, Shinya; Wang, Bing; Asanuma, Tetsuo; Onoda, Takayoshi; Sawa, Akiko; Tanaka, Kaoru; Takahashi, Ippei; Togami, Shotaro; Nenoi, Mitsuru; Inaba, Toshiya; Aoki, Shin

    2014-08-08

    Highlights: • A bidentate HQ derivative, AS-2, suppresses p53-dependent apoptosis by DNA damage. • AS-2 does not significantly affect nuclear p53 response. • UV-excited blue emission of AS-2 clearly showed its extranuclear localization. • AS-2 prevents mitochondrial dysfunction despite the increase of mitochondrial p53. • AS-2 protects mice from a radiation dose that causes lethal hematopoietic syndrome. - Abstract: In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-μ (PFTμ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also

  19. Disruption of G1-phase phospholipid turnover by inhibition of Ca2+-independent phospholipase A2 induces a p53-dependent cell-cycle arrest in G1 phase.

    PubMed

    Zhang, Xu Hannah; Zhao, Chunying; Seleznev, Konstantin; Song, Keying; Manfredi, James J; Ma, Zhongmin Alex

    2006-03-15

    The G1 phase of the cell cycle is characterized by a high rate of membrane phospholipid turnover. Cells regulate this turnover by coordinating the opposing actions of CTP:phosphocholine cytidylyltransferase and the group VI Ca2+-independent phospholipase A2 (iPLA2). However, little is known about how such turnover affects cell-cycle progression. Here, we show that G1-phase phospholipid turnover is essential for cell proliferation. Specific inhibition of iPLA2 arrested cells in the G1 phase of the cell cycle. This G1-phase arrest was associated with marked upregulation of the tumour suppressor p53 and the expression of cyclin-dependent kinase inhibitor p21cip1. Inactivation of iPLA2 failed to arrest p53-deficient HCT cells in the G1 phase and caused massive apoptosis of p21-deficient HCT cells, suggesting that this G1-phase arrest requires activation of p53 and expression of p21cip1. Furthermore, downregulation of p53 by siRNA in p21-deficient HCT cells reduced the cell death, indicating that inhibition of iPLA2 induced p53-dependent apoptosis in the absence of p21cip1. Thus, our study reveals hitherto unrecognized cooperation between p53 and iPLA2 to monitor membrane-phospholipid turnover in G1 phase. Disrupting the G1-phase phospholipid turnover by inhibition of iPLA2 activates the p53-p21cip1 checkpoint mechanism, thereby blocking the entry of G1-phase cells into S phase. PMID:16492706

  20. Activation of p53-dependent responses in tumor cells treated with a PARC-interacting peptide

    SciTech Connect

    Vitali, Roberta; Cesi, Vincenzo; Tanno, Barbara; Ferrari-Amorotti, Giovanna; Dominici, Carlo; Calabretta, Bruno; Raschella, Giuseppe

    2008-04-04

    We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.

  1. Activation of p53-dependent responses in tumor cells treated with a PARC-interacting peptide.

    PubMed

    Vitali, Roberta; Cesi, Vincenzo; Tanno, Barbara; Ferrari-Amorotti, Giovanna; Dominici, Carlo; Calabretta, Bruno; Raschellà, Giuseppe

    2008-04-01

    We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53. PMID:18230339

  2. AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation.

    PubMed

    Morita, Akinori; Ariyasu, Shinya; Wang, Bing; Asanuma, Tetsuo; Onoda, Takayoshi; Sawa, Akiko; Tanaka, Kaoru; Takahashi, Ippei; Togami, Shotaro; Nenoi, Mitsuru; Inaba, Toshiya; Aoki, Shin

    2014-08-01

    In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-μ (PFTμ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also protected mice that had been exposed to a lethal dose of ionizing radiation. Our findings indicate that some types of bidentate 8HQ chelators could serve as radioprotectors with no substantial toxicity in vivo. PMID:25026551

  3. Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

    PubMed Central

    Guha, Gunjan; Liang, Xiaobo; Kulesz-Martin, Molly F.; Mahmud, Taifo; Indra, Arup Kumar; Ganguli-Indra, Gitali

    2015-01-01

    Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G2/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21Cip1/WAF1, p27kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action. PMID:25938491

  4. A p53-dependent tumor suppressor network is induced by selective miR-125a-5p inhibition in multiple myeloma cells.

    PubMed

    Leotta, Marzia; Biamonte, Lavinia; Raimondi, Lavinia; Ronchetti, Domenica; Di Martino, Maria Teresa; Botta, Cirino; Leone, Emanuela; Pitari, Maria Rita; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco; Amodio, Nicola

    2014-12-01

    The analysis of deregulated microRNAs (miRNAs) is emerging as a novel approach to disclose the regulation of tumor suppressor or tumor promoting pathways in tumor cells. Targeting aberrantly expressed miRNAs is therefore a promising strategy for cancer treatment. By miRNA profiling of primary plasma cells from multiple myeloma (MM) patients, we previously reported increased miR-125a-5p levels associated to specific molecular subgroups. On these premises, we aimed at investigating the biological effects triggered by miR-125a-5p modulation in MM cells. Expression of p53 pathway-related genes was down-regulated in MM cells transfected with miR-125a-5p mimics. Luciferase reporter assays confirmed specific p53 targeting at 3'UTR level by miR-125a-5p mimics. Interestingly, bone marrow stromal cells (BMSCs) affected the miR-125a-5p/p53 axis, since adhesion of MM cells to BMSCs strongly up-regulated miR-125a-5p levels, while reduced p53 expression. Moreover, ectopic miR-125a-5p reduced, while miR-125-5p inhibitors promoted, the expression of tumor suppressor miR-192 and miR-194, transcriptionally regulated by p53. Lentiviral-mediated stable inhibition of miR-125a-5p expression in wild-type p53 MM cells dampened cell growth, increased apoptosis and reduced cell migration. Importantly, inhibition of in vitro MM cell proliferation and migration was also achieved by synthetic miR-125a-5p inhibitors and was potentiated by the co-expression of miR-192 or miR-194. Taken together, our data indicate that miR-125a-5p antagonism results in the activation of p53 pathway in MM cells, underlying the crucial role of this miRNA in the biopathology of MM and providing the molecular rationale for the combinatory use of miR-125a inhibitors and miR-192 or miR-194 mimics for MM treatment. PMID:24819167

  5. Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.

    PubMed

    Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

    2015-11-24

    Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention. PMID:26447614

  6. {sub p}53-Dependent Adaptive Responses in Human Cells Exposed to Space Radiations

    SciTech Connect

    Takahashi, Akihisa; Su Xiaoming; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki; Iwasaki, Toshiyasu; Ohnishi, Takeo

    2010-11-15

    Purpose: It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. Methods and Materials: Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. Results: In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. Conclusion: These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

  7. A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription

    PubMed Central

    Sammons, Morgan A.; Zhu, Jiajun; Berger, Shelley L.

    2016-01-01

    The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription. PMID:27334938

  8. A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription.

    PubMed

    Sammons, Morgan A; Zhu, Jiajun; Berger, Shelley L

    2016-01-01

    The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription. PMID:27334938

  9. Chemical Inhibition of Wild-Type p53-Induced Phosphatase 1 (WIP1/PPM1D) by GSK2830371 Potentiates the Sensitivity to MDM2 Inhibitors in a p53-Dependent Manner.

    PubMed

    Esfandiari, Arman; Hawthorne, Thomas A; Nakjang, Sirintra; Lunec, John

    2016-03-01

    Sensitivity to MDM2 inhibitors is widely different among responsive TP53 wild-type cell lines and tumors. Understanding the determinants of MDM2 inhibitor sensitivity is pertinent for their optimal clinical application. Wild-type p53-inducible phosphatase-1 (WIP1) encoded by PPM1D, is activated, gained/amplified in a range of TP53 wild-type malignancies, and is involved in p53 stress response homeostasis. We investigated cellular growth/proliferation of TP53 wild-type and matched mutant/null cell line pairs, differing in PPM1D genetic status, in response to Nutlin-3/RG7388 ± a highly selective WIP1 inhibitor, GSK2830371. We also assessed the effects of GSK2830371 on MDM2 inhibitor-induced p53(Ser15) phosphorylation, p53-mediated global transcriptional activity, and apoptosis. The investigated cell line pairs were relatively insensitive to single-agent GSK2830371. However, a non-growth-inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harboring PPM1D-activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also correlated with significant increase in MDM2 inhibitor-induced cell death endpoints that were preceded by a marked increase in a WIP1 negatively regulated substrate, phosphorylated p53(Ser15), known to increase p53 transcriptional activity. Microarray-based gene expression analysis showed that the combination treatment increases the subset of early RG7388-induced p53 transcriptional target genes. These findings demonstrate that potent and selective WIP1 inhibition potentiates the response to MDM2 inhibitors in TP53 wild-type cells, particularly those with PPM1D activation or gain, while highlighting the mechanistic importance of p53(Ser15) and its potential use as a biomarker for response to this combination regimen. Mol Cancer Ther; 15(3); 379-91. ©2016 AACR. PMID:26832796

  10. p53-Dependent Activation of microRNA-34a in Response to Etoposide-Induced DNA Damage in Osteosarcoma Cell Lines Not Impaired by Dominant Negative p53 Expression

    PubMed Central

    Novello, Chiara; Pazzaglia, Laura; Conti, Amalia; Quattrini, Irene; Pollino, Serena; Perego, Paola; Picci, Piero; Benassi, Maria Serena

    2014-01-01

    Osteosarcoma (OS) is the most common primary malignant bone tumor and prevalently occurs in the second decade of life. Etoposide, a chemotherapeutic agent used in combined treatments of recurrent human OS, belongs to the topoisomerase inhibitor family and causes DNA breakage. In this study we evaluated the cascade of events determined by etoposide-induced DNA damage in OS cell lines with different p53 status focusing on methylation status and expression of miR-34a that modulate tumor cell growth and cell cycle progression. Wild-type p53 U2-OS cells and U2-OS cells expressing dominant-negative form of p53 (U2- OS175) were more sensitive to etoposide than p53-deficient MG63 and Saos-2 cells, showing increased levels of unmethylated miR-34a, reduced expression of CDK4 and cell cycle arrest in G1 phase. In contrast, MG63 and Saos-2 cell lines presented aberrant methylation of miR-34a promoter gene with no miR-34a induction after etoposide treatment, underlining the close connection between p53 expression and miR-34a methylation status. Consistently, in p53siRNA transfected U2-OS cells we observed loss of miR-34a induction after etoposide exposure associated with a partial gain of gene methylation and cell cycle progress towards G2/M phase. Our results suggest that the open and unmethylated conformation of the miR-34a gene may be regulated by p53 able to bind the gene promoter. In conclusion, cell response to etoposide-induced DNA damage was not compromised in cells with dominant-negative p53 expression. PMID:25490093

  11. The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

  12. p53-dependent inhibition of mammalian cell survival by a genetically selected peptide aptamer that targets the regulatory subunit of protein kinase CK2.

    PubMed

    Martel, V; Filhol, O; Colas, P; Cochet, C

    2006-11-30

    Based on the perturbation of its expression in human cancers and on its involvement in transformation and tumorigenesis, protein kinase CK2 has recently attracted attention as a potential therapeutic target. To assess the value of CK2 as a target for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically the regulatory CK2beta subunit. Here, we use a two-hybrid approach to isolate from combinatorial libraries, peptide aptamers that specifically interact with CK2beta. One of these (P1), which has significant sequence homology to the cytomegalovirus IE2 protein, binds with high affinity to the N-terminal domain of CK2beta without disrupting the formation of the CK2 holoenzyme. Expression of green fluorescent protein (GFP)-P1 in different mammalian cell lines activates p53 phosphorylation on serine 15, induces an upregulation of p21 and the release of the Cyt-C and apoptosis-inducing factor proapoptotic proteins triggering caspase-dependent and caspase-independent apoptosis. GFP-P1-induced apoptosis is associated with a p53-dependent pathway as cell death was abrogated in p53 knocked out cells. In summary, our data show that genetically selected peptide aptamers that specifically target CK2beta can induce apoptosis in mammalian cells through the recruitment of a p53-dependent apoptosis pathway. They also emphasize the critical role of CK2beta for cell survival and might allow the design of novel proapoptotic agents targeting this protein. PMID:16751801

  13. Expression of cFLIPL Determines the Basal Interaction of Bcl-2 With Beclin-1 and Regulates p53 Dependent Ubiquitination of Beclin-1 During Autophagic Stress.

    PubMed

    Ranjan, Kishu; Pathak, Chandramani

    2016-08-01

    Autophagy and apoptosis are two different physiological processes, which is required for the maintenance of cellular homeostasis. The apoptosis associated proteins such as Bcl-2 and p53 have a close association with autophagic proteins HMGB1 and Beclin-1 to modulate autophagic signaling. We demonstrate here the involvement of anti-apoptotic protein cFLIPL in the regulation of autophagy during cellular stress. We found that ectopic expression of cFLIPL decreases the sensitivity of HEK 293T cells against rapamycin and H2 O2 induced autophagic stress. Notably, the selective knockdown of cFLIPL augments autophagic stress in the cells accompanied with JNK1 activation and p53 dependent ubiquitination of Beclin-1. However, re-expression of cFLIPL in cFLIP knockdown cells restores autophagic equilibrium collectively with reversible effects on JNK1 and Beclin-1 integrity. The co-immunoprecipitation analysis suggests that cFLIPL is essential to maintain the canonical interaction of Bcl-2 with Beclin-1 to regulate autophagic stress and cell death. Altogether, our findings suggest that expression of cFLIPL regulates the basal interaction of Bcl-2 with Beclin-1 and substantiates p53 dependent ubiquitination of Beclin-1 during autophagic stress to determine the fate of cell death or survival. J. Cell. Biochem. 117: 1757-1768, 2016. © 2015 Wiley Periodicals, Inc. PMID:26682748

  14. Implication of p53-dependent cellular senescence related gene, TARSH in tumor suppression

    SciTech Connect

    Wakoh, Takeshi; Uekawa, Natsuko; Terauchi, Kunihiko; Sugimoto, Masataka; Ishigami, Akihito; Shimada, Jun-ichi; Maruyama, Mitsuo

    2009-03-20

    A novel target of NESH-SH3 (TARSH) was identified as a cellular senescence related gene in mouse embryonic fibroblasts (MEFs) replicative senescence, the expression of which has been suppressed in primary clinical lung cancer specimens. However, the molecular mechanism underlying the regulation of TARSH involved in pulmonary tumorigenesis remains unclear. Here we demonstrate that the reduction of TARSH gene expression by short hairpin RNA (shRNA) system robustly inhibited the MEFs proliferation with increase in senescence-associated {beta}-galactosidase (SA-{beta}-gal) activity. Using p53{sup -/-} MEFs, we further suggest that this growth arrest by loss of TARSH is evoked by p53-dependent p21{sup Cip1} accumulation. Moreover, we also reveal that TARSH reduction induces multicentrosome in MEFs, which is linked in chromosome instability and tumor development. These results suggest that TARSH plays an important role in proliferation of replicative senescence and may serve as a trigger of tumor development.

  15. Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT

    PubMed Central

    Leszczynska, Katarzyna B.; Foskolou, Iosifina P.; Abraham, Aswin G.; Anbalagan, Selvakumar; Tellier, Céline; Haider, Syed; Span, Paul N.; O’Neill, Eric E.; Buffa, Francesca M.; Hammond, Ester M.

    2015-01-01

    Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent apoptosis is reliant on the DNA-binding and transactivation domains of p53 but not on the acetylation sites K120 and K164, which, in contrast, are essential for DNA damage–induced, p53-dependent apoptosis. Evaluation of hypoxia-induced transcripts in multiple cell lines identified a group of genes that are hypoxia-inducible proapoptotic targets of p53, including inositol polyphosphate-5-phosphatase (INPP5D), pleckstrin domain–containing A3 (PHLDA3), sulfatase 2 (SULF2), B cell translocation gene 2 (BTG2), cytoplasmic FMR1-interacting protein 2 (CYFIP2), and KN motif and ankyrin repeat domains 3 (KANK3). These targets were also regulated by p53 in human cancers, including breast, brain, colorectal, kidney, bladder, and melanoma cancers. Downregulation of these hypoxia-inducible targets associated with poor prognosis, suggesting that hypoxia-induced apoptosis contributes to p53-mediated tumor suppression and treatment response. Induction of p53 targets, PHLDA3, and a specific INPP5D transcript mediated apoptosis in response to hypoxia through AKT inhibition. Moreover, pharmacological inhibition of AKT led to apoptosis in the hypoxic regions of p53-deficient tumors and consequently increased radiosensitivity. Together, these results identify mediators of hypoxia-induced p53-dependent apoptosis and suggest AKT inhibition may improve radiotherapy response in p53-deficient tumors. PMID:25961455

  16. Tumor-specific activation of the C-JUN/MELK pathway regulates glioma stem cell growth in a p53-dependent manner.

    PubMed

    Gu, Chunyu; Banasavadi-Siddegowda, Yeshavanth K; Joshi, Kaushal; Nakamura, Yuko; Kurt, Habibe; Gupta, Snehalata; Nakano, Ichiro

    2013-05-01

    Accumulated evidence suggests that glioma stem cells (GSCs) may contribute to therapy resistance in high-grade glioma (HGG). Although recent studies have shown that the serine/threonine kinase maternal embryonic leucine-zipper kinase (MELK) is abundantly expressed in various cancers, the function and mechanism of MELK remain elusive. Here, we demonstrate that MELK depletion by shRNA diminishes the growth of GSC-derived mouse intracranial tumors in vivo, induces glial fibrillary acidic protein (+) glial differentiation of GSCs leading to decreased malignancy of the resulting tumors, and prolongs survival periods of tumor-bearing mice. Tissue microarray analysis with 91 HGG tumors demonstrates that the proportion of MELK (+) cells is a statistically significant indicator of postsurgical survival periods. Mechanistically, MELK is regulated by the c-Jun NH(2)-terminal kinase (JNK) signaling and forms a complex with the oncoprotein c-JUN in GSCs but not in normal progenitors. MELK silencing induces p53 expression, whereas p53 inhibition induces MELK expression, indicating that MELK and p53 expression are mutually exclusive. Additionally, MELK silencing-mediated GSC apoptosis is partially rescued by both pharmacological p53 inhibition and p53 gene silencing, indicating that MELK action in GSCs is p53 dependent. Furthermore, irradiation of GSCs markedly elevates MELK mRNA and protein expression both in vitro and in vivo. Clinically, recurrent HGG tumors following the failure of radiation and chemotherapy exhibit a statistically significant elevation of MELK protein compared with untreated newly diagnosed HGG tumors. Together, our data indicate that GSCs, but not normal cells, depend on JNK-driven MELK/c-JUN signaling to regulate their survival, maintain GSCs in an immature state, and facilitate tumor radioresistance in a p53-dependent manner. PMID:23339114

  17. Zebularine inhibits tumorigenesis and stemness of colorectal cancer via p53-dependent endoplasmic reticulum stress

    PubMed Central

    Yang, Pei-Ming; Lin, Yi-Ting; Shun, Chia-Tung; Lin, Shan-Hu; Wei, Tzu-Tang; Chuang, Shu-Hui; Wu, Ming-Shiang; Chen, Ching-Chow

    2013-01-01

    Aberrant DNA hypermethylation is frequently found in tumor cells and inhibition of DNA methylation is an effective anticancer strategy. In this study, the therapeutic effect of DNA methyltransferase (DNMT) inhibitor zebularine (Zeb) on colorectal cancer (CRC) was investigated. Zeb exhibited anticancer activity in cell cultures, tumor xenografts and mouse colitis-associated CRC model. It stabilizes p53 through ribosomal protein S7 (RPS7)/MDM2 pathways and DNA damage. Zeb-induced cell death was dependent on p53. Microarray analysis revealed that genes related to endoplasmic reticulum (ER) stress and unfolded protein response (UPR) were affected by Zeb. Zeb induced p53-dependent ER stress and autophagy. Pro-survival markers of ER stress/UPR (GRP78) and autophagy (p62) were increased in tumor tissues of CRC patients, AOM/DSS-induced CRC mice and HCT116-derived colonospheres. Zeb downregulates GRP78 and p62, and upregulates a pro-apoptotic CHOP. Our results reveal a novel mechanism for the anticancer activity of Zeb. PMID:24225777

  18. COH-203, a novel microtubule inhibitor, exhibits potent anti-tumor activity via p53-dependent senescence in hepatocellular carcinoma

    SciTech Connect

    Qi, Huan; Zuo, Dai-Ying; Bai, Zhao-Shi; Xu, Jing-Wen; Li, Zeng-Qiang; Shen, Qi-Rong; Wang, Zhi-Wei; Zhang, Wei-Ge; Wu, Ying-Liang

    2014-12-12

    Highlights: • COH-203 exhibits anti-hepatoma effects in vitro and in vivo with low toxicity. • COH-203 inhibits tubulin polymerization. • COH-203 induces mitotic arrest followed by mitotic slippage in BEL-7402 cells. • COH-203 induces p53-dependent senescence in BEL-7402 cells. - Abstract: 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3H-1, 2-dithiol-3-one (COH-203) is a novel synthesized analogue of combretastatin A-4 that can be classified as a microtubule inhibitor. In this study, we evaluated the anti-hepatoma effect of COH-203 in vitro and in vivo and explored the underlying molecular mechanisms. COH-203 was shown to be more effective in inhibiting the proliferation of liver cancer cells compared with normal liver cells. COH-203 also displayed potent anti-tumor activity in a hepatocellular carcinoma xenograft model without significant toxicity. Mechanistic studies demonstrated that treatment with COH-203 induced mitotic arrest by inhibiting tubulin polymerization in BEL-7402 liver cancer cells. Long-term COH-203 treatment in BEL-7402 cells led to mitotic slippage followed by senescence via the p14{sup Arf}–p53–p21 and p16{sup INK4α}–Rb pathways. Furthermore, suppression of p53 via pifithrin-α (p53 inhibitor) and p53-siRNA attenuated COH-203-induced senescence in BEL-7402 cells, suggesting that COH-203 induced senescence p53-dependently. In conclusion, we report for the first time that COH-203, one compound in the combretastatin family, promotes anti-proliferative activity through the induction of p-53 dependent senescence. Our findings will provide a molecular rationale for the development of COH-203 as a promising anti-tumor agent.

  19. Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

    PubMed

    Stępiński, Dariusz

    2016-08-01

    Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development. PMID:27142852

  20. MDM2 Inhibition Sensitizes Prostate Cancer Cells to Androgen Ablation and Radiotherapy in a p53-Dependent Manner12

    PubMed Central

    Feng, Felix Y.; Zhang, Yu; Kothari, Vishal; Evans, Joseph R.; Jackson, William C.; Chen, Wei; Johnson, Skyler B.; Luczak, Connor; Wang, Shaomeng; Hamstra, Daniel A.

    2016-01-01

    PURPOSE: Increased murine double minute 2 (MDM2) expression, independent of p53 status, is associated with increased cancer-specific mortality for men with prostate cancer treated with radiotherapy. We assessed MI-219, a small molecule inhibitor of MDM2 with improved pharmacokinetics over nutlin-3, for sensitization of prostate cancer cells to radiotherapy and androgen deprivation therapy, a standard treatment option for men with high-risk prostate cancer. EXPERIMENTAL DESIGN: The effect of MDM2 inhibition by MI-219 was assessed in vitro and in vivo with mouse xenograft models across multiple prostate cancer cell lines containing varying p53 functional status. RESULTS: MDM2 inhibition by MI-219 resulted in dose- and time-dependent p53 activation and decreased clonogenic cell survival after radiation in a p53-dependent manner. Mechanistically, radiosensitization following inhibition of MDM2 was largely the result of p53-dependent increases in apoptosis and DNA damage as evidenced by Annexin V flow cytometry and γ-H2AX foci immunofluorescence. Similarly, treatment with MI-219 enhanced response to antiandrogen therapy via a p53-dependent increase in apoptotic cell death. Lastly, triple therapy with radiation, androgen deprivation therapy, and MI-219 decreased xenograft tumor growth compared with any single- or double-agent treatment. CONCLUSION: MDM2 inhibition with MI-219 results in p53-dependent sensitization of prostate cancer cells to radiation, antiandrogen therapy, and the combination. These findings support MDM2 small molecule inhibitor therapy as a therapy intensification strategy to improve clinical outcomes in high-risk localized prostate cancer. TRANSLATIONAL RELEVANCE: The combination of radiotherapy and androgen deprivation therapy is a standard treatment option for men with high-risk prostate cancer. Despite improvements in outcomes when androgen deprivation therapy is added to radiation, men with high-risk prostate cancer have significant risk for

  1. Fast neutrons-induced apoptosis is Fas-independent in lymphoblastoid cells

    SciTech Connect

    Fischer, Barbara; Benzina, Sami; Jeannequin, Pierre; Dufour, Patrick; Bergerat, Jean-Pierre; Denis, Jean-Marc; Gueulette, John; Bischoff, Pierre L. . E-mail: Pierre.Bischoff@ircad.u-strasbg.fr

    2005-08-26

    We have previously shown that ionizing radiation-induced apoptosis in human lymphoblastoid cells differs according to their p53 status, and that caspase 8-mediated cleavage of BID is involved in the p53-dependent pathway. In the present study, we investigated the role of Fas signaling in caspase 8 activation induced by fast neutrons irradiation in these cells. Fas and FasL expression was assessed by flow cytometry and by immunoblot. We also measured Fas aggregation after irradiation by fluorescence microscopy. We found a decrease of Fas expression after irradiation, but no change in Fas ligand expression. We also showed that, in contrast to the stimulation of Fas by an agonistic antibody, Fas aggregation did not occur after irradiation. Altogether, our data strongly suggest that fast neutrons induced-apoptosis is Fas-independent, even in p53-dependent apoptosis.

  2. Ceramide Synthase 6 Is a Novel Target of Methotrexate Mediating Its Antiproliferative Effect in a p53-Dependent Manner

    PubMed Central

    Fekry, Baharan; Esmaeilniakooshkghazi, Amin; Krupenko, Sergey A.; Krupenko, Natalia I.

    2016-01-01

    We previously reported that ceramide synthase 6 (CerS6) is elevated in response to folate stress in cancer cells, leading to enhanced production of C16-ceramide and apoptosis. Antifolate methotrexate (MTX), a drug commonly used in chemotherapy of several types of cancer, is a strong inhibitor of folate metabolism. Here we investigated whether this drug targets CerS6. We observed that CerS6 protein was markedly elevated in several cancer cell lines treated with MTX. In agreement with the enzyme elevation, its product C16-ceramide was also strongly elevated, so as several other ceramide species. The increase in C16-ceramide, however, was eliminated in MTX-treated cells lacking CerS6 through siRNA silencing, while the increase in other ceramides sustained. Furthermore, the siRNA silencing of CerS6 robustly protected A549 lung adenocarcinoma cells from MTX toxicity, while the silencing of another ceramide synthase, CerS4, which was also responsive to folate stress in our previous study, did not interfere with the MTX effect. The rescue effect of CerS6 silencing upon MTX treatment was further confirmed in HCT116 and HepG2 cell lines. Interestingly, CerS6 itself, but not CerS4, induced strong antiproliferative effect in several cancer cell lines if elevated by transient transfection. The effect of MTX on CerS6 elevation was likely p53 dependent, which is in agreement with the hypothesis that the protein is a transcriptional target of p53. In line with this notion, lometrexol, the antifolate inducing cytotoxicity through the p53-independent mechanism, did not affect CerS6 levels. We have also found that MTX induces the formation of ER aggregates, enriched with CerS6 protein. We further demonstrated that such aggregation requires CerS6 and suggests that it is an indication of ER stress. Overall, our study identified CerS6 and ceramide pathways as a novel MTX target. PMID:26783755

  3. A Novel Anticancer Agent, 8-Methoxypyrimido[4′,5′:4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and -Independent Apoptotic Pathways

    PubMed Central

    Sahu, Upasana; Sidhar, Himakshi; Ghate, Pankaj S.; Advirao, Gopal M.; Raghavan, Sathees C.; Giri, Ranjit K.

    2013-01-01

    Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido [4′,5′:4,5]thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly (ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively

  4. Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways.

    PubMed

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Feng, Tsui-Hsia; Chen, Wen-Tsung; Chang, Phei-Lang; Chiang, Hou-Yu; Juang, Horng-Heng

    2014-01-01

    Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NFκB response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NFκB pathway. PMID:24981574

  5. Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways

    PubMed Central

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Feng, Tsui-Hsia; Chen, Wen-Tsung; Chang, Phei-Lang; Chiang, Hou-Yu; Juang, Horng-Heng

    2014-01-01

    Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NFκB response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NFκB pathway. PMID:24981574

  6. The energy blockers bromopyruvate and lonidamine lead GL15 glioblastoma cells to death by different p53-dependent routes.

    PubMed

    Davidescu, Magdalena; Macchioni, Lara; Scaramozzino, Gaetano; Cristina Marchetti, Maria; Migliorati, Graziella; Vitale, Rita; Corcelli, Angela; Roberti, Rita; Castigli, Emilia; Corazzi, Lanfranco

    2015-01-01

    The energy metabolism of tumor cells relies on aerobic glycolysis rather than mitochondrial oxidation. This difference between normal and cancer cells provides a biochemical basis for new therapeutic strategies aimed to block the energy power plants of cells. The effects produced by the energy blockers bromopyruvate (3BP) and lonidamine (LND) and the underlying biochemical mechanisms were investigated in GL15 glioblastoma cells. 3BP exerts early effects compared to LND, even though both drugs lead cells to death but by different routes. A dramatic decrease of ATP levels occurred after 1 hour treatment with 3BP, followed by cytochrome c and hexokinase II degradation, and by the decrease of both LC3I/LC3II ratio and p62, markers of an autophagic flux. In addition, Akt(Ser(473)) and p53(Ser(15)/Ser(315)) dephosphorylation occurred. In LND treatment, sustained ATP cellular levels were maintained up to 40 hours. The autophagic response of cells was overcome by apoptosis that was preceded by phosphatidylinositol disappearance and pAkt decrease. This last event favored p53 translocation to mitochondria triggering a p53-dependent apoptotic route, as observed at 48 and 72 hours. Adversely, in 3BP treatment, phospho-p53 dephosphorylation targeted p53 to MDM2-dependent proteolysis, thus channeling cells to irreversible autophagy. PMID:26387611

  7. The energy blockers bromopyruvate and lonidamine lead GL15 glioblastoma cells to death by different p53-dependent routes

    PubMed Central

    Davidescu, Magdalena; Macchioni, Lara; Scaramozzino, Gaetano; Cristina Marchetti, Maria; Migliorati, Graziella; Vitale, Rita; Corcelli, Angela; Roberti, Rita; Castigli, Emilia; Corazzi, Lanfranco

    2015-01-01

    The energy metabolism of tumor cells relies on aerobic glycolysis rather than mitochondrial oxidation. This difference between normal and cancer cells provides a biochemical basis for new therapeutic strategies aimed to block the energy power plants of cells. The effects produced by the energy blockers bromopyruvate (3BP) and lonidamine (LND) and the underlying biochemical mechanisms were investigated in GL15 glioblastoma cells. 3BP exerts early effects compared to LND, even though both drugs lead cells to death but by different routes. A dramatic decrease of ATP levels occurred after 1 hour treatment with 3BP, followed by cytochrome c and hexokinase II degradation, and by the decrease of both LC3I/LC3II ratio and p62, markers of an autophagic flux. In addition, Akt(Ser473) and p53(Ser15/Ser315) dephosphorylation occurred. In LND treatment, sustained ATP cellular levels were maintained up to 40 hours. The autophagic response of cells was overcome by apoptosis that was preceded by phosphatidylinositol disappearance and pAkt decrease. This last event favored p53 translocation to mitochondria triggering a p53-dependent apoptotic route, as observed at 48 and 72 hours. Adversely, in 3BP treatment, phospho-p53 dephosphorylation targeted p53 to MDM2-dependent proteolysis, thus channeling cells to irreversible autophagy. PMID:26387611

  8. p53-dependent expression of CXCR5 chemokine receptor in MCF-7 breast cancer cells

    PubMed Central

    Mitkin, Nikita A.; Hook, Christina D.; Schwartz, Anton M.; Biswas, Subir; Kochetkov, Dmitry V.; Muratova, Alisa M.; Afanasyeva, Marina A.; Kravchenko, Julia E.; Bhattacharyya, Arindam; Kuprash, Dmitry V.

    2015-01-01

    Elevated expression of chemokine receptors in tumors has been reported in many instances and is related to a number of survival advantages for tumor cells including abnormal activation of prosurvival intracellular pathways. In this work we demonstrated an inverse correlation between expression levels of p53 tumor suppressor and CXCR5 chemokine receptor in MCF-7 human breast cancer cell line. Lentiviral transduction of MCF-7 cells with p53 shRNA led to elevated CXCR5 at both mRNA and protein levels. Functional activity of CXCR5 in p53-knockdown MCF-7 cells was also increased as shown by activation of target gene expression and chemotaxis in response to B-lymphocyte chemoattractant CXCL13. Using deletion analysis and site-directed mutagenesis of the cxcr5 gene promoter and enhancer elements, we demonstrated that p53 appears to act upon cxcr5 promoter indirectly, by repressing the activity of NFκB transcription factors. Using chromatin immunoprecipitation and reporter gene analysis, we further demonstrated that p65/RelA was able to bind the cxcr5 promoter in p53-dependent manner and to directly transactivate it when overexpressed. Through the described mechanism, elevated CXCR5 expression may contribute to abnormal cell survival and migration in breast tumors that lack functional p53. PMID:25786345

  9. Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300

    PubMed Central

    Dornan, David; Hupp, Ted R.

    2001-01-01

    The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20 whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300–p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities. PMID:11258706

  10. Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300.

    PubMed

    Dornan, D; Hupp, T R

    2001-02-01

    The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20O whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300-p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities. PMID:11258706

  11. Rpl22 loss impairs the development of B lymphocytes by activating a p53-dependent checkpoint

    PubMed Central

    Fahl, Shawn P.; Harris, Bryan; Coffey, Francis; Wiest, David L.

    2014-01-01

    While ribosomal proteins facilitate the ribosome’s core function of translation, emerging evidence suggests that some ribosomal proteins are also capable of performing tissue restricted functions either from within specialized ribosomes or from outside of the ribosome. In particular, we have previously demonstrated that germline ablation of the gene encoding ribosomal protein Rpl22 causes a selective and p53 dependent arrest of αβ T cell progenitors at the β-selection checkpoint. We have now identified a crucial role for Rpl22 during early B cell development. Germline ablation of Rpl22 results in a reduction in the absolute number of B-lineage progenitors in the bone marrow beginning at the pro-B cell stage. Although Rpl22-deficient proB cells are hyporesponsive to IL-7, a key cytokine required for early B cell development, the arrest of B cell development does not result from disrupted IL-7 signaling. Instead, p53 induction appears to be responsible for the developmental defects, as Rpl22-deficiency causes increased expression of p53 and activation of downstream p53 target genes and p53-deficiency rescues the defect in B cell development in Rpl22-deficient mice. Interestingly, the requirement for Rpl22 in the B cell lineage appears to be developmentally restricted, since Rpl22-deficient splenic B cells proliferate normally in response to antigen receptor and toll receptor stimuli and undergo normal class switch recombination. These results indicate that Rpl22 performs a critical, developmentally restricted role in supporting early B cell development by preventing p53-induction. PMID:25416806

  12. PIM1 destabilization activates a p53-dependent response to ribosomal stress in cancer cells.

    PubMed

    Sagar, Vinay; Caldarola, Sara; Aria, Valentina; Monteleone, Valentina; Fuoco, Claudia; Gargioli, Cesare; Cannata, Stefano; Loreni, Fabrizio

    2016-04-26

    Defects in ribosome biogenesis triggers a stress response (ribosomal stress) that can lead to growth arrest and apoptosis. Signaling pathways activated by ribosomal stress are specifically involved in the pathological mechanism of a group of disorders defined as ribosomopathies. However, more generally, the quality control of ribosome synthesis is part of the regulatory circuits that control cell metabolism. A number of studies identified tumor suppressor p53 as a central player in ribosomal stress. We have previously reported that the kinase PIM1 plays a role as a sensor for ribosome deficiency. In this report we address the relationship between PIM1 and p53 in cancer cell lines after depletion of a ribosomal protein. We identified a novel signaling pathway that includes the kinase AKT and the ubiquitin ligase MDM2. In fact, our results indicate that the lower level of PIM1, induced by ribosomal stress, causes inactivation of AKT, inhibition of MDM2 and a consequent p53 stabilization. Therefore, we propose that activation of p53 in response to ribosomal stress, is dependent on the pathway PIM1-AKT-MDM2. In addition, we report evidence that PIM1 level may be relevant to assess the sensitivity of cancer cells to chemotherapeutic drugs that induce ribosomal stress. PMID:26993775

  13. Hdm2 and Nitric Oxide Radicals Contribute to the P53-Dependent Radioadaptive Response

    SciTech Connect

    Takahashi, Akihisa; Matsumoto, Hideki; Ohnishi, Takeo

    2008-06-01

    Purpose: The aim of this work was to characterize the radioadaptive response at the molecular level. Methods and Materials: We used wild-type (wt) p53 and mutated (m) p53-containing cells derived from the human lung cancer H1299 cell line, which is p53-null. Cellular radiation sensitivities were determined with a colony-forming assay. The accumulations of p53, the human homolog of endogenous murine double minute 2 (Hdm2), and inducible nitric oxide synthase were analyzed with Western blotting. Quantification of chromosomal aberrations was estimated by scoring dicentrics per cell. Results: In wtp53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low-dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wtp53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were seen to increase about two- to fourfold after challenging irradiation subsequent to a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wtp53 cells, induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of 5, 5'-(2, 5-Furanidiyl)bis-2-thiophenemethanol (RITA) or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an inducible nitric oxide synthase inhibitor), and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover radioresistance developed when wtp53 cells were treated with isosorbide dinitrate (an NO-generating agent) alone. Conclusions: These findings suggest that NO radicals are initiators of the radioadaptive response, acting through the activation of Hdm2 and the depression of p53 accumulations.

  14. Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells.

    PubMed

    Seth, Rohit; Corniola, Rikki S; Gower-Winter, Shannon D; Morgan, Thomas J; Bishop, Brian; Levenson, Cathy W

    2015-04-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin-μ, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency. PMID:25467851

  15. CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

    PubMed Central

    Marjanović, Marko; Sánchez-Huertas, Carlos; Terré, Berta; Gómez, Rocío; Scheel, Jan Frederik; Pacheco, Sarai; Knobel, Philip A.; Martínez-Marchal, Ana; Aivio, Suvi; Palenzuela, Lluís; Wolfrum, Uwe; McKinnon, Peter J.; Suja, José A.; Roig, Ignasi; Costanzo, Vincenzo; Lüders, Jens; Stracker, Travis H.

    2015-01-01

    CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63 deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63 deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination. PMID:26158450

  16. Protocatechuic Acid Prevents oxLDL-Induced Apoptosis by Activating JNK/Nrf2 Survival Signals in Macrophages

    PubMed Central

    Varì, Rosaria; Scazzocchio, Beatrice; Santangelo, Carmela; Filesi, Carmelina; Galvano, Fabio; D'Archivio, Massimo; Masella, Roberta; Giovannini, Claudio

    2015-01-01

    Protocatechuic acid (PCA), one of the main metabolites of complex polyphenols, exerts numerous biological activities including antiapoptotic, anti-inflammatory, and antiatherosclerotic effects. Oxidised LDL have atherogenic properties by damaging arterial wall cells and inducing p53-dependent apoptosis in macrophages. This study was aimed at defining the molecular mechanism responsible for the protective effects of PCA against oxidative and proapoptotic damage exerted by oxLDL in J774 A.1 macrophages. We found that the presence of PCA in cells treated with oxLDL completely inhibited the p53-dependent apoptosis induced by oxLDL. PCA decreased oxLDL-induced ROS overproduction and in particular prevented the early increase of ROS. This decrease seemed to be the main signal responsible for maintaining the intracellular redox homeostasis hindering the activation of p53 induced by ROS, p38MAPK, and PKCδ. Consequently the overexpression of the proapoptotic p53-target genes such as p66Shc protein did not occur. Finally, we demonstrated that PCA induced the activation of JNK, which, in turn, determined the increase of nuclear Nrf2, leading to inhibition of the early ROS overproduction. We concluded that the antiapoptotic mechanism of PCA was most likely related to the activation of the JNK-mediated survival signals that strengthen the cellular antioxidant defences rather than to the PCA antioxidant power. PMID:26180584

  17. Protocatechuic Acid Prevents oxLDL-Induced Apoptosis by Activating JNK/Nrf2 Survival Signals in Macrophages.

    PubMed

    Varì, Rosaria; Scazzocchio, Beatrice; Santangelo, Carmela; Filesi, Carmelina; Galvano, Fabio; D'Archivio, Massimo; Masella, Roberta; Giovannini, Claudio

    2015-01-01

    Protocatechuic acid (PCA), one of the main metabolites of complex polyphenols, exerts numerous biological activities including antiapoptotic, anti-inflammatory, and antiatherosclerotic effects. Oxidised LDL have atherogenic properties by damaging arterial wall cells and inducing p53-dependent apoptosis in macrophages. This study was aimed at defining the molecular mechanism responsible for the protective effects of PCA against oxidative and proapoptotic damage exerted by oxLDL in J774 A.1 macrophages. We found that the presence of PCA in cells treated with oxLDL completely inhibited the p53-dependent apoptosis induced by oxLDL. PCA decreased oxLDL-induced ROS overproduction and in particular prevented the early increase of ROS. This decrease seemed to be the main signal responsible for maintaining the intracellular redox homeostasis hindering the activation of p53 induced by ROS, p38MAPK, and PKCδ. Consequently the overexpression of the proapoptotic p53-target genes such as p66Shc protein did not occur. Finally, we demonstrated that PCA induced the activation of JNK, which, in turn, determined the increase of nuclear Nrf2, leading to inhibition of the early ROS overproduction. We concluded that the antiapoptotic mechanism of PCA was most likely related to the activation of the JNK-mediated survival signals that strengthen the cellular antioxidant defences rather than to the PCA antioxidant power. PMID:26180584

  18. GAMT, a p53-Inducible Modulator of Apoptosis, Is Critical for the Adaptive Response to Nutrient Stress

    PubMed Central

    Ide, Takao; Brown-Endres, Lauren; Chu, Kiki; Ongusaha, Pat P.; Ohtsuka, Takao; El-Deiry, Wafik S.; Aaronson, Stuart A.; Lee, Sam W.

    2009-01-01

    SUMMARY The p53 tumor suppressor protein has a well-established role in cell fate decision-making processes. However, recent discoveries indicate that p53 has a non-tumor-suppressive role. Here, we identify GAMT (guanidinoacetate methyltransferase), an enzyme involved in creatine synthesis, as a p53 target gene and a key downstream effector of adaptive response to nutrient stress. We show that GAMT is not only involved in p53-dependent apoptosis in response to genotoxic stress but is important for apoptosis induced by glucose deprivation. Additionally, p53→GAMT up-regulates fatty acid oxidation (FAO) induced by glucose starvation, utilizing this pathway as an alternate ATP-generating energy source. These results highlight that p53-dependent regulation of GAMT allows cells to maintain energy levels sufficient to undergo apoptosis or survival under conditions of nutrient stress. p53→GAMT pathway represents a new link between cellular stress responses and processes of creatine synthesis and FAO, demonstrating a further role of p53 in cellular metabolism. PMID:19917247

  19. POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) inhibits endothelial cell senescence through a p53 dependent pathway

    PubMed Central

    Cho, J H; Kim, M J; Kim, K J; Kim, J-R

    2012-01-01

    Vascular cell senescence, induced by the DNA damage response or inflammatory stress, contributes to age-associated vascular disease. Using complementary DNA microarray technology, we found that the level of POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) is downregulated during endothelial cell (EC) senescence. PATZ1 may have an important role as a transcriptional repressor in chromatin remodeling and transcription regulation; however, the role of PATZ1 in EC senescence and vascular aging remains unidentified. Knockdown of PATZ1 in young cells accelerated premature EC senescence, which was confirmed by growth arrest, increased p53 protein level and senescence-associated β-galactosidase (SA-β-gal) activity, and repression of EC tube formation. In contrast, overexpression of PATZ1 in senescent cells reversed senescent phenotypes. Cellular senescence induced by PATZ1 knockdown in young cells was rescued by knockdown of p53, but not by knockdown of p16INK4a. PATZ1 knockdown increased ROS levels, and pretreatment with N-acetylcysteine abolished EC senescence induced by PATZ1 knockdown. Notably, PATZ1 immunoreactivity was lower in ECs of atherosclerotic tissues than those of normal arteries in LDLR−/− mice, and immunoreactivity also decreased in ECs of old human arteries. These results suggest that PATZ1 may have an important role in the regulation of EC senescence through an ROS-mediated p53-dependent pathway and contribute to vascular diseases associated with aging. PMID:22052190

  20. CAPE Analogs Induce Growth Arrest and Apoptosis in Breast Cancer Cells.

    PubMed

    Beauregard, Annie-Pier; Harquail, Jason; Lassalle-Claux, Grégoire; Belbraouet, Mehdi; Jean-Francois, Jacques; Touaibia, Mohamed; Robichaud, Gilles A

    2015-01-01

    Breast cancer is the second leading cause of death amongst women worldwide. As a result, many have turned their attention to new alternative approaches to treat this disease. Caffeic acid phenylethyl ester (CAPE), a well-known active compound from bee propolis, has been previously identified as a strong antioxidant, anti-inflammatory, antiviral and anticancer molecule. In fact, CAPE is well documented as inducing cell death by inhibiting NFκB and by inducing pro-apoptotic pathways (i.e., p53). With the objective of developing stronger anticancer compounds, we studied 18 recently described CAPE derivatives for their ability to induce apoptosis in breast cancer cell lines. Five of the said compounds, including CAPE, were selected and subsequently characterised for their anticancer mechanism of action. We validated that CAPE is a potent inducer of caspase-dependent apoptosis. Interestingly, some newly synthesized CAPE derivatives also showed greater cell death activity than the lead CAPE structure. Similarly to CAPE, analog compounds elicited p53 activation. Interestingly, one compound in particular, analog 10, induced apoptosis in a p53-mutated cell line. These results suggest that our new CAPE analog compounds may display the capacity to induce breast cancer apoptosis in a p53-dependent and/or independent manner. These CAPE analogs could thus provide new therapeutic approaches for patients with varying genotypic signatures (such as p53 mutations) in a more specific and targeted fashion. PMID:26184141

  1. Overexpression of TDP-43 causes partially p53-dependent G2/M arrest and p53-independent cell death in HeLa cells.

    PubMed

    Lee, Kikyo; Suzuki, Hiroaki; Aiso, Sadakazu; Matsuoka, Masaaki

    2012-01-11

    It has been hypothesized that the dysregulation of transactive response DNA-binding protein-43 (TDP-43) in neurons is closely linked to the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. However, it remains undefined whether the dysregulation of TDP-43 in non-neuronal cells, such as glial cells, contributes to the pathogenesis of these neurodegenerative diseases. Primarily using HeLa cells, we show that a low-grade overexpression of TDP-43, 2- to 5-fold greater than endogenous expression, which is thought to mimic the gain of function of TDP-43, induced cell cycle arrest at the G2/M phase and cell death in cultured non-neuronal cells. Since the activation of p53 may induce G2/M arrest and/or cell death in many abnormal situations, we examined the mechanism underlying G2/M arrest from the standpoint of p53 regulation. It was determined that the TDP-43-induced G2/M arrest was attenuated, while TDP-43-induced death was not attenuated, in cells in which the p53 function was compromised. These data collectively indicate that TDP-43 causes G2/M arrest in a partially p53-dependent manner and it causes cell death in a p53-independent manner in cycling cells. Because it is likely that the impaired proliferation in glial cells causes a decrease in the neuron-supporting ability, these findings further suggests that the gain of function of TDP-43 may cause neurotoxicity by inducing cell cycle arrest and death in glial cells. PMID:22133803

  2. p53-dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts

    SciTech Connect

    Cao Feng |; Zhou Tong; Simpson, Dennis; Zhou Yingchun; Boyer, Jayne; Chen Bo |; Jin Taiyi; Cordeiro-Stone, Marila; Kaufmann, William . E-mail: wkarlk@med.unc.edu

    2007-01-15

    This study focused on the activation of cell cycle checkpoint responses in diploid human fibroblasts that were treated with cadmium chloride and the potential roles of ATM and p53 signaling pathways in cadmium-induced responses. The alkaline comet assay indicated that cadmium caused a dose-dependent increase in DNA damage. Cells that were rendered p53-defective by expression of a dominant-negative p53 allele or knockdown of p53 mRNA were more resistant to cadmium-induced inactivation of colony formation than normal and ataxia telangiectasia (AT) cells. Synchronized fibroblasts in S were more sensitive to cadmium toxicity than cells in G1, suggesting that cadmium may target some element of DNA replication. Cadmium produced a dose- and time-dependent inhibition of DNA synthesis. An immediate inhibition was associated with severe delay in progression through S phase and a delayed inhibition seen 24 h after treatment was associated with accumulation of cells in G2. AT and normal cells displayed similar patterns of inhibition of DNA synthesis and G2 delay after treatment with cadmium, while p53-defective cells displayed significantly less of the delayed inhibition of DNA synthesis and accumulation in G2 post-treatment. Total p53 protein and ser15-phosphorylated p53 were induced by cadmium in normal and AT cells. The p53 transactivation target Gadd45{alpha} was induced in both p53-effective and p53-defective cells after 4 h cadmium treatment, and this was associated with an acute inhibition of mitosis. Cadmium produced a very unusual pattern of toxicity in human fibroblasts, inhibiting DNA replication and inducing p53-dependent growth arrest but without induction of p21{sup Cip1/Waf1} or activation of Chk1.

  3. Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

    The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

  4. PUMA is invovled in ischemia/reperfusion-induced apoptosis of mouse cerebral astrocytes.

    PubMed

    Chen, H; Tian, M; Jin, L; Jia, H; Jin, Y

    2015-01-22

    PUMA (p53-upregulated modulator of apoptosis), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and p53-independent forms of apoptosis. PUMA has been invovled in the onset and progress of several diseases, including cancer, acquired immunodeficiency syndrome, and ischemic brain disease. Although many studies have shown that ischemia and reperfusion (I/R) can induce the apoptosis of astrocytes, the role of PUMA in I/R-mediated apoptosis of cerebral astrocyte apoptosis remains unclear. To mimic in vivo I/R conditions, primary mouse cerebral astrocytes were incubated in a combinational cultural condition of oxygen, glucose, and serum deprivation (OSGD) for 1 h followed by reperfusion (OSGD/R). Cell death determination assays and cell viability assays indicated that OSGD and OSGD/R induce the apoptosis of primary cerebral astrocytes. The expression of PUMA was significantly elevated in primary cerebral astrocytes during OSGD/R. Moreover, targeted down-regulation of PUMA by siRNA transfection significantly decreased the OSGD/R-induced apoptosis of primary cerebral astrocytes. We also found that OSGD and OSGD/R triggered the release of cytochrome c in astrocytes, indicating the dependence on a mitochondrial apoptotic pathway. Reactive oxygen species (ROS) was extremely generated during OSGD and OSGD/R, and the elimination of ROS by treated with N-acetyl-L-cysteine (NAC) remarkably inhibited the expression of PUMA and the apoptosis of primary cerebral astrocytes. The activation of Caspase 3 and Caspase 9 was extremely elevated in primary cerebral astrocytes during OSGD. In addition, we found that knockdown of PUMA led to the depressed expression of Bax, cleaved caspase-9 and caspase-3 during OSGD/R. These results indicate that PUMA is invovled in the apoptosis of cerebral astrocytes upon I/R injury. PMID:25451294

  5. Terpinen-4-ol Induces Apoptosis in Human Nonsmall Cell Lung Cancer In Vitro and In Vivo

    PubMed Central

    Wu, Chieh-Shan; Chen, Yun-Ju; Chen, Jeremy J. W.; Shieh, Jeng-Jer; Huang, Chia-Hsin; Lin, Pei-Shan; Chang, Gee-Chen; Chang, JingHua-Tsai; Lin, Chi-Chen

    2012-01-01

    Terpinen-4-ol, a monoterpene component of the essential oils of several aromatic plants, exhibits antitumor effects. In this study, the antitumor effects of terpinen-4-ol and the cellular and molecular mechanisms responsible for it were evaluated and studied, respectively on human nonsmall cell lung cancer (NSCLC) cells. Our results indicated that terpinen-4-ol elicited a dose-dependent cytotoxic effect, as determined by MTT assay. Increased sub-G1 population and annexin-V binding, activation of caspases 9 and 3, cleavage of poly(ADPribose) polymerase (PARP), and a decrease of mitochondrial membrane potential (MMP) indicated involvement of the mitochondrial apoptotic pathway in terpinen-4-ol-treated A549 and CL1-0 cells. Elevation of the Bax/Bcl-2 ratio and a decrease in IAP family proteins XIAP and survivin were also observed following terpinen-4-ol treatment. Notably, terpinen-4-ol was able to increase p53 levels in A549 and CL1-0 cells. Diminution of p53 by RNA interference induced necrosis instead of apoptosis in A549 cells following terpinen-4-ol treatment, indicating that terpinen-4-ol-elicited apoptosis is p53-dependent. Moreover, intratumoral administration of terpinen-4-ol significantly suppressed the growth of s.c. A549 xenografts by inducing apoptosis, as confirmed by TUNEL assay. Collectively, these data provide insight into the molecular mechanisms underlying terpinen-4-ol-induced apoptosis in NSCLC cells, rendering this compound a potential anticancer drug for NSCLC. PMID:21760828

  6. Climacostol reduces tumour progression in a mouse model of melanoma via the p53-dependent intrinsic apoptotic programme

    PubMed Central

    Perrotta, Cristiana; Buonanno, Federico; Zecchini, Silvia; Giavazzi, Alessio; Proietti Serafini, Francesca; Catalani, Elisabetta; Guerra, Laura; Belardinelli, Maria Cristina; Picchietti, Simona; Fausto, Anna Maria; Giorgi, Simone; Marcantoni, Enrico; Clementi, Emilio; Ortenzi, Claudio; Cervia, Davide

    2016-01-01

    Climacostol, a compound produced by the ciliated protozoan Climacostomum virens, displayed cytotoxic properties in vitro. This study demonstrates that it has anti-tumour potential. Climacostol caused a reduction of viability/proliferation of B16-F10 mouse melanoma cells, a rapidly occurring DNA damage, and induced the intrinsic apoptotic pathway characterised by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of Cytochrome c from the mitochondria, and the activation of Caspase 9-dependent cleavage of Caspase 3. The apoptotic mechanism of climacostol was found to rely on the up-regulation of p53 and its targets Noxa and Puma. In vivo analysis of B16-F10 allografts revealed a persistent inhibition of tumour growth rate when melanomas were treated with intra-tumoural injections of climacostol. In addition, it significantly improved the survival of transplanted mice, decreased tumour weight, induced a remarkable reduction of viable cells inside the tumour, activated apoptosis and up-regulated the p53 signalling network. Importantly, climacostol toxicity was more selective against tumour than non-tumour cells. The anti-tumour properties of climacostol and the molecular events associated with its action indicate that it is a powerful agent that may be considered for the design of pro-apoptotic drugs for melanoma therapy. PMID:27271364

  7. Climacostol reduces tumour progression in a mouse model of melanoma via the p53-dependent intrinsic apoptotic programme.

    PubMed

    Perrotta, Cristiana; Buonanno, Federico; Zecchini, Silvia; Giavazzi, Alessio; Proietti Serafini, Francesca; Catalani, Elisabetta; Guerra, Laura; Belardinelli, Maria Cristina; Picchietti, Simona; Fausto, Anna Maria; Giorgi, Simone; Marcantoni, Enrico; Clementi, Emilio; Ortenzi, Claudio; Cervia, Davide

    2016-01-01

    Climacostol, a compound produced by the ciliated protozoan Climacostomum virens, displayed cytotoxic properties in vitro. This study demonstrates that it has anti-tumour potential. Climacostol caused a reduction of viability/proliferation of B16-F10 mouse melanoma cells, a rapidly occurring DNA damage, and induced the intrinsic apoptotic pathway characterised by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of Cytochrome c from the mitochondria, and the activation of Caspase 9-dependent cleavage of Caspase 3. The apoptotic mechanism of climacostol was found to rely on the up-regulation of p53 and its targets Noxa and Puma. In vivo analysis of B16-F10 allografts revealed a persistent inhibition of tumour growth rate when melanomas were treated with intra-tumoural injections of climacostol. In addition, it significantly improved the survival of transplanted mice, decreased tumour weight, induced a remarkable reduction of viable cells inside the tumour, activated apoptosis and up-regulated the p53 signalling network. Importantly, climacostol toxicity was more selective against tumour than non-tumour cells. The anti-tumour properties of climacostol and the molecular events associated with its action indicate that it is a powerful agent that may be considered for the design of pro-apoptotic drugs for melanoma therapy. PMID:27271364

  8. Marijuana smoke condensate induces p53-mediated apoptosis in human lung epithelial cells.

    PubMed

    Kim, Ha Ryong; Jung, Mi Hyun; Lee, Soo Yeun; Oh, Seung Min; Chung, Kyu Hyuck

    2013-01-01

    Since the largely abused worldwide used of marijuana, there have been many ongoing debates regarding the adverse health effects of marijuana smoking. Marijuana smoking was recently proved to cause pulmonary toxicity by inducing genotoxic effects or generating reactive oxygen species. Because p53, a tumor suppressor gene, has an important pathophysiologic role in the regulation of lung epithelial cell DNA damage responses, we hypothesized that p53 may be involved in the oxidative stress-mediated apoptosis induced by marijuana smoking. First, we confirmed that marijuana smoke condensate (MSC) induces oxidative stress in BEAS-2B cells. We observed that reactive oxygen species (ROS) generation was increased by MSC in the DCFH-DA assay. Also, antioxidant enzyme (superoxide dismutase, catalase) activity and their mRNA expressions were up-regulated by MSC. Second, we investigated p53 involvement in the MSC-induced apoptotic pathway in BEAS-2B cells. The results showed that MSC increased caspase-3 activation and DNA fragmentation as markers of apoptosis. In addition, the mRNA levels of apoptosis-related genes (p53 and Bax) were increased by MSC and phospho-p53, along with the increase of Bax protein expression by MSC. Apoptosis and apoptosis-related gene expression were partially blocked by an inhibitor of p53-dependent transcriptional activation (pifithrin-α). The results indicate that p53 plays a role in MSC-induced apoptosis. Taken together, the findings of the present study suggest that MSC partially induces p53-mediated apoptosis through ROS generation in human lung epithelial cells and this may have broader implications for our understanding of pulmonary diseases. PMID:23665932

  9. Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells.

    PubMed

    Yoneshima, Yasuto; Abolhassani, Nona; Iyama, Teruaki; Sakumi, Kunihiko; Shiomi, Naoko; Mori, Masahiko; Shiomi, Tadahiro; Noda, Tetsuo; Tsuchimoto, Daisuke; Nakabeppu, Yusaku

    2016-01-01

    Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. PMID:27618981

  10. A Temperature Sensitive Variant of p53 Drives p53-Dependent MicroRNA Expression without Evidence of Widespread Post-Transcriptional Gene Silencing

    PubMed Central

    Cabrita, Miguel A.; Vanzyl, Erin J.; Hamill, Jeff D.; Pan, Elysia; Marcellus, Kristen A.; Tolls, Victoria J.; Alonzi, Rhea C.; Pastic, Alyssa; Rambo, Teeghan M. E.; Sayed, Hadil; McKay, Bruce C.

    2016-01-01

    The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional

  11. In several cell types tumour suppressor p53 induces apoptosis largely via Puma but Noxa can contribute.

    PubMed

    Michalak, E M; Villunger, A; Adams, J M; Strasser, A

    2008-06-01

    The ability of p53 to induce apoptosis in cells with damaged DNA is thought to contribute greatly to its tumour suppressor function. P53 has been proposed to induce apoptosis via numerous transcriptional targets or even by direct cytoplasmic action. Two transcriptional targets shown to mediate its apoptotic role in several cell types encode Noxa and Puma, BH3-only members of the Bcl-2 family. To test if their functions in p53-dependent apoptosis overlap, we generated mice lacking both. These mice develop normally and no tumours have yet arisen. In embryonic fibroblasts, the absence of both Noxa and Puma prevented induction of apoptosis by etoposide. Moreover, following whole body gamma-irradiation, the loss of both proteins protected thymocytes better than loss of Puma alone. Indeed, their combined deficiency protected thymocytes as strongly as loss of p53 itself. These results indicate that, at least in fibroblasts and thymocytes, p53-induced apoptosis proceeds principally via Noxa and Puma, with Puma having the predominant role in diverse cell types. The absence of tumours in the mice suggests that tumour suppression by p53 requires functions in addition to induction of apoptosis. PMID:18259198

  12. Macrophage Migration Inhibitory Factor: A Novel Inhibitor of Apoptosis Signal-Regulating Kinase 1-p38-Xanthine Oxidoreductase-Dependent Cigarette Smoke-Induced Apoptosis.

    PubMed

    Fallica, Jonathan; Varela, Lidenys; Johnston, Laura; Kim, Bo; Serebreni, Leonid; Wang, Lan; Damarla, Mahendra; Kolb, Todd M; Hassoun, Paul M; Damico, Rachel

    2016-04-01

    Cigarette smoke (CS) exposure is the leading cause of emphysema. CS mediates pathologic emphysematous remodeling of the lung via apoptosis of lung parenchymal cells resulting in enlargement of the airspaces, loss of the capillary bed, and diminished surface area for gas exchange. Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, is reduced both in a preclinical model of CS-induced emphysema and in patients with chronic obstructive pulmonary disease, particularly those with the most severe disease and emphysematous phenotype. MIF functions to antagonize CS-induced DNA damage, p53-dependent apoptosis of pulmonary endothelial cells (EndoCs) and resultant emphysematous tissue remodeling. Using primary alveolar EndoCs and a mouse model of CS-induced lung damage, we investigated the capacity and molecular mechanism(s) by which MIF modifies oxidant injury. Here, we demonstrate that both the activity of xanthine oxidoreductase (XOR), a superoxide-generating enzyme obligatory for CS-induced DNA damage and EndoC apoptosis, and superoxide concentrations are increased after CS exposure in the absence of MIF. Both XOR hyperactivation and apoptosis in the absence of MIF occurred via a p38 mitogen-activated protein kinase-dependent mechanism. Furthermore, a mitogen-activated protein kinase kinase kinase family member, apoptosis signal-regulating kinase 1 (ASK1), was necessary for CS-induced p38 activation and EndoC apoptosis. MIF was sufficient to directly suppress ASK1 enzymatic activity. Taken together, MIF suppresses CS-mediated cytotoxicity in the lung, in part by antagonizing ASK1-p38-XOR-dependent apoptosis. PMID:26390063

  13. PUMA promotes Bax translocation in FOXO3a-dependent pathway during STS-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Yingjie; Chen, Qun

    2009-08-01

    PUMA (p53 up-regulated modulator of apoptosis, also called Bbc3) was first identified as a BH3-only Bcl-2 family protein that is transcriptionally up-regulated by p53 and activated upon p53-dependent apoptotic stimuli, such as treatment with DNA-damaging drugs or UV irradiation. Recently studies have been shown that Puma is also up-regulated in response to certain p53-independent apoptotic stimuli, such as growth factor deprivation or treatment with glucocorticoids or STS (staurosporine). However, the molecular mechanisms of PUMA up-regulation and how PUMA functions in response to p53-independent apoptotic stimuli remain poorly understood. In this study, based on real-time single cell analysis, flow cytometry and western blotting technique, we investigated the function of PUMA in living human lung adenocarcinoma cells (ASTC-a-1) after STS treatment. Our results show that FOXO3a was activated by STS stimulation and then translocated from cytosol to nucleus. The expression of PUMA was up-regulated via a FOXO3a-dependent manner after STS treatment, while p53 had little function in this process. Moreover, cell apoptosis and Bax translocation induced by STS were not blocked by Pifithrin-α (p53 inhibitor), which suggested that p53 was not involved in this signaling pathway. Taken together, these results indicate that PUMA promoted Bax translocation in a FOXO3a-dependment pathway during STS-induced apoptosis, while p53 was dispensable in this process.

  14. Calmodulin antagonists induce platelet apoptosis.

    PubMed

    Wang, Zhicheng; Li, Suping; Shi, Quanwei; Yan, Rong; Liu, Guanglei; Dai, Kesheng

    2010-04-01

    Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis. PMID:20172594

  15. Fluoxetine protects against IL-1β-induced neuronal apoptosis via downregulation of p53.

    PubMed

    Shan, Han; Bian, Yaqi; Shu, Zhaoma; Zhang, Linxia; Zhu, Jialei; Ding, Jianhua; Lu, Ming; Xiao, Ming; Hu, Gang

    2016-08-01

    Fluoxetine, a selective serotonin reuptake inhibitor, exerts neuroprotective effects in a variety of neurological diseases including stroke, but the underlying mechanism remains obscure. In the present study, we addressed the molecular events in fluoxetine against ischemia/reperfusion-induced acute neuronal injury and inflammation-induced neuronal apoptosis. We showed that treatment of fluoxetine (40 mg/kg, i.p.) with twice injections at 1 h and 12 h after transient middle cerebral artery occlusion (tMCAO) respectively alleviated neurological deficits and neuronal apoptosis in a mouse ischemic stroke model, accompanied by inhibiting interleukin-1β (IL-1β), Bax and p53 expression and upregulating anti-apoptotic protein Bcl-2 level. We next mimicked neuroinflammation in ischemic stroke with IL-1β in primary cultured cortical neurons and found that pretreatment with fluoxetine (1 μM) prevented IL-1β-induced neuronal apoptosis and upregulation of p53 expression. Furthermore, we demonstrated that p53 overexpression in N2a cell line abolished the anti-apoptotic effect of fluoxetine, indicating that p53 downregulation is required for the protective role of fluoxetine in IL-1β-induced neuronal apoptosis. Fluoxetine downregulating p53 expression could be mimicked by SB203580, a specific inhibitor of p38, but blocked by anisomycin, a p38 activator. Collectively, our findings have revealed that fluoxetine protects against IL-1β-induced neuronal apoptosis via p38-p53 dependent pathway, which give us an insight into the potential of fluoxetine in terms of opening up novel therapeutic avenues for neurological diseases including stroke. PMID:26976669

  16. PUMA Binding Induces Partial Unfolding within BCL-xL to Disrupt p53 Binding and Promote Apoptosis

    PubMed Central

    Follis, Ariele Viacava; Chipuk, Jerry E.; Fisher, John C.; Yun, Mi-Kyung; Grace, Christy R.; Nourse, Amanda; Baran, Katherine; Ou, Li; Min, Lie; White, Stephen W.; Green, Douglas R.; Kriwacki, Richard W.

    2012-01-01

    Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the anti-apoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique amongst BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules, BAX and BAK. Structural investigations using nuclear magnetic resonance spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the anti-apoptotic BCL-2 repertoire to sensitize for death receptor (DR)-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways. PMID:23340338

  17. Targeting Pro-Apoptotic TRAIL Receptors Sensitizes HeLa Cervical Cancer Cells to Irradiation-Induced Apoptosis

    SciTech Connect

    Maduro, John H.; Vries, Elisabeth de; Meersma, Gert-Jan; Hougardy, Brigitte; Zee, Ate G.J. van der; Jong, Steven de

    2008-10-01

    Purpose: To investigate the potential of irradiation in combination with drugs targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor (DR)4 and DR5 and their mechanism of action in a cervical cancer cell line. Methods and Materials: Recombinant human TRAIL (rhTRAIL) and the agonistic antibodies against DR4 and DR5 were added to irradiated HeLa cells. The effect was evaluated with apoptosis and cytotoxicity assays and at the protein level. Membrane receptor expression was measured with flow cytometry. Small-interfering RNA against p53, DR4, and DR5 was used to investigate their function on the combined effect. Results: rhTRAIL and the agonistic DR4 and DR5 antibodies strongly enhanced 10-Gy-induced apoptosis. This extra effect was 22%, 23%, and 29% for rhTRAIL, DR4, and DR5, respectively. Irradiation increased p53 expression and increased the membrane expression of DR5 and DR4. p53 suppression, as well as small-interfering RNA against DR5, resulted in a significant downregulation of DR5 membrane expression but did not affect apoptosis induced by irradiation and rhTRAIL. After small-interfering RNA against DR4, rhTRAIL-induced apoptosis and the additive effect of irradiation on rhTRAIL-induced apoptosis were abrogated, implicating an important role for DR4 in apoptosis induced through irradiation in combination with rhTRAIL. Conclusion: Irradiation-induced apoptosis is strongly enhanced by targeting the pro-apoptotic TRAIL receptors DR4 or DR5. Irradiation results in a p53-dependent increase in DR5 membrane expression. The sensitizing effect of rhTRAIL on irradiation in the HeLa cell line is, however especially mediated through the DR4 receptor.

  18. E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

    PubMed

    Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

    2015-10-01

    Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

  19. The potassium ion channel opener NS1619 inhibits proliferation and induces apoptosis in A2780 ovarian cancer cells

    SciTech Connect

    Han Xiaobing; Xi Ling; Wang Hui; Huang Xiaoyuan; Ma Xiangyi; Han Zhiqiang; Wu Peng; Ma Xiaoli; Lu Yunping; Wang, Gang Zhou Jianfeng; Ma Ding

    2008-10-17

    Diverse types of voltage-gated potassium (K{sup +}) channels have been shown to be involved in regulation of cell proliferation. The maxi-conductance Ca{sup 2+}-activated K{sup +} channels (BK channels) may play an important role in the progression of human cancer. To explore the role of BK channels in regulation of apoptosis in human ovarian cancer cells, the effects of the specific BK channel activator NS1619 on induction of apoptosis in A2780 cells were observed. Following treatment with NS1619, cell proliferation was measured by MTT assay. Apoptosis of A2780 cells pretreated with NS1619 was detected by agarose gel electrophoresis of cellular DNA and flow cytometry. Our data demonstrate that NS1619 inhibits the proliferation of A2780 cells in a dosage and time dependent manner IC{sub 50} = 31.1 {mu}M, for 48 h pretreatment and induces apoptosis. Western blot analyses showed that the anti-proliferation effect of NS1619 was associated with increased expression of p53, p21, and Bax. These results indicate that BK channels play an important role in regulating proliferation of human ovarian cancer cells and may induce apoptosis through induction of p21{sup Cip1} expression in a p53-dependent manner.

  20. NML-mediated rRNA base methylation links ribosomal subunit formation to cell proliferation in a p53-dependent manner.

    PubMed

    Waku, Tsuyoshi; Nakajima, Yuka; Yokoyama, Wataru; Nomura, Naoto; Kako, Koichiro; Kobayashi, Akira; Shimizu, Toshiyuki; Fukamizu, Akiyoshi

    2016-06-15

    Ribosomal RNAs (rRNAs) act as scaffolds and ribozymes in ribosomes, and these functions are modulated by post-transcriptional modifications. However, the biological role of base methylation, a well-conserved modification of rRNA, is poorly understood. Here, we demonstrate that a nucleolar factor, nucleomethylin (NML; also known as RRP8), is required for the N(1)-methyladenosine (m(1)A) modification in 28S rRNAs of human and mouse cells. NML also contributes to 60S ribosomal subunit formation. Intriguingly, NML depletion increases 60S ribosomal protein L11 (RPL11) levels in the ribosome-free fraction and protein levels of p53 through an RPL11-MDM2 complex, which activates the p53 pathway. Consequently, the growth of NML-depleted cells is suppressed in a p53-dependent manner. These observations reveal a new biological function of rRNA base methylation, which links ribosomal subunit formation to p53-dependent inhibition of cell proliferation in mammalian cells. PMID:27149924

  1. Activation of p53-Dependent Growth Suppression in Human Cells by Mutations in PTEN or PIK3CA▿

    PubMed Central

    Kim, Jung-Sik; Lee, Carolyn; Bonifant, Challice L.; Ressom, Habtom; Waldman, Todd

    2007-01-01

    In an effort to identify genes whose expression is regulated by activated phosphatidylinositol 3-kinase (PI3K) signaling, we performed microarray analysis and subsequent quantitative reverse transcription-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN−/− cells via an Akt1-dependent and p14ARF-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescence-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic, but not wild-type, PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on phosphatidylinositol (3,4,5)-triphosphate-induced mitogenesis during human cancer pathogenesis. PMID:17060456

  2. Grifola frondosa Glycoprotein GFG-3a Arrests S phase, Alters Proteome, and Induces Apoptosis in Human Gastric Cancer Cells.

    PubMed

    Cui, Fengjie; Zan, Xinyi; Li, Yunhong; Sun, Wenjing; Yang, Yan; Ping, Lifeng

    2016-01-01

    GFG-3a is a novel glycoprotein previously purified from the fermented mycelia of Grifola frondosa with novel sugar compositions and protein sequencing. The present study aims to investigate its effects on the cell cycle, differential proteins expression, and apoptosis of human gastric cancer SGC-7901 cells. Our findings revealed that GFG-3a induced the cell apoptosis and arrested cell cycle at S phase. GFG-3a treatment resulted in the differential expression of 21 proteins in SGC-7901 cells by upregulating 10 proteins including RBBP4 associated with cell cycle arrest and downregulating 11 proteins including RUVBL1, NPM, HSP90AB1, and GRP78 involved in apoptosis and stress response. qRT-PCR and Western blot analysis also suggested that GFG-3a could increase the expressions of Caspase-8/-3, p53, Bax, and Bad while decrease the expressions of Bcl2, Bcl-xl, PI3K, and Akt1. These results indicated that the stress response, p53-dependent mitochondrial-mediated, Caspase-8/-3-dependent, and PI3k/Akt pathways were involved in the GFG-3a-induced apoptosis process in SGC-7901 cells. These findings might provide a basis to prevent or treat human gastric cancer with GFG-3a and understand the tumor-inhibitory molecular mechanisms of mushroom glycoproteins. PMID:27040446

  3. p53-Dependent Senescence in Mesenchymal Stem Cells under Chronic Normoxia Is Potentiated by Low-Dose γ-Irradiation

    PubMed Central

    Ingawale, Yashodhara; Hertlein, Heidi; Nelson, Peter J.

    2016-01-01

    Mesenchymal stem cells (MSCs) are a source of adult multipotent cells important in tissue regeneration. Murine MSCs are known to proliferate poorly in vitro under normoxia. The aim of this study is to analyze the interaction of nonphysiological high oxygen and low-dose γ-irradiation onto growth, senescence, and DNA damage. Tri-potent bone marrow-derived MSCs from p53 wildtype and p53−/− mice were cultured under either 21% or 2% O2. Long-term observations revealed a decreasing ability of wildtype mMSCs to proliferate and form colonies under extended culture in normoxia. This was accompanied by increased senescence under normoxia but not associated with telomere shortening. After low-dose γ-irradiation, the normoxic wildtype cells further increased the level of senescence. The number of radiation-induced γH2AX DNA repair foci was higher in mMSCs kept under normoxia but not in p53−/− cells. P53-deficient MSCs additionally showed higher clonogeneity, lower senescence levels, and fewer γH2AX repair foci per cell as compared to their p53 wildtype counterparts irrespective of oxygen levels. These results reveal that oxygen levels together with γ-irradiation and p53 status are interconnected factors modulating growth capacity of BM MSCs in long-term culture. These efforts help to better understand and optimize handling of MSCs prior to their therapeutic use. PMID:26788069

  4. A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure.

    PubMed

    Ear, Jason; Hsueh, Jessica; Nguyen, Melinda; Zhang, QingHua; Sung, Victoria; Chopra, Rajesh; Sakamoto, Kathleen M; Lin, Shuo

    2016-05-20

    5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype. PMID:27216296

  5. Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells.

    PubMed

    Matsumoto, Masaru; Nakajima, Wataru; Seike, Masahiro; Gemma, Akihiko; Tanaka, Nobuyuki

    2016-04-29

    Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-cell lung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs. Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression of proapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53-dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells, cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the specific inhibitor of Bcl-2 and Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system in cisplatin-induced NSCLC cell apoptosis, and a candidate efficient combination chemotherapy method against lung cancers. PMID:26996126

  6. Decitabine and SAHA-Induced Apoptosis Is Accompanied by Survivin Downregulation and Potentiated by ATRA in p53-Deficient Cells

    PubMed Central

    Brodská, Barbora; Otevřelová, Petra; Holoubek, Aleš

    2014-01-01

    While p53-dependent apoptosis is triggered by combination of methyltransferase inhibitor decitabine (DAC) and histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in leukemic cell line CML-T1, reactive oxygen species (ROS) generation as well as survivin and Bcl-2 deregulation participated in DAC + SAHA-induced apoptosis in p53-deficient HL-60 cell line. Moreover, decrease of survivin expression level is accompanied by its delocalization from centromere-related position in mitotic cells suggesting that both antiapoptotic and cell cycle regulation roles of survivin are affected by DAC + SAHA action. Addition of subtoxic concentration of all-trans-retinoic acid (ATRA) increases the efficiency of DAC + SAHA combination on viability, apoptosis induction, and ROS generation in HL-60 cells but has no effect in CML-T1 cell line. Peripheral blood lymphocytes from healthy donors showed no damage induced by DAC + SAHA + ATRA combination. Therefore, combination of ATRA with DAC and SAHA represents promising tool for therapy of leukemic disease with nonfunctional p53 signalization. PMID:25140197

  7. Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

    SciTech Connect

    Yu Xiaozhong Robinson, Joshua F.; Gribble, Elizabeth; Hong, Sung Woo; Sidhu, Jaspreet S.; Faustman, Elaine M.

    2008-12-15

    Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As{sup 3+}) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53{sup +/+} and p53{sup -/-} mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53{sup -/-} cells than in the p53{sup +/+} cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As{sup 3+}. A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53{sup +/+} MEFs, As{sup 3+} induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53{sup -/-} MEFs, As{sup 3+} induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.

  8. Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

    PubMed Central

    Yu, Xiaozhong; Robinson, Joshua F.; Gribble, Elizabeth; Hong, Sung Woo; Sidhu, Jaspreet S; Faustman, Elaine M

    2008-01-01

    Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As3+) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53+/+ and p53−/− mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53−/− cells than in the p53+/+ cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As3+. A significant alteration in the Nrf2-mediated oxidative stress response pathway were found in both genotypes. In p53+/+ MEFs, As3+ induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53−/− MEFs, As3+ induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic’s dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent. PMID:18929588

  9. Hypoxia downregulates p53 but induces apoptosis and enhances expression of BAD in cultures of human syncytiotrophoblasts.

    PubMed

    Chen, Baosheng; Longtine, Mark S; Sadovsky, Yoel; Nelson, D Michael

    2010-11-01

    Hypoxia is commonly assigned a role in the placental dysfunction characteristic of preeclampsia and intrauterine growth restriction. We previously showed that hypoxia upregulates p53 and enhances apoptosis in primary cultures of human cytotrophoblasts. Here we tested the hypothesis that hypoxia also induces apoptosis in syncytiotrophoblasts by upregulation of p53. Primary cultures of human cytotrophoblasts that had differentiated into syncytiotrophoblasts by 52 h were exposed for ≤24 h to 20% or <1% oxygen in the presence or absence of staurosporine or the p53 modulators nutlin-3, pifithrin-α, and pifithrin-μ. Proteins were detected by Western blot analysis or immunofluorescence. Compared with 20% oxygen, exposure of syncytiotrophoblasts to <1% oxygen upregulated hypoxia-inducible factor (HIF)-1α and rapidly downregulated p53. Activity of p53 in hypoxic syncytiotrophoblasts was reduced by the higher expression of the negative p53 regulator MDMX and by the reduction of phosphorylation of p53 at Ser(392), which reduces p53 activity. Conversely, staurosporine, a kinase inhibitor, and nutlin-3, a drug that enhances p53 expression, both raised p53 levels and increased the rate of apoptosis in syncytiotrophoblasts compared with vehicle controls. Immunofluorescence staining showed p53 immunolocalized to both cytoplasm and nuclei of nutlin-3-exposed syncytiotrophoblasts. The hypoxia-induced apoptosis in syncytiotrophoblasts correlated with enhanced expression of the proapoptotic BAD and a reduced level of antiapoptotic BAD phosphorylated on Ser(112). We surmise that cell death induced by extreme hypoxia in syncytiotrophoblasts follows a non-p53-dependent pathway, unlike that of a nonhypoxic stimulus and unlike hypoxic cytotrophoblasts. We speculate that downregulation of p53 activity in response to hypoxia reduces or eliminates the apoptosis transduced by the p53 pathway in syncytiotrophoblasts, thereby limiting cell death and maintaining the integrity of this

  10. Hypoxia downregulates p53 but induces apoptosis and enhances expression of BAD in cultures of human syncytiotrophoblasts

    PubMed Central

    Chen, Baosheng; Longtine, Mark S.; Sadovsky, Yoel

    2010-01-01

    Hypoxia is commonly assigned a role in the placental dysfunction characteristic of preeclampsia and intrauterine growth restriction. We previously showed that hypoxia upregulates p53 and enhances apoptosis in primary cultures of human cytotrophoblasts. Here we tested the hypothesis that hypoxia also induces apoptosis in syncytiotrophoblasts by upregulation of p53. Primary cultures of human cytotrophoblasts that had differentiated into syncytiotrophoblasts by 52 h were exposed for ≤24 h to 20% or <1% oxygen in the presence or absence of staurosporine or the p53 modulators nutlin-3, pifithrin-α, and pifithrin-μ. Proteins were detected by Western blot analysis or immunofluorescence. Compared with 20% oxygen, exposure of syncytiotrophoblasts to <1% oxygen upregulated hypoxia-inducible factor (HIF)-1α and rapidly downregulated p53. Activity of p53 in hypoxic syncytiotrophoblasts was reduced by the higher expression of the negative p53 regulator MDMX and by the reduction of phosphorylation of p53 at Ser392, which reduces p53 activity. Conversely, staurosporine, a kinase inhibitor, and nutlin-3, a drug that enhances p53 expression, both raised p53 levels and increased the rate of apoptosis in syncytiotrophoblasts compared with vehicle controls. Immunofluorescence staining showed p53 immunolocalized to both cytoplasm and nuclei of nutlin-3-exposed syncytiotrophoblasts. The hypoxia-induced apoptosis in syncytiotrophoblasts correlated with enhanced expression of the proapoptotic BAD and a reduced level of antiapoptotic BAD phosphorylated on Ser112. We surmise that cell death induced by extreme hypoxia in syncytiotrophoblasts follows a non-p53-dependent pathway, unlike that of a nonhypoxic stimulus and unlike hypoxic cytotrophoblasts. We speculate that downregulation of p53 activity in response to hypoxia reduces or eliminates the apoptosis transduced by the p53 pathway in syncytiotrophoblasts, thereby limiting cell death and maintaining the integrity of this critical

  11. Parental exposure to natural mixtures of persistent organic pollutants (POP) induced changes in transcription of apoptosis-related genes in offspring zebrafish embryos.

    PubMed

    Lyche, Jan L; Grześ, Irena M; Karlsson, Camilla; Nourizadeh-Lillabadi, Rasoul; Aleström, Peter; Ropstad, Erik

    2016-01-01

    Apoptosis is an integral element of development that may also be initiated by environmental contaminants. The aim of the present study was to assess potential changes in the regulation of apoptotic genes in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POP). The mixture from Lake Mjøsa contained exceptionally high concentrations of polybrominated diphenyl ethers (PBDE), as well as relatively high levels of polychlorinated biphenyls (PCB) and dichlorodiphenyltrichloroethane (DDT). The mixture from Lake Losna contained background concentrations of POP. Genes involved in the apoptotic machinery were screened for their expression profile at four time points during embryonic development. Thirteen and 15 genes involved in apoptosis were found to be significantly upregulated in the high-exposure and background exposure groups, respectively, compared with controls. Modulation of apoptotic genes was restricted only to the first time point, which corresponds with the blastula stage. Although there were substantial differences in POP concentrations between mixtures, genes underlying the apoptosis process showed almost similar responses to the two mixtures. In both exposure groups the main executors of apoptosis p53, casp 2, casp 6, cassp 8, and BAX displayed upregulation compared to controls, suggesting that these POP induce apoptosis via a p53-dependent mechanism. Upregulation of genes that play a critical role in apoptosis suggests that disturbance of normal apoptotic signaling during gametogenesis and embryogenesis may be one of the central mechanisms involved in adverse reproductive effects produced by POP in zebrafish. PMID:27484141

  12. LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

    PubMed Central

    Kramer, Holly B.; Lai, Chun-Fui; Patel, Hetal; Periyasamy, Manikandan; Lin, Meng-Lay; Feller, Stephan M.; Fuller-Pace, Frances V.; Meek, David W.; Ali, Simak; Buluwela, Laki

    2016-01-01

    Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53. PMID:26400164

  13. Stapled α−helical peptide drug development: A potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy

    PubMed Central

    Chang, Yong S.; Graves, Bradford; Guerlavais, Vincent; Tovar, Christian; Packman, Kathryn; To, Kwong-Him; Olson, Karen A.; Kesavan, Kamala; Gangurde, Pranoti; Mukherjee, Aditi; Baker, Theresa; Darlak, Krzysztof; Elkin, Carl; Filipovic, Zoran; Qureshi, Farooq Z.; Cai, Hongliang; Berry, Pamela; Feyfant, Eric; Shi, Xiangguo E.; Horstick, James; Annis, D. Allen; Manning, Anthony M.; Fotouhi, Nader; Nash, Huw; Vassilev, Lyubomir T.; Sawyer, Tomi K.

    2013-01-01

    Stapled α−helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein–protein interaction and may offer a viable modality for cancer therapy. PMID:23946421

  14. Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

    PubMed

    Chang, Yong S; Graves, Bradford; Guerlavais, Vincent; Tovar, Christian; Packman, Kathryn; To, Kwong-Him; Olson, Karen A; Kesavan, Kamala; Gangurde, Pranoti; Mukherjee, Aditi; Baker, Theresa; Darlak, Krzysztof; Elkin, Carl; Filipovic, Zoran; Qureshi, Farooq Z; Cai, Hongliang; Berry, Pamela; Feyfant, Eric; Shi, Xiangguo E; Horstick, James; Annis, D Allen; Manning, Anthony M; Fotouhi, Nader; Nash, Huw; Vassilev, Lyubomir T; Sawyer, Tomi K

    2013-09-01

    Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy. PMID:23946421

  15. Involvement of S100A14 protein in cell invasion by affecting expression and function of matrix metalloproteinase (MMP)-2 via p53-dependent transcriptional regulation.

    PubMed

    Chen, Hongyan; Yuan, Yi; Zhang, Chunpeng; Luo, Aiping; Ding, Fang; Ma, Jianlin; Yang, Shouhui; Tian, Yanyan; Tong, Tong; Zhan, Qimin; Liu, Zhihua

    2012-05-18

    S100 proteins have been implicated in tumorigenesis and metastasis. As a member of S100 proteins, the role of S100A14 in carcinogenesis has not been fully understood. Here, we showed that ectopic overexpression of S100A14 promotes motility and invasiveness of esophageal squamous cell carcinoma cells. We investigated the underlying mechanisms and found that the expression of matrix metalloproteinase (MMP)-2 is obviously increased after S100A14 gene overexpression. Inhibition of MMP2 by a specific MMP2 inhibitor at least partly reversed the invasive phenotype of cells overexpressing S100A14. By serendipity, we found that S100A14 could affect p53 transactivity and stability. Thus, we further investigated whether the effect of MMP2 by S100A14 is dependent on p53. A series of biochemical assays showed that S100A14 requires functional p53 to affect MMP2 transcription, and p53 potently transrepresses the expression of MMP2. Finally, RT-quantitative PCR analysis of human breast cancer specimens showed a significant correlation between S100A14 mRNA expression and MMP2 mRNA expression in cases with wild-type p53 but not in cases with mutant p53. Collectively, our data strongly suggest that S100A14 promotes cell motility and invasiveness by regulating the expression and function of MMP2 in a p53-dependent manner. PMID:22451655

  16. LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner.

    PubMed

    Kramer, Holly B; Lai, Chun-Fui; Patel, Hetal; Periyasamy, Manikandan; Lin, Meng-Lay; Feller, Stephan M; Fuller-Pace, Frances V; Meek, David W; Ali, Simak; Buluwela, Laki

    2016-01-29

    Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53. PMID:26400164

  17. 53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis

    PubMed Central

    Fong, Chii Shyang; Mazo, Gregory; Das, Tuhin; Goodman, Joshua; Kim, Minhee; O'Rourke, Brian P; Izquierdo, Denisse; Tsou, Meng-Fu Bryan

    2016-01-01

    Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency. DOI: http://dx.doi.org/10.7554/eLife.16270.001 PMID:27371829

  18. Inhibitory role of cAMP on doxorubicin-induced apoptosis in pre-B ALL cells through dephosphorylation of p53 serine residues.

    PubMed

    Safa, Majid; Kazemi, Ahmad; Zand, Hamid; Azarkeivan, Azita; Zaker, Farhad; Hayat, Parisa

    2010-02-01

    Exposure of cells to chemotherapeutic drug doxorubicin, a DNA-damaging agent, induces an increase in the levels and activity of the wild-type p53 protein. Less well appreciated was the effect of cAMP levels on posttranslational modifications of p53 in response to doxorubicin. Here we show that elevation of cAMP in pre-B acute lymphoblastic leukemia NALM-6 cells significantly attenuated phosphorylation state of p53 at Ser6, Ser9, Ser15, Ser20, Ser37, Ser46 and Ser392 upon exposure to doxorubicin. Increased cAMP levels also shifted the ratio of the death promoter to death repressor genes via alteration of Bcl-2 and Bax proteins expression. In conclusion, our results suggest that activation of cAMP-signaling system may repress p53-dependent apoptosis in malignant cells exposed to doxorubicin. PMID:19882354

  19. Cyclin-dependent kinase inhibitors sensitize tumor cells to nutlin-induced apoptosis: a potent drug combination.

    PubMed

    Cheok, Chit Fang; Dey, Anwesha; Lane, David P

    2007-11-01

    Current chemotherapy focuses on the use of genotoxic drugs that may induce general DNA damage in cancer cells but also high levels of toxicity in normal tissues. Nongenotoxic activation of p53 by targeting specific molecular pathways therefore provides an attractive therapeutic strategy in cancers with wild-type p53. Here, we explored the antitumor potential of cyclin-dependent kinase (CDK) inhibitors in combination with a small molecule inhibitor of p53-murine double minute 2 (MDM2) interaction. We show that low doses of CDK inhibitors roscovitine and DRB synergize with the MDM2 antagonist nutlin-3a in the induction of p53 activity and promote p53-dependent apoptosis in a dose- and time-dependent manner. Statistical measurement of the combination effects shows that the drug combination is additive on the reduction of cell viability and synergistic on inducing apoptosis, a critical end point of cytotoxic drugs. The degree of apoptosis observed 24 to 48 h after drug treatment correlated with the accumulation of p53 protein and concomitant induction of proapoptotic proteins Puma and PIG3. The antiproliferative and cytotoxic effects of this drug combination are validated in a range of tumor-derived cells including melanoma, colon carcinoma, breast adenocarcinoma, and hepatocarcinoma cells. Furthermore, this drug combination does not induce phosphorylation of Ser(15) on p53 and does not induce genotoxic stress in the cell. Given that many cytotoxic drugs rely on their ability to induce apoptosis via DNA damage-mediated activation of p53, the data presented here may provide a new therapeutic approach for the use of CDK inhibitors and MDM2 antagonists in combinatorial drug therapy. PMID:18025259

  20. Radiation-induced apoptosis in SCID mice spleen after low dose irradiation

    NASA Astrophysics Data System (ADS)

    Takahashi, A.; Kondo, N.; Inaba, H.; Uotani, K.; Kiyohara, Y.; Ohnishi, K.; Ohnishi, T.

    To assess the radioadaptive response of the whole body system in mice, we examined the temporal effect of low dose priming as an indicator of challenging irradiation-induced apoptosis through a p53 tumor suppressor protein- mediated signal transduction pathway. The p53 protein also plays an important role both in cell cycle control and DNA repair through cellular signal transduction. Using severe combined immunodeficiency mice defective in DNA-dependent protein kinase catalytic subunit, we examined the role of DNA-dependent protein kinase activity in radioadaptation induced by low dose irradiation. Specific pathogen free 5-week-old female severe combined immunodeficiency mice and the parental mice (CB-17 Icr +/ + were irradiated with X-ray at 3.0 C3y at 1, 2, 3 or 4 weeks after the conditioning irradiation at 0.15, 0.30, 0.45 or 0.60 Gy. The mice spleens were fixed for immunohistochemistry 12 h after the challenging irradiation. The p53-dependent apoptosis related Bax proteins on formalin-fixed paraffin-embedded sections were stained by the avidin-biotin peroxidase complex method The apoptosis incidence in the sections was measured by hematoxylin-eosin staining. The frequency of Bax- and apoptosis-positive cells increased up to 12 h after the challenging irradiation in the spleen of both mice. However, these cells were not observed after a low dose irradiation at 0.15-0.60 Gy When pre-irradiation at 0.45 Gy 2 weeks before the challenging irradiation at 3.0 Gy was performed, Bax accumulation and apoptosis induced by challenging irradiation were depressed in the spleens of CB-17 Icr +/ + mice, but not in severe combined immunodeficiency mice. These data suggest that DNA-dependent protein kinase might play a major role in radioadaptation induced by pre-irradiation with a low dose in mice spleen. We expect that the present findings will provide useful information in the health care of space crews.

  1. Repression of the antiapoptotic molecule galectin-3 by homeodomain-interacting protein kinase 2-activated p53 is required for p53-induced apoptosis.

    PubMed

    Cecchinelli, Barbara; Lavra, Luca; Rinaldo, Cinzia; Iacovelli, Stefano; Gurtner, Aymone; Gasbarri, Alessandra; Ulivieri, Alessandra; Del Prete, Fabrizio; Trovato, Maria; Piaggio, Giulia; Bartolazzi, Armando; Soddu, Silvia; Sciacchitano, Salvatore

    2006-06-01

    Galectin 3 (Gal-3), a member of the beta-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3. PMID:16738336

  2. Cyclosporine A Suppressed Glucose Oxidase Induced P53 Mitochondrial Translocation and Hepatic Cell Apoptosis through Blocking Mitochondrial Permeability Transition

    PubMed Central

    Yu, Weihua; Zhang, Xiaodi; Liu, Jiangzheng; Wang, Xin; Li, Shuang; Liu, Rui; Liao, Nai; Zhang, Tao; Hai, Chunxu

    2016-01-01

    P53 is known as a transcription factor to control apoptotic cell death through regulating a series of target genes in nucleus. There is accumulating evidences show that p53 can directly induce cell apoptosis through transcription independent way at mitochondria. However, the mechanism by which p53 translocation into mitochondria in response to oxidative stress remains unclear. Here, glucose oxidase (GOX) was used to induce ROS generation in HepG2 cells and liver tissues of mice. The results showed that p53 was stabilized and translocated to mitochondria in a time and dose dependent manner after GOX exposure. Interestingly, as an inhibitor of mitochondrial permeability transition, cyclosporine A (CsA) was able to effectively reduce GOX mediated mitochondrial p53 distribution without influencing on the expression of p53 target genes including Bcl-2 and Bax. These indicated that CsA could just block p53 entering into mitochondria, but not affect p53-dependent transcription. Meanwhile, CsA failed to inhibit the ROS generation induced by GOX, which indicated that CsA had no antioxidant function. Moreover, GOX induced typical apoptosis characteristics including, mitochondrial dysfunction, accumulation of Bax and release of cytochrome C in mitochondria, accompanied with activation of caspase-9 and caspase-3. These processions were suppressed after pretreatment with CsA and pifithrin-μ (PFT-μ, a specific inhibitor of p53 mitochondrial translocation). In vivo, CsA was able to attenuate p53 mitochondrial distribution and protect mice liver against from GOX mediated apoptotic cell death. Taken together, these suggested that CsA could suppress ROS-mediated p53 mitochondrial distribution and cell apoptosis depended on its inhibition effect to mitochondrial permeability transition. It might be used to rescue the hepatic cell apoptosis in the patients with acute liver injury. PMID:26884717

  3. Adenovirus type 5 early region 4 is responsible for E1A-induced p53-independent apoptosis.

    PubMed Central

    Marcellus, R C; Teodoro, J G; Wu, T; Brough, D E; Ketner, G; Shore, G C; Branton, P E

    1996-01-01

    In the absence of E1B, the 289- and 243-residue E1A products of human adenovirus type 5 induce p53-dependent apoptosis. However, our group has shown recently that the 289-residue E1A protein is also able to induce apoptosis by a p53-independent mechanism (J. G. Teodoro, G. C. Shore, and P. E. Branton, Oncogene 11:467-474, 1995). Preliminary results suggested that p53-independent cell death required expression of one or more additional adenovirus early gene products. Here we show that both the E1B 19-kDa protein and cellular Bcl-2 inhibit or significantly delay p53-independent apoptosis. Neither early region E2 or E3 appeared to be necessary for such cell death. Analysis of a series of E1A mutants indicated that mutations in the transactivation domain and other regions of E1A correlated with E1A-mediated transactivation of E4 gene expression. Furthermore, p53-deficient human SAOS-2 cells infected with a mutant which expresses E1B but none of the E4 gene products remained viable for considerably longer times than those infected with wild-type adenovirus type 5. In addition, an adenovirus vector lacking both E1 and E4 was unable to induce DNA degradation and cell killing in E1A-expressing cell lines. These data showed that an E4 product is essential for E1A-induced p53-independent apoptosis. PMID:8709247

  4. Cyclosporine A Suppressed Glucose Oxidase Induced P53 Mitochondrial Translocation and Hepatic Cell Apoptosis through Blocking Mitochondrial Permeability Transition.

    PubMed

    Yu, Weihua; Zhang, Xiaodi; Liu, Jiangzheng; Wang, Xin; Li, Shuang; Liu, Rui; Liao, Nai; Zhang, Tao; Hai, Chunxu

    2016-01-01

    P53 is known as a transcription factor to control apoptotic cell death through regulating a series of target genes in nucleus. There is accumulating evidences show that p53 can directly induce cell apoptosis through transcription independent way at mitochondria. However, the mechanism by which p53 translocation into mitochondria in response to oxidative stress remains unclear. Here, glucose oxidase (GOX) was used to induce ROS generation in HepG2 cells and liver tissues of mice. The results showed that p53 was stabilized and translocated to mitochondria in a time and dose dependent manner after GOX exposure. Interestingly, as an inhibitor of mitochondrial permeability transition, cyclosporine A (CsA) was able to effectively reduce GOX mediated mitochondrial p53 distribution without influencing on the expression of p53 target genes including Bcl-2 and Bax. These indicated that CsA could just block p53 entering into mitochondria, but not affect p53-dependent transcription. Meanwhile, CsA failed to inhibit the ROS generation induced by GOX, which indicated that CsA had no antioxidant function. Moreover, GOX induced typical apoptosis characteristics including, mitochondrial dysfunction, accumulation of Bax and release of cytochrome C in mitochondria, accompanied with activation of caspase-9 and caspase-3. These processions were suppressed after pretreatment with CsA and pifithrin-μ (PFT-μ, a specific inhibitor of p53 mitochondrial translocation). In vivo, CsA was able to attenuate p53 mitochondrial distribution and protect mice liver against from GOX mediated apoptotic cell death. Taken together, these suggested that CsA could suppress ROS-mediated p53 mitochondrial distribution and cell apoptosis depended on its inhibition effect to mitochondrial permeability transition. It might be used to rescue the hepatic cell apoptosis in the patients with acute liver injury. PMID:26884717

  5. Mono(2-ethylhexyl) phthalate induces apoptosis in p53-silenced L02 cells via activation of both mitochondrial and death receptor pathways.

    PubMed

    Yang, Guangtao; Zhang, Wenjuan; Qin, Qizhi; Wang, Jing; Zheng, Hongyan; Xiong, Wei; Yuan, Jing

    2015-09-01

    Mono(2-ethylhexyl) phthalate (MEHP) is one of the main metabolites of di(2-ethylhexyl) phthalate. The evidence shows that DEHP may exert its toxic effects primarily via MEHP, which is 10-fold more potent than its parent compound in toxicity in vitro. MEHP-induced apoptosis is mediated by either p53-dependent or -independent pathway. However, the detailed mechanism of its toxicity remains unclear. In this study, immortalized normal human liver cell line L02 was chosen, as an in vitro model of nonmalignant liver, to elucidate the role of p53 in MEHP-induced apoptosis. The cells were treated with MEHP (6.25, 12.50, 25.00, 50.00, and 100.00 μM) for 24 and 36 h, then small interfering RNA (siRNA) was used to specifically silence p53 gene of L02 cells. The results indicated that MEHP caused oxidative DNA damage and apoptosis in L02 cells were associated with the p53 signaling pathway. Further study found that MEHP (50.00 and 100.00 μM) induced apoptosis in p53-silenced L02 cells, along with the up-regulations of Fas and FasL proteins as well as increased the Bax/Bcl-2 ratio and Caspase 3, 8, and 9 activities. Additionally, both FasL inhibitor (AF-016) and Caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp- fluoromethylketone (Z-VAD-FMK) could prevent the cell apoptosis induced by MEHP. The findings suggested that MEHP-induced apoptosis in L02 cells involving a Caspases-mediated mitochondrial signaling pathway and/or death receptor pathway. p53 was not absolutely necessary for MEHP-induced L02 cell apoptosis. PMID:24706461

  6. Apoptosis inducers in chronic lymphocytic leukemia

    PubMed Central

    Billard, Christian

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by a typical defect in apoptosis and is still an incurable disease. Numerous apoptosis inducers have been described. These synthetic compounds and natural products (mainly derived from plants) display antileukemic properties in vitro and in vivo and some have even been tested in the clinic in CLL. They act through several different mechanisms. Most of them involve proteins of the Bcl-2 family, which are the key regulators in triggering the mitochondrial pathway of caspase-dependent apoptosis. Thus, the Mcl-1/Noxa axis appeared as a target. Here I overview natural and synthetic apoptosis inducers and their mechanisms of action in CLL cells. Opportunities for developing novel, apoptosis-based therapeutics are presented. PMID:24525395

  7. Activation of p53 with Ilimaquinone and Ethylsmenoquinone, Marine Sponge Metabolites, Induces Apoptosis and Autophagy in Colon Cancer Cells

    PubMed Central

    Lee, Hyun-Young; Chung, Kyu Jin; Hwang, In Hyun; Gwak, Jungsuk; Park, Seoyoung; Ju, Bong Gun; Yun, Eunju; Kim, Dong-Eun; Chung, Young-Hwa; Na, MinKyun; Song, Gyu-Yong; Oh, Sangtaek

    2015-01-01

    The tumor suppressor, p53, plays an essential role in the cellular response to stress through regulating the expression of genes involved in cell cycle arrest, apoptosis and autophagy. Here, we used a cell-based reporter system for the detection of p53 response transcription to identify the marine sponge metabolites, ilimaquinone and ethylsmenoquinone, as activators of the p53 pathway. We demonstrated that ilimaquinone and ethylsmenoquinone efficiently stabilize the p53 protein through promotion of p53 phosphorylation at Ser15 in both HCT116 and RKO colon cancer cells. Moreover, both compounds upregulate the expression of p21WAF1/CIP1, a p53-dependent gene, and suppress proliferation of colon cancer cells. In addition, ilimaquinone and ethylsmenoquinone induced G2/M cell cycle arrest and increased caspase-3 cleavage and the population of cells that positively stained with Annexin V-FITC, both of which are typical biochemical markers of apoptosis. Furthermore, autophagy was elicited by both compounds, as indicated by microtubule-associated protein 1 light chain 3 (LC3) puncta formations and LC3-II turnover in HCT116 cells. Our findings suggest that ilimaquinone and ethylsmenoquinone exert their anti-cancer activity by activation of the p53 pathway and may have significant potential as chemo-preventive and therapeutic agents for human colon cancer. PMID:25603347

  8. p53 mediates cigarette smoke-induced apoptosis of pulmonary endothelial cells: inhibitory effects of macrophage migration inhibitor factor.

    PubMed

    Damico, Rachel; Simms, Tiffany; Kim, Bo S; Tekeste, Zenar; Amankwan, Henry; Damarla, Mahendra; Hassoun, Paul M

    2011-03-01

    Exposure to cigarette smoke (CS) is the most common cause of emphysema, a debilitating pulmonary disease histopathologically characterized by the irreversible destruction of lung architecture. Mounting evidence links enhanced endothelial apoptosis causally to the development of emphysema. However, the molecular determinants of human endothelial cell apoptosis and survival in response to CS are not fully defined. Such determinants could represent clinically relevant targets for intervention. We show here that CS extract (CSE) triggers the death of human pulmonary macrovascular endothelial cells (HPAECs) through a caspase 9-dependent apoptotic pathway. Exposure to CSE results in the increased expression of p53 in HPAECs. Using the p53 inhibitor, pifithrin-α (PFT-α), and RNA interference (RNAi) directed at p53, we demonstrate that p53 function and expression are required for CSE-mediated apoptosis. The expression of macrophage migration inhibitory factor (MIF), an antiapoptotic cytokine produced by HPAECs, also increases in response to CSE exposure. The addition of recombinant human MIF prevents cell death from exposure to CSE. Further, the suppression of MIF or its receptor/binding partner, Jun activation domain-binding protein 1 (Jab-1), with RNAi enhances the sensitivity of human pulmonary endothelial cells to CSE via a p53-dependent (PFT-α-inhibitable) pathway. Finally, we demonstrate that MIF is a negative regulator of p53 expression in response to CSE, placing MIF upstream of p53 as an antagonist of CSE-induced apoptosis. We conclude that MIF can protect human vascular endothelium from the toxic effects of CSE via the antagonism of p53-mediated apoptosis. PMID:20448056

  9. Phytochemical regulation of the tumor suppressive microRNA, miR-34a, by p53-dependent and independent responses in human breast cancer cells.

    PubMed

    Hargraves, Kris G; He, Lin; Firestone, Gary L

    2016-05-01

    The tumor suppressive microRNA miR-34a is transcriptionally regulated by p53 and shown to inhibit breast cancer cell proliferation as well as being a marker of increased disease free survival. Indole-3-carbinol (I3C) derived from cruciferous vegetables, artemisinin, extracted from the sweet wormwood plant, and artesunate, a semi-synthetic derivative of artemisinin, are phytochemicals with anti-tumorigenic properties however, little is known about the role of microRNAs in their mechanism of action. Human breast cancer cells expressing wild-type (MCF-7) or mutant p53 (T47D) were treated with a concentration range and time course of each phytochemical under conditions of cell cycle arrest as detected by flow cytometry to examine the potential connection between miR-34a expression and their anti-proliferative responses. Real-time PCR and western blot analysis of extracted RNA and total protein revealed artemsinin and artesunate increased miR-34a expression in a dose-dependent manner correlating with down-regulation of the miR-34a target gene, CDK4. I3C stimulation of miR-34a expression required functional p53, whereas, both artemisinin and artesunate up-regulated miR-34a expression regardless of p53 mutational status or in the presence of dominant negative p53. Phytochemical treatments inhibited the luciferase activity of a construct containing the wild-type 3'UTR of CDK4, but not those with a mutated miR-34a binding site, whereas, transfection of miR-34a inhibitors ablated the phytochemical mediated down-regulation of CDK4 and induction of cell cycle arrest. Our results suggest that miR-34a is an essential component of the anti-proliferative activities of I3C, artemisinin, and artesunate and demonstrate that both wild-type p53 dependent and independent pathways are responsible for miR-34a induction. © 2015 Wiley Periodicals, Inc. PMID:25789847

  10. Treatment with a Small Synthetic Compound, KMU-193, induces Apoptosis in A549 Human Lung Carcinoma Cells through p53 Up-Regulation.

    PubMed

    Choi, Eun Young; Shin, Kyeong-Cheol; Lee, Jinho; Kwon, Taeg Kyu; Kim, Shin; Park, Jong-Wook

    2015-01-01

    Despite recent advances in therapeutic strategies for lung cancer, mortality still is increasing. In the present study, we investigated the anti-cancer effects of KMU-193, 2-(4-Ethoxy-phenyl)-N-{5-[2-fluoro-4-(4-methyl- piperazine-1-carbonyl)-phenylamino]-1H-indazol-3-yl}-acetamide in a human non-small cell lung cancer cell line A549. KMU-193 strongly inhibited the proliferation of A549 cells, but it did not have anti-proliferative effect in other types of cancer cell lines. KMU-193 further induced apoptosis in association with activation of caspase-3 and cleavage of PLC-γ1. However, KMU-193 had no apoptotic effect in untransformed cells such as TMCK-1 and BEAS-2B. Interestingly, pretreatment with z-VAD-fmk, a pan-caspase inhibitor, strongly abrogated KMU- 193-induced apoptosis. KMU-193 treatment enhanced the expression levels of p53 and PUMA. Importantly, p53 siRNA transfection attenuated KMU-193-induced apoptosis. Collectively, these results for the first time demonstrate that KMU-193 has strong apoptotic effects on A549 cells and these are largely mediated through caspase-3- and p53-dependent pathways. PMID:26320467

  11. Crude aqueous extracts of Pluchea indica (L.) Less. inhibit proliferation and migration of cancer cells through induction of p53-dependent cell death

    PubMed Central

    2012-01-01

    Background Pluchea indica (L.) Less. (Asteraceae) is a perennial shrub plant with anti-inflammatory and antioxidant medicinal properties. However, the anti-cancer properties of its aqueous extracts have not been studied. The aim of this study was to investigate the anti-proliferation, anti-migration, and pro-apoptotic properties of crude aqueous extracts of P. indica leaf and root on human malignant glioma cancer cells and human cervical cancer cells, and the underlying molecular mechanism. Methods GBM8401 human glioma cells and HeLa cervical carcinoma cells were treated with various concentrations of crude aqueous extracts of P. indica leaf and root and cancer cell proliferation and viability were measured by cell growth curves, trypan blue exclusions, and the tetrazolium reduction assay. Effects of the crude aqueous extracts on focus formation, migration, and apoptosis of cancer cells were studied as well. The molecular mechanism that contributed to the anti-cancer activities of crude aqueous extracts of P. indica root was also examined using Western blotting analysis. Results Crude aqueous extracts of P. indica leaf and root suppressed proliferation, viability, and migration of GBM8401 and HeLa cells. Treatment with crude aqueous extracts of P. indica leaf and root for 48 hours resulted in a significant 75% and 70% inhibition on proliferation and viability of GBM8401 and HeLa cancer cells, respectively. Crude aqueous extracts of P. indica root inhibited focus formation and promoted apoptosis of HeLa cells. It was found that phosphorylated-p53 and p21 were induced in GBM8401 and HeLa cells treated with crude aqueous extracts of P. indica root. Expression of phosphorylated-AKT was decreased in HeLa cells treated with crude aqueous extracts of P. indica root. Conclusion The in vitro anti-cancer effects of crude aqueous extracts of P. indica leaf and root indicate that it has sufficient potential to warrant further examination and development as a new anti

  12. Lysosomal destabilization in p53-induced apoptosis

    PubMed Central

    Yuan, Xi-Ming; Li, Wei; Dalen, Helge; Lotem, Joseph; Kama, Rachel; Sachs, Leo; Brunk, Ulf T.

    2002-01-01

    The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders. PMID:11959917

  13. Absence of a p53 allele delays nitrogen mustard-induced early apoptosis and inflammation of murine skin

    PubMed Central

    Inturi, Swetha; Tewari-Singh, Neera; Jain, Anil K.; Roy, Srirupa; White, Carl W.; Agarwal, Rajesh

    2013-01-01

    Bifunctional alkylating agent sulfur mustard (SM) and its analog nitrogen mustard (NM) cause DNA damage leading to cell death, and potentially activating inflammation. Transcription factor p53 plays a critical role in DNA damage by regulating cell cycle progression and apoptosis. Earlier studies by our laboratory demonstrated phosphorylation of p53 at Ser15 and an increase in total p53 in epidermal cells both in vitro and in vivo following NM exposure. To elucidate the role of p53 in NM-induced skin toxicity, we employed SKH-1 hairless mice harboring wild type (WT) or heterozygous p53 (p53+/−). Exposure to NM (3.2 mg) caused a more profound increase in epidermal thickness and apoptotic cell death in WT relative to p53+/− mice at 24 h. However, by 72 h after exposure, there was a comparable increase in NM-induced epidermal cell death in both WT and p53+/− mice. Myeloperoxidase activity data showed that neutrophil infiltration was strongly enhanced in NM-exposed WT mice at 24 h persisting through 72 h of exposure. Conversely, robust NM-induced neutrophil infiltration (comparable to WT mice) was seen only at 72 h after exposure in p53+/− mice. Similarly, NM-exposure strongly induced macrophage and mast cell infiltration in WT, but not p53+/− mice. Together, these data indicate that early apoptosis and inflammation induced by NM in mouse skin are p53-dependent. Thus, targeting this pathway could be a novel strategy for developing countermeasures against vesicants-induced skin injury. PMID:23845566

  14. Training-induced apoptosis in skeletal muscle.

    PubMed

    Boffi, F M; Cittar, J; Balskus, G; Muriel, M; Desmaras, E

    2002-09-01

    Apoptosis or programmed cell death is a genetically controlled response of cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include Ca2+i and oxygen free radicals/oxidative stress, which are also implicated in the pathogenesis of exercise-induced myopathies. To examine training-induced apoptosis, Thoroughbred horses were subjected to 3 months training programme on a treadmill. At the end of the training programme venous blood samples were taken for a creatine kinase (CK) assay. In addition, muscle biopsy samples were obtained for a membrane lipid peroxidation measurement by malondialdehyde (MDA) assay and for apoptosis detection. Apoptosis was studied by visualising the apoptotic myocytes on the paraffin sections by the modified TUNEL method. DNA laddering was evaluated by subjecting the DNA obtained from the biopsies to 1.5% agarose gel electrophoresis. There was a significant increase (P<0.05) of protein-bound MDA, and a nonsignificant trend (P = 0.14) for the control group to have higher levels of CK compared to the trained group. Under light microscopy, percentage of the TUNEL positive cells was higher (P<0.001) in the training group. This result was corroborated with the findings of DNA fragmentation by gel electrophoresis, which showed higher ladders of DNA band at the same group. In conclusion, these results clearly demonstrate that there is training-induced apoptosis in skeletal muscle. It is probable that apoptosis allows the work/recovery/rebound/supercompensation cycle, when unaccustomed muscle cells activate programmed cell death and are replaced by new and stronger cells, which is the mechanism for training-induced increases in fitness. PMID:12405700

  15. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    NASA Astrophysics Data System (ADS)

    Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng

    2013-02-01

    Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (ΔΨm). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ΔΨm. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.

  16. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  17. LiCl induces TNF-α and FasL production, thereby stimulating apoptosis in cancer cells

    PubMed Central

    2011-01-01

    Background The incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours. Results Here we show that LiCl induces apoptosis of tumour cells both in vitro and in vivo. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-α and FasL expression. Inhibition of TNF-α induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth. Conclusions Induction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-α and FasL. Key words: LiCl, TNF-α, FasL, apoptosis, GSK-3, FasL PMID:21609428

  18. Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells.

    PubMed

    Banerjee, Mithu; Singh, Parminder; Panda, Dulal

    2010-08-01

    In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules. PMID:20646066

  19. Supercritical carbon dioxide extract of Physalis peruviana induced cell cycle arrest and apoptosis in human lung cancer H661 cells.

    PubMed

    Wu, Shu-Jing; Chang, Shun-Pang; Lin, Doung-Liang; Wang, Shyh-Shyan; Hou, Fwu-Feuu; Ng, Lean-Teik

    2009-06-01

    Physalis peruviana L. (PP) is a popular folk medicine used for treating cancer, leukemia, hepatitis, rheumatism and other diseases. In this study, our objectives were to examine the total flavonoid and phenol content of different PP extracts (aqueous: HWEPP; ethanolic: EEPP; supercritical carbon dioxide: SCEPP-0, SCEPP-4 and SCEPP-5) and their antiproliferative effects in human lung cancer H661 cells. Among all the extracts tested, results showed that SCEPP-5 possessed the highest total flavonoid (226.19 +/- 4.15 mg/g) and phenol (100.82 +/- 6.25 mg/g) contents. SCEPP-5 also demonstrated the most potent inhibitory effect on H661 cell proliferation. Using DNA ladder and flow cytometry analysis, SCEPP-5 effectively induced H661 cell apoptosis as demonstrated by the accumulation of Sub-G1 peak and fragmentation of DNA. SCEPP-5 not only induced cell cycle arrest at S phase, it also up-regulated the expression of pro-apoptotic protein (Bax) and down-regulated the inhibitor of apoptosis protein (IAP). Furthermore, the apoptotic induction in H661 cells was found to associate with an elevated p53 protein expression, cytochrome c release, caspase-3 activation and PARP cleavage. Taken together, these results conclude that SCEPP-5 induced cell cycle arrest at S phase, and its apoptotic induction could be mediated through the p53-dependent pathway and modification of Bax and XIAP proteins expression. The results have also provided important pharmacological backgrounds for the potential use of PP supercritical fluid extract as products for cancer prevention. PMID:19425186

  20. Benzo(a)pyrene Induced p53 Mediated Male Germ Cell Apoptosis: Synergistic Protective Effects of Curcumin and Resveratrol.

    PubMed

    Banerjee, Bhaswati; Chakraborty, Supriya; Ghosh, Debidas; Raha, Sanghamitra; Sen, Parimal C; Jana, Kuladip

    2016-01-01

    Benzo(a)pyrene (B(a)P) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, and Apaf1. B(a)P induced testicular reactive oxygen species (ROS) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 (Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ

  1. Benzo(a)pyrene Induced p53 Mediated Male Germ Cell Apoptosis: Synergistic Protective Effects of Curcumin and Resveratrol

    PubMed Central

    Banerjee, Bhaswati; Chakraborty, Supriya; Ghosh, Debidas; Raha, Sanghamitra; Sen, Parimal C.; Jana, Kuladip

    2016-01-01

    Benzo(a)pyrene (B(a)P) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, and Apaf1. B(a)P induced testicular reactive oxygen species (ROS) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 (Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ

  2. The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells.

    PubMed

    Priyadarsini, R Vidya; Murugan, R Senthil; Sripriya, P; Karunagaran, D; Nagini, S

    2010-06-01

    Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-kappaB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs. PMID:20429769

  3. Lithium protects ethanol-induced neuronal apoptosis

    SciTech Connect

    Zhong Jin . E-mail: jizhong@iupui.edu; Yang Xianlin; Yao Weiguo; Lee Weihua

    2006-12-01

    Lithium is widely used for the treatment of bipolar disorder. Recent studies have demonstrated its neuroprotective effect. Ethanol is a potent neurotoxin that is particularly harmful to the developing nervous system. In this study, we evaluated lithium's neuroprotection against ethanol-induced apoptosis. Transient exposure of infant mice to ethanol caused apoptotic cell death in brain, which was prevented significantly by administering a low dose of lithium 15 min later. In cultured cerebellar granule neurons, ethanol-induced apoptosis and activation of caspase-3/9, both of which were prevented by lithium. However, lithium's protection is not mediated by its commonly known inhibition of glycogen synthase3{beta}, because neither ethanol nor lithium has significant effects on the phosphorylation of Akt (ser473) or GSK3{beta} (ser9). In addition, the selective GSK-3{beta} inhibitor SB-415286 was unable to prevent ethanol-induced apoptosis. These data suggest lithium may be used as a potential preventive measure for ethanol-induced neurological deficits.

  4. Loss of p19(Arf) facilitates the angiogenic switch and tumor initiation in a multi-stage cancer model via p53-dependent and independent mechanisms.

    PubMed

    Ulanet, Danielle B; Hanahan, Douglas

    2010-01-01

    The Arf tumor suppressor acts as a sensor of oncogenic signals, countering aberrant proliferation in large part via activation of the p53 transcriptional program, though a number of p53-independent functions have been described. Mounting evidence suggests that, in addition to promoting tumorigenesis via disruptions in the homeostatic balance between cell proliferation and apoptosis of overt cancer cells, genetic alterations leading to tumor suppressor loss of function or oncogene gain of function can also incite tumor development via effects on the tumor microenvironment. In a transgenic mouse model of multi-stage pancreatic neuroendocrine carcinogenesis (PNET) driven by inhibition of the canonical p53 and Rb tumor suppressors with SV40 large T-antigen (Tag), stochastic progression to tumors is limited in part by a requirement for initiation of an angiogenic switch. Despite inhibition of p53 by Tag in this mouse PNET model, concomitant disruption of Arf via genetic knockout resulted in a significantly accelerated pathway to tumor formation that was surprisingly not driven by alterations in tumor cell proliferation or apoptosis, but rather via earlier activation of the angiogenic switch. In the setting of a constitutional p53 gene knockout, loss of Arf also accelerated tumor development, albeit to a lesser degree. These findings demonstrate that Arf loss of function can promote tumorigenesis via facilitating angiogenesis, at least in part, through p53-independent mechanisms. PMID:20805995

  5. Indirect p53-dependent transcriptional repression of Survivin, CDC25C, and PLK1 genes requires the cyclin-dependent kinase inhibitor p21/CDKN1A and CDE/CHR promoter sites binding the DREAM complex

    PubMed Central

    Nickel, Annina; Engeland, Kurt

    2015-01-01

    The transcription factor p53 is central to cell cycle control by downregulation of cell cycle-promoting genes upon cell stress such as DNA damage. Survivin (BIRC5), CDC25C, and PLK1 encode important cell cycle regulators that are repressed following p53 activation. Here, we provide evidence that p53-dependent repression of these genes requires activation of p21 (CDKN1A, WAF1, CIP1). Chromatin immunoprecipitation (ChIP) data indicate that promoter binding of B-MYB switches to binding of E2F4 and p130 resulting in a replacement of the MMB (Myb-MuvB) by the DREAM complex. We demonstrate that this replacement depends on p21. Furthermore, transcriptional repression by p53 requires intact DREAM binding sites in the target promoters. The CDE and CHR cell cycle promoter elements are the sites for DREAM binding. These elements as well as the p53 response of Survivin, CDC25C, and PLK1 are evolutionarily conserved. No binding of p53 to these genes is detected by ChIP and mutation of proposed p53 binding sites does not alter the p53 response. Thus, a mechanism for direct p53-dependent transcriptional repression is not supported by the data. In contrast, repression by DREAM is consistent with most previous findings and unifies models based on p21-, E2F4-, p130-, and CDE/CHR-dependent repression by p53. In conclusion, the presented data suggest that the p53-p21-DREAM-CDE/CHR pathway regulates p53-dependent repression of Survivin, CDC25C, and PLK1. PMID:26595675

  6. Farnesol-induced apoptosis in Candida albicans.

    PubMed

    Shirtliff, Mark E; Krom, Bastiaan P; Meijering, Roelien A M; Peters, Brian M; Zhu, Jingsong; Scheper, Mark A; Harris, Megan L; Jabra-Rizk, Mary Ann

    2009-06-01

    Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival. PMID:19364863

  7. Loss of oocytes due to conditional ablation of Murine double minute 2 (Mdm2) gene is p53-dependent and results in female sterility.

    PubMed

    Livera, Gabriel; Uzbekov, Rustem; Jarrier, Peggy; Fouchécourt, Sophie; Duquenne, Clotilde; Parent, Anne-Simone; Marine, Jean-Christophe; Monget, Philippe

    2016-08-01

    Murine double minute 2 and 4 (Mdm2, Mdm4) are major p53-negative regulators, preventing thus uncontrolled apoptosis induction in numerous cell types, although their function in the female germ line has received little attention. In the present work, we have generated mice with specific invalidation of Mdm2 and Mdm4 genes in the mouse oocyte (Mdm2(Ocko) and Mdm4(Ocko) mice), to test their implication in survival of these germ cells. Most of the Mdm2(Ocko) but not Mdm4(Ocko) mice were sterile, with a dramatic reduction of the weight of ovaries and genital tract, a strong increase in follicle-stimulating hormone and luteinizing hormone serum levels, and a reduction of anti-mullerian hormone serum levels. Histological analyses revealed an obvious decrease of the number of growing follicles beyond the primary stage in Mdm2(Ocko) ovaries in comparison to controls, with a pronounced increase in the apparition of primary atretic follicles, most being devoid of oocyte. Similar phenotypes were observed with Mdm2(Ocko) Mdm4(Ocko) ovaries, with no worsening of the phenotype. However, we failed to detect any increase in p53 level in mutant oocytes, nor any other apoptotic marker, introgression of this targeted invalidation in p53-/- mice restored the fertility of females. This study is the first to show that Mdm2, but not Mdm4, has a critical role in oocyte survival and would be involved in premature ovarian insufficiency phenotype. PMID:27364741

  8. Artesunate induces AIF-dependent apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  9. The enhancing effect of genistein on apoptosis induced by trichostatin A in lung cancer cells with wild type p53 genes is associated with upregulation of histone acetyltransferase.

    PubMed

    Wu, Tzu-Chin; Lin, Yi-Chin; Chen, Hsiao-Ling; Huang, Pei-Ru; Liu, Shang-Yu; Yeh, Shu-Lan

    2016-02-01

    Genistein has been shown to enhance the antitumor activity of trichostatin A (TSA) in human lung carcinoma A549 cells. However, whether the combined treatment exerts the same effect in other lung cancer cells is unclear. In the present study we first compared the enhancing effect of genistein on the antitumor effect of TSA in ABC-1, NCI-H460 (H460) and A549 cells. Second, we investigated whether the effects of genistein are associated with increased histone/non-histone protein acetylation. We found that the enhancing effect of genistein on cell-growth-arrest in ABC-1 cells (p53 mutant) was less than in A549 and H460 cells. Genistein enhanced TSA induced apoptosis in A549 and H460 cells rather than in ABC-1 cells. After silencing p53 expression in A549 and H460 cells, the enhancing effect of genistein was diminished. In addition, genistein increased TSA-induced histone H3/H4 acetylation in A549 and H460 cells. Genistein also increased p53 acetylation in H460 cells. The inhibitor of acetyltransferase, anacardic acid, diminished the enhancing effect of genistein on all TSA-induced histone/p53 acetylation and apoptosis. Genistein in combination with TSA increased the expression of p300 protein, an acetyltransferase, in A549 and NCI-H460 cells. Furthermore, we demonstrated that genistein also enhanced the antitumor effect of genistein in A549-tumor-bearing mice. Taken together, these results suggest that the enhancing effects of genistein on TSA-induced apoptosis in lung cancer cells were p53-dependent and were associated with histone/non-histone protein acetylation. PMID:26768552

  10. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  11. p53-dependent chemokine production by senescent tumor cells supports NKG2D-dependent tumor elimination by natural killer cells

    PubMed Central

    Iannello, Alexandre; Thompson, Thornton W.; Ardolino, Michele; Lowe, Scott W.

    2013-01-01

    The induction of cellular senescence is an important mechanism by which p53 suppresses tumorigenesis. Using a mouse model of liver carcinoma, where cellular senescence is triggered in vivo by inducible p53 expression, we demonstrated that NK cells participate in the elimination of senescent tumors. The elimination of senescent tumor cells is dependent on NKG2D. Interestingly, p53 restoration neither increases ligand expression nor increases the sensitivity to lysis by NK cells. Instead, p53 restoration caused tumor cells to secrete various chemokines with the potential to recruit NK cells. Antibody-mediated neutralization of CCL2, but not CCL3, CCL4 or CCL5, prevented NK cell recruitment to the senescent tumors and reduced their elimination. Our findings suggest that elimination of senescent tumors by NK cells occurs as a result of the cooperation of signals associated with p53 expression or senescence, which regulate NK cell recruitment, and other signals that induce NKG2D ligand expression on tumor cells. PMID:24043758

  12. Minnelide/Triptolide Impairs Mitochondrial Function by Regulating SIRT3 in P53-Dependent Manner in Non-Small Cell Lung Cancer.

    PubMed

    Kumar, Ajay; Corey, Catherine; Scott, Iain; Shiva, Sruti; D'Cunha, Jonathan

    2016-01-01

    Minnelide/Triptolide (TL) has recently emerged as a potent anticancer drug in non-small cell lung cancer (NSCLC). However, the precise mechanism of its action remains ambiguous. In this study, we elucidated the molecular basis for TL-induced cell death in context to p53 status. Cell death was attributed to dysfunction of mitochondrial bioenergetics in p53-deficient cells, which was characterized by decreased mitochondrial respiration, steady-state ATP level and membrane potential, but augmented reactive oxygen species (ROS). Increased ROS production resulted in oxidative stress in TL-treated cells. This was exhibited by elevated nuclear levels of a redox-sensitive transcriptional factor, NF-E2-related factor-2 (NRF2), along with diminished cellular glutathione (GSH) content. We further demonstrated that in the absence of p53, TL blunted the expression of mitochondrial SIRT3 triggering increased acetylation of NDUAF9 and succinate dehydrogenase, components of complexes I and II of the electron transport chain (ETC). TL-mediated hyperacetylation of complexes I and II proteins and these complexes displayed decreased enzymatic activities. We also provide the evidence that P53 regulate steady-state level of SIRT3 through Proteasome-Pathway. Finally, forced overexpression of Sirt3, but not deacetylase-deficient mutant of Sirt3 (H243Y), restored the deleterious effect of TL on p53-deficient cells by rescuing mitochondrial bioenergetics. On contrary, Sirt3 deficiency in the background of wild-type p53 triggered TL-induced mitochondrial impairment that echoed TL effect in p53-deficeint cells. These findings illustrate a novel mechanism by which TL exerts its potent effects on mitochondrial function and ultimately the viability of NSCLC tumor. PMID:27501149

  13. Minnelide/Triptolide Impairs Mitochondrial Function by Regulating SIRT3 in P53-Dependent Manner in Non-Small Cell Lung Cancer

    PubMed Central

    Kumar, Ajay; Corey, Catherine; Scott, Iain; Shiva, Sruti; D’Cunha, Jonathan

    2016-01-01

    Minnelide/Triptolide (TL) has recently emerged as a potent anticancer drug in non-small cell lung cancer (NSCLC). However, the precise mechanism of its action remains ambiguous. In this study, we elucidated the molecular basis for TL-induced cell death in context to p53 status. Cell death was attributed to dysfunction of mitochondrial bioenergetics in p53-deficient cells, which was characterized by decreased mitochondrial respiration, steady-state ATP level and membrane potential, but augmented reactive oxygen species (ROS). Increased ROS production resulted in oxidative stress in TL-treated cells. This was exhibited by elevated nuclear levels of a redox-sensitive transcriptional factor, NF-E2-related factor-2 (NRF2), along with diminished cellular glutathione (GSH) content. We further demonstrated that in the absence of p53, TL blunted the expression of mitochondrial SIRT3 triggering increased acetylation of NDUAF9 and succinate dehydrogenase, components of complexes I and II of the electron transport chain (ETC). TL-mediated hyperacetylation of complexes I and II proteins and these complexes displayed decreased enzymatic activities. We also provide the evidence that P53 regulate steady-state level of SIRT3 through Proteasome-Pathway. Finally, forced overexpression of Sirt3, but not deacetylase-deficient mutant of Sirt3 (H243Y), restored the deleterious effect of TL on p53-deficient cells by rescuing mitochondrial bioenergetics. On contrary, Sirt3 deficiency in the background of wild-type p53 triggered TL-induced mitochondrial impairment that echoed TL effect in p53-deficeint cells. These findings illustrate a novel mechanism by which TL exerts its potent effects on mitochondrial function and ultimately the viability of NSCLC tumor. PMID:27501149

  14. Oridonin induces apoptosis in SW1990 pancreatic cancer cells via p53- and caspase-dependent induction of p38 MAPK.

    PubMed

    Bu, He-Qi; Liu, Dian-Lei; Wei, Wei-Tian; Chen, Liang; Huang, Hai; Li, Ye; Cui, Jun-Hui

    2014-02-01

    Oridonin, an active component isolated from Rabdosia rubescens, has been reported to exhibit antitumor effects. In the present study, we evaluated the antitumor activity and the mechanisms of action of oridonin in pancreatic cancer. Oridonin treatment significantly induced apoptotic cell death in SW1990 pancreatic cancer cells in a dose-dependent manner. Additionally, cell apoptosis was markedly inhibited by PFT α (pifithrin α), a p53-specific inhibitor, which was applied to evaluate the function of p53, showing that p53 was responsible for the cytotoxity of oridonin. Moreover, oridonin increased the expression of p-p53 with a concomitant increase in p21 in the SW1990 cells. Following treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor), the cytotoxity of oridonin was not influenced by JNK (SP600125) and ERK (PD98059), but these effects were opposite to the cytotoxity of oridonin observed with SP203580 treatment. These findings confirmed that orodonin-induced apoptosis was p38-dependent, but JNK- and ERK-independent. Furthermore, the activation of the p38 kinase promoted the activation of p53 and its downstream target p21, and further caused caspase-9 and -3 activation, as demonstrated by evidence showing that the p38 inhibitor SB203580 not only blocked the phosphorylation of p38 but also reduced the activation of p53, p21 and caspase-9 and -3. Collectively, these results suggest that p53-dependent and caspase-dependent induction of p38 MAPK directly participates in apoptosis induced by oridonin. PMID:24297112

  15. Selective IAP inhibition results in sensitization of unstimulated but not CD40-stimulated chronic lymphocytic leukaemia cells to TRAIL-induced apoptosis

    PubMed Central

    Zhuang, Jianguo; Laing, Naomi; Oates, Melanie; Lin, Ke; Johnson, Gillian; Pettitt, Andrew R

    2014-01-01

    Despite recent advances in therapy, chronic lymphocytic leukaemia (CLL) remains incurable and new treatment strategies are therefore urgently required. Inhibitor of apoptosis proteins (IAPs) are over-expressed in CLL, suggesting both a role in disease pathogenesis and the potential for therapeutic targeting. To explore these questions, we evaluated the effects on primary CLL cells of AZD5582, a novel potent and selective inhibitor of IAPs. AZD5582 at nanomolar concentrations induced extensive degradation of cIAP-1 and cIAP-2, but minimally of X chromosome-linked IAP (XIAP). However, these effects of AZD5582 produced little or no direct cytotoxicity, nor did they sensitize CLL cells to p53-dependent killing by fludarabine or p53-independent killing by dexamethasone. In contrast, AZD5582 significantly enhanced apoptosis induced by the death receptor (DR) agonist tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, killing by TRAIL plus AZD5582 was independent of adverse prognostic features including TP53 deletion which is strongly associated with chemoresistance in CLL. Coculture experiments involving transfected mouse fibroblasts expressing human CD40L (CD154) to mimic the effect of T cells at sites of tissue involvement showed that CD40 stimulation almost completely prevented the killing of CLL cells by TRAIL plus AZD5582 despite up-regulating TRAIL receptors 1 and 2. In conclusion, our findings confirm the rate-limiting, upstream involvement of IAPs in the extrinsic but not intrinsic apoptotic pathway of CLL cells and suggest that drug combinations that simultaneously activate DRs and inhibit IAPs may have therapeutic potential in patients with CLL who have failed T-cell-depleting chemotherapy. PMID:25505620

  16. Selective IAP inhibition results in sensitization of unstimulated but not CD40-stimulated chronic lymphocytic leukaemia cells to TRAIL-induced apoptosis.

    PubMed

    Zhuang, Jianguo; Laing, Naomi; Oates, Melanie; Lin, Ke; Johnson, Gillian; Pettitt, Andrew R

    2014-12-01

    Despite recent advances in therapy, chronic lymphocytic leukaemia (CLL) remains incurable and new treatment strategies are therefore urgently required. Inhibitor of apoptosis proteins (IAPs) are over-expressed in CLL, suggesting both a role in disease pathogenesis and the potential for therapeutic targeting. To explore these questions, we evaluated the effects on primary CLL cells of AZD5582, a novel potent and selective inhibitor of IAPs. AZD5582 at nanomolar concentrations induced extensive degradation of cIAP-1 and cIAP-2, but minimally of X chromosome-linked IAP (XIAP). However, these effects of AZD5582 produced little or no direct cytotoxicity, nor did they sensitize CLL cells to p53-dependent killing by fludarabine or p53-independent killing by dexamethasone. In contrast, AZD5582 significantly enhanced apoptosis induced by the death receptor (DR) agonist tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, killing by TRAIL plus AZD5582 was independent of adverse prognostic features including TP53 deletion which is strongly associated with chemoresistance in CLL. Coculture experiments involving transfected mouse fibroblasts expressing human CD40L (CD154) to mimic the effect of T cells at sites of tissue involvement showed that CD40 stimulation almost completely prevented the killing of CLL cells by TRAIL plus AZD5582 despite up-regulating TRAIL receptors 1 and 2. In conclusion, our findings confirm the rate-limiting, upstream involvement of IAPs in the extrinsic but not intrinsic apoptotic pathway of CLL cells and suggest that drug combinations that simultaneously activate DRs and inhibit IAPs may have therapeutic potential in patients with CLL who have failed T-cell-depleting chemotherapy. PMID:25505620

  17. Rabies virus matrix protein induces apoptosis by targeting mitochondria.

    PubMed

    Zan, Jie; Liu, Juan; Zhou, Jian-Wei; Wang, Hai-Long; Mo, Kai-Kun; Yan, Yan; Xu, Yun-Bin; Liao, Min; Su, Shuo; Hu, Rong-Liang; Zhou, Ji-Yong

    2016-09-10

    Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination. PMID:27426727

  18. B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box

    SciTech Connect

    Liang Xin; Xu Ke; Xu Yufang; Liu Jianwen Qian Xuhong

    2011-10-01

    The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P{sub 2} promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment. - Research Highlights: > B1 induced apoptosis in MCF-7 cells, following a transcriptional decrease in Bcl-2. > B1 treatment triggered p53 activation and leads to a p53-dependent down-regulation of Bcl-2. > B1 induced significant increase of p53 binding to Bcl-2 P{sub 2} promoter TATA box.

  19. Research Advances on Pathways of Nickel-Induced Apoptosis

    PubMed Central

    Guo, Hongrui; Chen, Lian; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Wu, Bangyuan

    2015-01-01

    High concentrations of nickel (Ni) are harmful to humans and animals. Ni targets a number of organs and produces multiple toxic effects. Apoptosis is important in Ni-induced toxicity of the kidneys, liver, nerves, and immune system. Apoptotic pathways mediated by reactive oxygen species (ROS), mitochondria, endoplasmic reticulum (ER), Fas, and c-Myc participate in Ni-induced cell apoptosis. However, the exact mechanism of apoptosis caused by Ni is still unclear. Understanding the mechanism of Ni-induced apoptosis may help in designing measures to prevent Ni toxicity. PMID:26703593

  20. Quercetin-induced apoptosis prevents EBV infection

    PubMed Central

    Lee, Minjung; Son, Myoungki; Ryu, Eunhyun; Shin, Yu Su; Kim, Jong Gwang; Kang, Byung Woog; Sung, Gi-Ho; Cho, Hyosun; Kang, Hyojeung

    2015-01-01

    Epstein-Barr virus (EBV) is a human gamma-1 herpesvirus that establishes a lifelong latency in over 90% of the world's population. During latency, virus exists predominantly as a chromatin-associated, multicopy episome in the nuclei of a variety of tumor cells derived from B cells, T cells, natural killer (NK) cells, and epithelial cells. Licorice is the root of Glycyrrhiza uralensis or G. glabra that has traditionally cultivated in eastern part of Asia. Licorice was reported to have anti-viral, anti-inflammatory, anti-atopic, hepatoprotective, anti-neurodegenerative, anti-tumor, anti-diabetic effects and so forth. Quercetin and isoliquiritigenin are produced from licorice and highly similar in molecular structure. They have diverse bioactive effects such as antiviral activity, anti-asthmatic activity, anti-cancer activity, anti-inflammation activity, monoamine-oxidase inhibitor, and etc. To determine anti-EBV and anti-EBVaGC (Epstein-Barr virus associated gastric carcinoma) effects of licorice, we investigated antitumor and antiviral effects of quercetin and isoliquiritigenin against EBVaGC. Although both quercetin and isoliquiritigenin are cytotoxic to SNU719 cells, quercetin induced more apoptosis in SNU719 cells than isoliquiritigenin, more completely eliminated DNMT1 and DNMT3A expressions than isoliquiritigenin, and more strongly affects the cell cycle progression of SNU719 than isoliquiritigenin. Both quercetin and isoliquiritigenin induce signal transductions to stimulate apoptosis, and induce EBV gene transcription. Quercetin enhances frequency of F promoter use, whereas isoliquiritigenin enhances frequency of Q promoter use. Quercetin reduces EBV latency, whereas isoliquiritigenin increases the latency. Quercetin increases more the EBV progeny production, and inhibits more EBV infection than isoliquiritigenin. These results indicate that quercetin could be a promising candidate for antiviral and antitumor agents against EBV and human gastric carcinoma

  1. Participation of cyclin A in Myc-induced apoptosis.

    PubMed Central

    Hoang, A T; Cohen, K J; Barrett, J F; Bergstrom, D A; Dang, C V

    1994-01-01

    The involvement of c-Myc in cellular proliferation or apoptosis has been linked to differential cyclin gene expression. We observed that in both proliferating cells and cells undergoing apoptosis, cyclin A (but not B, C, D1, and E) mRNA level was elevated in unsynchronized Myc-overexpressing cells when compared with parental Rat1a fibroblasts. We further demonstrated that Zn(2+)-inducible cyclin A expression was sufficient to cause apoptosis. When Myc-induced apoptosis was blocked by coexpression of Bcl-2, the levels of cyclin C, D1, and E mRNAs were also elevated. Thus, while apoptosis induced by c-Myc is associated with an elevated cyclin A mRNA level, protection from apoptosis by coexpressed Bcl-2 is associated with a complementary increase in cyclin C, D1, and E mRNAs. Images PMID:8041712

  2. Platelets induce apoptosis via membrane-bound FasL

    PubMed Central

    Schleicher, Rebecca I.; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O’Reilly, Lorraine; Meuth, Sven G.; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank

    2015-01-01

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. PMID:26232171

  3. PRMT6 mediates CSE induced inflammation and apoptosis.

    PubMed

    Kang, Naixing; Chen, Ping; Chen, Yan; Zeng, Huihui; He, Xue; Zhu, Yingqun

    2015-01-01

    Cigarette smoke extract (CSE) induces apoptosis and inflammation, but the mechanism is unknown. Arginine methyltransferase (PRMT6) catalyzes the asymmetric di-methylation of histone H3 arginine 2 (H3R2me2a) to control global level transcription. We hypothesized that PRMT6 mediates CSE induced apoptosis and inflammation through H3R2me2a. The apoptosis after CSE treatment in human umbilical vein endothelial cells (HUVECs) was fully measured with real-time reverse transcription PCR, western blotting and Annexin-V staining. Meanwhile, the inflammation in HUVECs after CSE exposure was detected with real-time reverse transcription PCR, western blotting and ELISA. CSE treatment promoted apoptosis and inflammation in HUVECs, coinciding with the decreased protein abundance of PRMT6. Meanwhile, HUVECs transfected with PRMT6 expressing plasmid inhibited the CSE-induced apoptosis and inflammation. Also, the inhibition of PRMT6 promoted the apoptosis and inflammation in HUVECs induced by CSE. Notably, H3R2me2a was associated with the modulation of PRMT6 in CSE induced apoptosis and inflammation in HUVECs. In conclusion, PRMT6 mediates CSE induced apoptosis and inflammation through H3R2me2a in HUVECs. PMID:25481537

  4. Role of PUMA in methamphetamine-induced neuronal apoptosis.

    PubMed

    Chen, Chuanxiang; Qincao, Litao; Xu, Jingtao; Du, Sihao; Huang, Enping; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

    2016-01-01

    Exposure to methamphetamine (METH), a widely used illicit drug, has been shown to cause neuron apoptosis. p53 upregulated modulator of apoptosis (PUMA) is a key mediator in neuronal apoptosis. This study aimed to examine the effects of PUMA in METH-induced neuronal apoptosis. We determined PUMA protein expression in PC12 cells and SH-SY5Y cells after METH exposure using western blot. We also observed the effect of METH on neuronal apoptosis after silencing PUMA expression with siRNA using TUNEL staining and flow cytometry. Additionally, to investigate possible mechanisms of METH-induced PUMA-mediated neuronal apoptosis, we measured the protein expression of apoptotic markers, including cleaved caspase-3, cleaved PARP, Bax, B-cell leukemia/lymphoma-2 (Bcl-2) and cytochrome c (cyto c), after METH treatment with or without PUMA knockdown. Results showed that METH exposure induced cell apoptosis, increased PUMA protein levels, activated caspase-3 and PARP, elevated Bax and reduced Bcl-2 expression, as well as increased the release of cyto c from mitochondria to the cytoplasm in both PC12 and SH-SY5Y cells. All these effects were attenuated or reversed after silencing PUMA. A schematic depicting the role of PUMA in METH-induced mitochondrial apoptotic pathway was proposed. Our results suggest that PUMA plays an important role in METH-triggered apoptosis and it may be a potential target for ameliorating neuronal injury and apoptosis caused by METH. PMID:26524635

  5. Marine Drugs Regulating Apoptosis Induced by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)

    PubMed Central

    Elmallah, Mohammed I. Y.; Micheau, Olivier

    2015-01-01

    Marine biomass diversity is a tremendous source of potential anticancer compounds. Several natural marine products have been described to restore tumor cell sensitivity to TNF-related apoptosis inducing ligand (TRAIL)-induced cell death. TRAIL is involved during tumor immune surveillance. Its selectivity for cancer cells has attracted much attention in oncology. This review aims at discussing the main mechanisms by which TRAIL signaling is regulated and presenting how marine bioactive compounds have been found, so far, to overcome TRAIL resistance in tumor cells. PMID:26580630

  6. Retinoids induce Nur77-dependent apoptosis in mouse thymocytes.

    PubMed

    Kiss, Beáta; Tóth, Katalin; Sarang, Zsolt; Garabuczi, Éva; Szondy, Zsuzsa

    2015-03-01

    Nur77 is a transcription factor, which plays a determinant role in mediating T cell receptor-induced cell death of thymocytes. In addition to regulation of transcription, Nur77 contributes to apoptosis induction by targeting mitochondria, where it can convert Bcl-2, an anti-apoptotic protein into a proapoptotic molecule. Previous studies have demonstrated that retinoids are actively produced in the mouse thymus and can induce a transcription-dependent apoptosis in mouse thymocytes. Here we show that retinoic acids induce the expression of Nur77, and retinoid-induced apoptosis is completely dependent on Nur77, as retinoids were unable to induce apoptosis in Nur77 null thymocytes. In wild-type thymocytes retinoids induced enhanced expression of the apoptosis-related genes FasL, TRAIL, NDG-1, Gpr65 and Bid, all of them in a Nur77-dependent manner. The combined action of these proteins led to Caspase 8-dependent Bid cleavage in the mitochondria. In addition, we could demonstrate the Nur77-dependent induction of STAT1 leading to enhanced Bim expression, and the mitochondrial translocation of Nur77 leading to the exposure of the Bcl-2/BH3 domain. The retinoid-induced apoptosis was dependent on both Caspase 8 and STAT1. Our data together indicate that retinoids induce a Nur77-dependent cell death program in thymocytes activating the mitochondrial pathway of apoptosis. PMID:25576519

  7. Mitochondrion-mediated apoptosis induced by photofrin-PDT

    NASA Astrophysics Data System (ADS)

    Wu, Yunxia; Xing, Da

    2007-05-01

    Apoptosis is an important cellular event that plays a key role in pathogeny and therapy of many diseases. The mechanisms of the initiation and regulation of PDT-induced apoptosis are complex. Some PDT-associated apoptosis pathways involved plasma membrane death receptors, mitochondria, lysosomes and endoplasmic reticulum (ER). In order to determine the apoptosis pathway induced by Photofrin-PDT, we used fluorescence resonance energy transfer (FRET) technique and probe SCAT3 to monitor the dynamics of caspase-3 activation after PDT treatment and also measured caspase-8 activity. With laser scanning confocal microscopy, we found that Photofrin were localized primarily in mitochondria, the primary targets of Photofrin-PDT. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes after PDT treatment. This was followed by mitochondrial membrane potential (ΔΨm), cytochrome c release, caspase-9 activity, caspase-3 activity and apoptosis. After PDT treatment, caspase-3 was activated rapidly while caspase-8 remained inactivated. Our results indicated that PDT-induced apoptosis was initiated from mitochondria pathway and independent of caspase-8 activation. The activation of caspase-3 by PDT started 20 minutes after treatment and completed in about 15 minutes. PDT-induced apoptosis is directly initiated from mitochondria pathway and not involved in the death receptors-dependent pathway. Our results demonstrated that FRET could be an effective tool to determine PDT-induced apoptosis and other cell death mechanism.

  8. Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation

    SciTech Connect

    Yun, Hong Shik; Baek, Jeong-Hwa; Yim, Ji-Hye; Lee, Su-Jae; Lee, Chang-Woo; Song, Jie-Young; Um, Hong-Duck; Park, Jong Kuk; Park, In-Chul; Hwang, Sang-Gu

    2014-07-11

    Highlights: • HRP-3 is a radiation- and anticancer drug-responsive protein in H1299 cells. • Depletion of HRP-3 induces apoptosis of radio- and chemoresistant H1299 cells. • Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. • ROS generation enhances NF-κB activity, which acts as an upstream signal in the c-Myc/Noxa apoptotic pathway. - Abstract: We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates the radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.

  9. The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-κB inhibition.

    PubMed

    Vidya Priyadarsini, R; Senthil Murugan, R; Maitreyi, S; Ramalingam, K; Karunagaran, D; Nagini, S

    2010-12-15

    With increasing use of plant-derived cancer chemotherapeutic agents, exploring the antiproliferative effects of phytochemicals has gained increasing momentum for anticancer drug design. The dietary phytochemical quercetin, modulates several signal transduction pathways associated with cell proliferation and apoptosis. The present study was undertaken to examine the effect of quercetin on cell viability, and to determine the molecular mechanism of quercetin-induced cell death by investigating the expression of Bcl-2 family proteins (Bcl-2, Bcl-xL, Mcl1, Bax, Bad, p-Bad), cytochrome C, Apaf-1, caspases, and survivin as well as the cell cycle regulatory proteins (p53, p21, cyclin D1), and NF-κB family members (p50, p65, IκB, p-IκB-α, IKKβ and ubiquitin ligase) in human cervical cancer (HeLa) cells. The results demonstrate that quercetin suppressed the viability of HeLa cells in a dose-dependent manner by inducing G2/M phase cell cycle arrest and mitochondrial apoptosis through a p53-dependent mechanism. This involved characteristic changes in nuclear morphology, phosphatidylserine externalization, mitochondrial membrane depolarization, modulation of cell cycle regulatory proteins and NF-κB family members, upregulation of proapoptotic Bcl-2 family proteins, cytochrome C, Apaf-1 and caspases, and downregulation of antiapoptotic Bcl-2 proteins and survivin. Quercetin that exerts opposing effects on different signaling networks to inhibit cancer progression is a classic candidate for anticancer drug design. PMID:20858478

  10. Trauma patients’ elevated Tumor Necrosis Related Apoptosis Inducing Ligand (TRAIL) contributes to increased T cell apoptosis

    PubMed Central

    Bandyopadhyay, Gautam; Bankey, Paul E.; Miller-Graziano, Carol L.

    2012-01-01

    Immunosuppression resulting from excessive post-trauma apoptosis of hyperactivated Tcells is controversial. TRAIL mediated Tcell apoptosis decreases highly activated Tcells’ responses. Caspase-10, a particular TRAIL target, was increased in trauma patients’ Tcells with concomitantly elevated plasma TRAIL levels. These patients’ Tcells developed anergy, implicating increased TRAIL-mediated Tcell apoptosis in post-trauma Tcell anergy. Control Tcells cultured with patients’ sera containing high TRAIL levels increased their Caspase-10 activity and apoptosis. Stimulated primary Tcells are TRAIL apoptosis resistant. Increased plasma Thrombospondin-1 and Tcell expression of CD47, a Thrombospondin-1 receptor, preceded patients’ Tcell anergy. CD47 triggering of Tcells increased their sensitivity to TRAIL-induced apoptosis. Augmentation of Tcell TRAIL-induced apoptosis was secondary to CD47 triggered activation of the Src homology-containing phosphatase-1(SHP-1) and was partially blocked by a SHP-1 inhibitor. We suggest that combined post-trauma CD47 triggering, SHP-1 mediated NFκB suppression, and elevated TRAIL levels increase patients’ CD47 expressing Tcell apoptosis, thus contributing to subsequent Tcell anergy. PMID:22926077

  11. UXT plays dual opposing roles on SARM-induced apoptosis.

    PubMed

    Sethurathinam, Shalini; Singh, Laishram Pradeepkumar; Panneerselvam, Porkodi; Byrne, Bernadette; Ding, Jeak Ling

    2013-10-11

    Apoptosis is a vital defense mechanism for the clearance of infected cells. Ubiquitously expressed transcript (UXT), which exists in two isoforms (V1 and V2), interact with both apoptotic and cellular proteins. By yeast two-hybrid analysis, we found that UXT interacts with SARM (sterile α and HEAT armadillo motif-containing protein). Since SARM is a TLR adaptor which induces intrinsic apoptosis following immune activation, we were prompted to query whether UXT and SARM might co-regulate apoptosis. We found that the UXT isoforms elicit dual opposing regulatory effects on SARM-induced apoptosis; while UXT V1, co-expressed with SARM, caused a reduction in caspase 8 activity, UXT V2 strongly increased caspase 8 activity and enhanced SARM-induced apoptosis by activating the extrinsic pathway and depolarizing the mitochondria. PMID:24021647

  12. TRPV1 receptors mediate particulate matter-induced apoptosis.

    PubMed

    Agopyan, N; Head, J; Yu, S; Simon, S A

    2004-03-01

    Exposure to airborne particulate matter (PM) is a world-wide health problem mainly because it produces adverse cardiovascular and respiratory effects that frequently result in morbidity. Despite many years of epidemiological and basic research, the mechanisms underlying PM toxicity remain largely unknown. To understand some of these mechanisms, we measured PM-induced apoptosis and necrosis in normal human airway epithelial cells and sensory neurons from both wild-type mice and mice lacking TRPV1 receptors using Alexa Fluor 488-conjugated annexin V and propidium iodide labeling, respectively. Exposure of environmental PMs containing residual oil fly ash and ash from Mount St. Helens was found to induce apoptosis, but not necrosis, as a consequence of sustained calcium influx through TRPV1 receptors. Apoptosis was completely prevented by inhibiting TRPV1 receptors with capsazepine or by removing extracellular calcium or in sensory neurons from TRPV1(-/-) mice. Binding of either one of the PMs to the cell membrane induced a capsazepine-sensitive increase in cAMP. PM-induced apoptosis was augmented upon the inhibition of PKA. PKA inhibition on its own also induced apoptosis, thereby suggesting that this pathway may be endogenously protective against apoptosis. In summary, it was found that inhibiting TRPV1 receptors prevents PM-induced apoptosis, thereby providing a potential mechanism to reduce their toxicity. PMID:14633515

  13. Characterization of radiation-induced Apoptosis in rodent cell lines

    SciTech Connect

    Guo, Min; Chen, Changhu; Ling, C.C.

    1997-03-01

    For REC:myc(ch1), Rat1 and Rat1:myc{sub b} cells, we determined the events in the development of radiation-induced apoptosis to be in the following order: cell division followed by chromatin condensation, membrane blebbing, loss of adhesion and the uptake of vital dye. Experimental data which were obtained using {sup 4}He ions of well defined energies and which compared the dependence of apoptosis and clonogenic survival on {sup 4}He range strongly suggested that in our cells both apoptosis and loss of clonogenic survival resulted from radiation damage to the cell nucleus. Corroboratory evidence was that BrdU incorporation sensitized these cells to radiation-induced apoptosis. Comparing the dose response for apoptosis and the clonogenic survival curves for Rat1 and Rat1:myc{sub b} cells, we concluded that radiation-induced cell inactivation as assayed by clonogenic survival, and that a modified linear-quadratic model, proposed previously, modeled such a contribution effectively. In the same context, the selective increase in radiation-induced apoptosis. Comparing the dose response for apoptosis and the clonogenic survival curves for Rat1 and Rat1:myc{sub b} cells, we concluded that radiation-induced apoptosis contributed to the overall radiation-induced cell inactivation as assayed by clonogenic survival, and that a modified linear-quadratic model, proposed previously, modeled such a contribution effectively. In the same context, the selective increase in radiation-induced apoptosis during late S and G{sub 2} phases reduced the relative radioresistance observed for clonogenic survival during late S and G{sub 2} phases. 30 refs., 8 figs.

  14. Modulation of Radiation-Induced Apoptosis by Thiolamines

    NASA Technical Reports Server (NTRS)

    Warters, R. L.; Roberts, J. C.; Wilmore, B. H.; Kelley, L. L.

    1997-01-01

    Exposure to the thiolamine radioprotector N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) induced apoptosis in the mouse TB8-3 hybridoma after 60-minute (LD(sub50) = 4.5mM) or during a 20-hour (LD(sub50) = 0.15 mM) exposure. In contrast, a 20-hour exposure to 17 mM L-cysteine or 10 mM cysteamine was required to induce 50 percent apoptosis within 20 hours. Apoptosis was not induced by either a 60-minute or 20-hour exposure to 10 mM of the thiazolidime prodrugs ribose-cysteine (RibCys) or ribose-cysteamine (RibCyst). Thiolamine-induced apoptosis appeared to be a p53-independent process since it was induced by WR-1065 exposure in human HL60 cells. Exposure to WR-1065 (4mM for 15 minutes) or cysteine (10mM for 60 minutes) before and during irradiation protected cells against the induction of both DNA double-strand breaks and apoptosis, while exposure to RibCys (10 mM for 3 hours) did not. Treatment with either WR-1065, cysteine, RibCys or RibCyst for 60 minutes beginning 60 minutes after irradiation did not affect the level of radiation-induced apoptosis. In contrast, treatment with either cysteine, cysteamine or RibCys for 20 hours beginning 60 minutes after irradiation enhanced radiation-induced apoptosis. Similar experiments could not be conducted with WR-1065 because of its extreme toxicity. Our results indicate that thiolamine enhancement of radiation-induced apoptosis is not involved in their previously reported capacity to reduce radiation-induced mutations.

  15. Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells.

    PubMed

    Kato, Satsuki; Sugimura, Norihiko; Nakashima, Keisuke; Nishihara, Tatsuji; Kowashi, Yusuke

    2005-03-01

    It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans-induced

  16. Preventive effects of bicarbonate on cerivastatin-induced apoptosis.

    PubMed

    Kobayashi, Masaki; Kaido, Fumie; Kagawa, Toshiki; Itagaki, Shirou; Hirano, Takeshi; Iseki, Ken

    2007-08-16

    Although HMG-CoA reductase inhibitors such as statins are the most widely used cholesterol-lowering agents, there is a risk of myopathy or rhabdmyolysis occurring in patients taking these drugs. It has been reported that a number of lipophilic statins cause apoptosis in various cells, but it is still not clear whether intracellular acidification is involved in statin-induced apoptosis. There have been few studies aimed at identifying compounds that suppress statin-induced myotoxicity. In the present study, we examined the relationship between cerivastatin-induced apoptosis and intracellular acidification and the effect of bicarbonate on cerivastatin-induced apoptosis using an RD cell line as a model of in vitro skeletal muscle. Cerivastatin reduced the number of viable cells and caused dramatic morphological changes and DNA fragmentation in a concentration-dependent manner. Moreover, cerivastatin-induced apoptosis was associated with intracellular acidification and caspase-9 and -3/7 activation. On the other hand, bicarbonate suppressed cerivastatin-induced pH alteration, caspase activation, morphological change and reduction of cell viability. Accordingly, bicarbonate suppressed statin-induced apoptosis. The strategy to combine statins with bicarbonate can lead to reduction in the chance of the severe adverse events including myopathy or rhabdmyolysis. PMID:17553641

  17. Phellinus linteus sensitises apoptosis induced by doxorubicin in prostate cancer

    PubMed Central

    Collins, L; Zhu, T; Guo, J; Xiao, Z J; Chen, C-Y

    2006-01-01

    It has been demonstrated that the Phellinus linteus (PL) mushroom, which mainly consists of polysaccharides, possesses antitumour activity. The mechanisms of PL against malignant growth remain unknown. The anticancer drug doxorubicin (Dox) has been shown to induce apoptosis via initiating a caspase cascade. In this investigation, we tested the effect of PL on Dox-induced apoptosis in prostate cancer LNCaP cells. We showed that PL or Dox, at relatively low doses, does not induce apoptosis in the cells. However, combination treatment with low doses of PL and Dox results in a synergistic effect on the induction of apoptosis. In this apoptotic process, caspases 8, 3 and BID are cleaved, and the addition of caspase inhibitor z-VADfmk completely blocks apoptosis. In addition, JNK is activated in response to PL or the combination treatment in LNCaP cells. The suppression of JNK partially inhibits the induction of apoptosis elicited by the co-treatment. These findings indicate that PL has a synergistic effect with Dox to activate caspases in prostate cancer LNCaP cells. Our study also suggests that PL has therapeutic potential to augment the magnitude of apoptosis induced by antiprostate cancer drugs. PMID:16868541

  18. Involvement of endoplasmic reticulum stress and p53 in lncRNA MEG3-induced human hepatoma HepG2 cell apoptosis.

    PubMed

    Chen, Rui-Pei; Huang, Zhen-Lun; Liu, Li-Xuan; Xiang, Meng-Qi; Li, Guo-Ping; Feng, Jia-Lin; Liu, Bin; Wu, Ling-Fei

    2016-09-01

    Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. Although downregulation of lncRNA maternally expressed gene 3 (MEG3) has been identified in several types of cancers, little is known concerning its biological role and regulatory mechanism in hepatoma. Our previous studies demonstrated that MEG3 induces apoptosis in a p53-dependent manner. The aim of the present study was to determine whether endoplasmic reticulum (ER) stress is involved in MEG3‑induced apoptosis. Recombinant lentiviral vectors containing MEG3 (Lv‑MEG3) were constructed and transfected into HepG2 cells. A 3‑(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, RT‑PCR, flow cytometry, western blot analysis, immunofluorescence and immunohistochemistry were applied. Transfected HepG2 cells were also transplanted into nude mice, and the tumor growth curves were determined. The results showed that the recombinant lentivirus of MEG3 was transfected successfully into the HepG2 cells and the expression level of MEG3 was significantly increased. Ectopic expression of MEG3 inhibited HepG2 cell proliferation in vitro and in vivo, and also induced apoptosis. Ectopic expression of MEG3 increased ER stress‑related proteins 78‑kDa glucose‑regulated protein (GRP78), inositol‑requiring enzyme 1 (IRE1), RNA‑dependent protein kinase‑like ER kinase (PERK), activating transcription factor 6 (ATF6), C/EBP homologous protein (CHOP), caspase‑3, as well as p53 and NF‑κB expression accompanied by NF‑κB translocation from the cytoplasm to the nucleus. Furthermore, inhibition of NF‑κB with Bay11‑7082 decreased p53 expression in the MEG3‑transfected cells. These results indicate that MEG3 inhibits cell proliferation and induces apoptosis, partially via the activation of the ER stress and p53 pathway, in which NF‑κB signaling is required for p53 activation in ER stress. PMID:27432655

  19. ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

    PubMed

    Stefanelli, C; Bonavita, F; Stanic', I; Farruggia, G; Falcieri, E; Robuffo, I; Pignatti, C; Muscari, C; Rossoni, C; Guarnieri, C; Caldarera, C M

    1997-03-15

    In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some

  20. The Interplays between Autophagy and Apoptosis Induced by Enterovirus 71

    PubMed Central

    Wang, Bei; Wang, Tao; Wang, Ji; Huang, He; Wang, Jianwei; Jin, Qi; Zhao, Zhendong

    2013-01-01

    Background Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear. Methodology/Principal Findings In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A) cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II and degradation of sequestosome 1 (SQSTM1/P62). Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles. Conclusions/Significance In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection. PMID:23437282

  1. ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

    PubMed Central

    Stefanelli, C; Bonavita, F; Stanic', I; Farruggia, G; Falcieri, E; Robuffo, I; Pignatti, C; Muscari, C; Rossoni, C; Guarnieri, C; Caldarera, C M

    1997-01-01

    In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some

  2. Apoptosis in vascular cells induced by cold atmospheric plasma treatment

    NASA Astrophysics Data System (ADS)

    Sladek, Raymond; Stoffels, Eva

    2006-10-01

    Apoptosis is a natural mechanism of cellular self-destruction. It can be triggered by moderate, yet irreversible damage. Apoptosis plays a major role in tissue renewal. Artificial apoptosis induction will become a novel therapy that meets all requirements for tissue-saving surgery. Diseased tissues can disappear without inflammation and scarring. This is particularly important in treatment of blockages in body tracts (e.g. cardiovascular diseases). Artificial induction of apoptosis can be achieved by means of cold plasma treatment. In this work an atmospheric micro-plasma operated in helium/air has been used to induce apoptosis in vascular cells. Parametric studies of apoptosis induction have been conducted; the efficiency is almost 100%. The apoptotic factors are ROS/RNS (reactive oxygen and nitrogen species). Their densities in the plasma have been measured by mass spectrometry. For apoptosis induction, RNS seem to be more important than ROS, because of their relative abundance. Moreover, addition of a ROS scavenger (ascorbic acid) to the cell culture medium does not reduce the occurrence of apoptosis. Cold plasma is a very efficient tool for fundamental studies of apoptosis, and later, for controlled tissue removal in vivo.

  3. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    PubMed Central

    Kuo, Chen-Tzu; Chen, Bing-Chang; Yu, Chung-Chi; Weng, Chih-Ming; Hsu, Ming-Jen; Chen, Chien-Chih; Chen, Mei-Chieh; Teng, Che-Ming; Pan, Shiow-Lin; Bien, Mauo-Ying; Shih, Chung-Hung; Lin, Chien-Huang

    2009-01-01

    In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis. PMID:19405983

  4. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    SciTech Connect

    Aguilar, David; Strom, Joshua; Chen, Qin M.

    2014-04-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA.

  5. Recovering drug-induced apoptosis subnetwork from Connectivity Map data.

    PubMed

    Yu, Jiyang; Putcha, Preeti; Silva, Jose M

    2015-01-01

    The Connectivity Map (CMAP) project profiled human cancer cell lines exposed to a library of anticancer compounds with the goal of connecting cancer with underlying genes and potential treatments. Since the therapeutic goal of most anticancer drugs is to induce tumor-selective apoptosis, it is critical to understand the specific cell death pathways triggered by drugs. This can help to better understand the mechanism of how cancer cells respond to chemical stimulations and improve the treatment of human tumors. In this study, using CMAP microarray data from breast cancer cell line MCF7, we applied a Gaussian Bayesian network modeling approach and identified apoptosis as a major drug-induced cellular-pathway. We then focused on 13 apoptotic genes that showed significant differential expression across all drug-perturbed samples to reconstruct the apoptosis network. In our predicted subnetwork, 9 out of 15 high-confidence interactions were validated in the literature, and our inferred network captured two major cell death pathways by identifying BCL2L11 and PMAIP1 as key interacting players for the intrinsic apoptosis pathway and TAXBP1 and TNFAIP3 for the extrinsic apoptosis pathway. Our inferred apoptosis network also suggested the role of BCL2L11 and TNFAIP3 as "gateway" genes in the drug-induced intrinsic and extrinsic apoptosis pathways. PMID:25883971

  6. Molecular mechanisms of nutlin-induced apoptosis in multiple myeloma

    PubMed Central

    Saha, Manujendra N; Jiang, Hua

    2010-01-01

    Multiple myeloma (MM) is an incurable plasma cell malignancy in which p53 is rarely mutated. Thus, activation of the p53 pathway by a small molecule inhibitor of the p53-MDM2 interaction, nutlin, in MM cells retaining wild type p53 is an attractive therapeutic strategy. Recently we reported that nutlin plus velcade (a proteasome inhibitor) displayed a synergistic response in MM. However, the mechanism of the p53-mediated apoptosis in MM has not been fully understood. Our data show that nutlin-induced apoptosis correlated with reduction in cell viability, upregulation of p53, p21 and MDM2 protein levels with a simultaneous increase in pro-apoptotic targets PUMA, Bax and Bak and downregulation of anti-apoptotic targets Bcl2 and survivin and activation of caspase in MM cells harboring wild type p53. Nutlin-induced apoptosis was inhibited when activation of caspase was blocked by the caspase inhibitor. Nutlin caused mitochondrial translocation of p53 where it binds with Bcl2, leading to cytochrome C release. Moreover, blocking the transcriptional arm of p53 by the p53-specific transcriptional inhibitor, pifithrin-α, not only inhibited nutlin-induced upregulation of p53-transcriptional targets but also augmented apoptosis in MM cells, suggesting an association of transcription-independent pathway of apoptosis. However, inhibitor of mitochondrial translocation of p53, PFT-µ, did not prevent nutlin-induced apoptosis, suggesting that the p53 transcription-dependent pathway was also operational in nutlin-induced apoptosis in MM. Our study provides the evidence that nutlin-induced apoptosis in MM cells is mediated by transcription-dependent and -independent pathways and supports further clinical evaluation of nutlin as a novel therapeutic agent in MM. PMID:20595817

  7. Catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents by accelerating the degradation of p53.

    PubMed

    Bai, Jingxiang; Cederbaum, Arthur I

    2003-02-14

    Oxidants such as H(2)O(2) play a role in the toxicity of certain DNA-damaging agents, a process that often involves the tumor suppressor p53. H(2)O(2) is rapidly degraded by catalase, which protects cells against oxidant injury. To study the effect of catalase on apoptosis induced by DNA-damaging agents, HepG2 cells were infected with adenovirus containing the cDNA of catalase (Ad-Cat). Forty-eight hours after infection, catalase protein and activity was increased 7-10-fold compared with control cells infected with Ad-LacZ. After treatment with Vp16 or mitomycin C, control cells underwent apoptosis in a p53-dependent manner; however, overexpression of catalase inhibited this apoptosis. Basal levels as well as Vp16- or mitomycin C-stimulated levels of p53 and p21 protein were decreased in the catalase-overexpressing cells as compared with control cells; however, p53 mRNA levels were not decreased by catalase. There was no difference in p53 protein synthesis between catalase-overexpressing cells and control cells. However, pulse-chase experiments indicated that p53 protein degradation was enhanced in the catalase-overexpressing cells. Proteasome inhibitors but not calpeptin prevented the catalase-mediated decrease of p53 content. Whereas Vp16 increased, catalase overexpression decreased the phosphorylation of p53. The protein phosphatase inhibitor okadaic acid did not prevent the catalase-mediated down-regulation of p53 or phosphorylated p53. These results demonstrate that catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents in association with decreasing p53 phosphorylation; the latter may lead to an acceleration in the degradation of p53 protein by the proteasome complex. This suggests that the level of catalase may play a critical role in cell-induced resistance to the effects of anti-cancer drugs which up-regulate p53. PMID:12468545

  8. Ketamine-induced apoptosis in cultured rat cortical neurons

    SciTech Connect

    Takadera, Tsuneo . E-mail: t-takadera@hokuriku-u.ac.jp; Ishida, Akira; Ohyashiki, Takao

    2006-01-15

    Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cell death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons.

  9. Differential Apoptosis Radiosensitivity of Neural Progenitors in Adult Mouse Hippocampus.

    PubMed

    Li, Yu-Qing; Cheng, Zoey; Wong, Shun

    2016-01-01

    Mammalian tissue-specific stem cells and progenitors demonstrate differential DNA damage response. Neural progenitors in dentate gyrus of the hippocampus are known to undergo apoptosis after irradiation. Using a mouse model of hippocampal neuronal development, we characterized the apoptosis sensitivity of the different neural progenitor subpopulations in adult mouse dentate gyrus after irradiation. Two different bromodeoxyuridine incorporation paradigms were used for cell fate mapping. We identified two apoptosis sensitive neural progenitor subpopulations after irradiation. The first represented non-proliferative and non-newborn neuroblasts and immature neurons that expressed doublecortin, calretinin or both. The second consisted of proliferative intermediate neural progenitors. The putative radial glia-like neural stem cells or type-1 cells, regardless of proliferation status, were apoptosis resistant after irradiation. There was no evidence of radiation-induced apoptosis in the absence of the Trp53 (p53) gene but absence of Cdkn1a (p21) did not alter the apoptotic response. Upregulation of nuclear p53 was observed in neuroblasts after irradiation. We conclude that adult hippocampal neural progenitors may demonstrate differential p53-dependent apoptosis sensitivity after irradiation. PMID:27331809

  10. Differential Apoptosis Radiosensitivity of Neural Progenitors in Adult Mouse Hippocampus

    PubMed Central

    Li, Yu-Qing; Cheng, Zoey; Wong, Shun

    2016-01-01

    Mammalian tissue-specific stem cells and progenitors demonstrate differential DNA damage response. Neural progenitors in dentate gyrus of the hippocampus are known to undergo apoptosis after irradiation. Using a mouse model of hippocampal neuronal development, we characterized the apoptosis sensitivity of the different neural progenitor subpopulations in adult mouse dentate gyrus after irradiation. Two different bromodeoxyuridine incorporation paradigms were used for cell fate mapping. We identified two apoptosis sensitive neural progenitor subpopulations after irradiation. The first represented non-proliferative and non-newborn neuroblasts and immature neurons that expressed doublecortin, calretinin or both. The second consisted of proliferative intermediate neural progenitors. The putative radial glia-like neural stem cells or type-1 cells, regardless of proliferation status, were apoptosis resistant after irradiation. There was no evidence of radiation-induced apoptosis in the absence of the Trp53 (p53) gene but absence of Cdkn1a (p21) did not alter the apoptotic response. Upregulation of nuclear p53 was observed in neuroblasts after irradiation. We conclude that adult hippocampal neural progenitors may demonstrate differential p53-dependent apoptosis sensitivity after irradiation. PMID:27331809

  11. Apoptosis induced by dioscin in Hela cells.

    PubMed

    Cai, Jing; Liu, Mingjie; Wang, Zhao; Ju, Yong

    2002-02-01

    Dioscin, a saponin extracted from the root of Polygonatum Zanlanscianense Pamp, markedly inhibited proliferation of Hela cells. The results indicated that Hela cells underwent apoptosis in dose- and time-dependent manners when treated with Dioscin. Caspase-3, -8 and -9 activities were also detected. The low enzymatic activity of caspase-8 and high activity of caspase-9 showed that the mitochondrial pathway was activated in apoptosis. The reduced expression of the survival protein Bcl-2 also confirmed this result. These studies may be significant in finding a new drug to treat human cervical cancer. PMID:11853164

  12. Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells.

    PubMed

    Kurokawa, K; Akaike, Y; Masuda, K; Kuwano, Y; Nishida, K; Yamagishi, N; Kajita, K; Tanahashi, T; Rokutan, K

    2014-03-13

    Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth. PMID:23503458

  13. X-ray-induced cell death: Apoptosis and necrosis

    SciTech Connect

    Nakano, Hisako; Shinohara, Kunio

    1994-10-01

    X-ray-induced cell death in MOLT-4N1, a subclone of MOLT-4 cells, and M10 cells was studied with respect to their modes of cell death, apoptosis and necrosis. MOLT-4N1 cells showed radiosensitivity similar to that of M10 cells, a radiosensitive mutant of L5178Y, as determined by the colony formation assay. Analysis of cell size demonstrated that MOLT-4N1 cells increased in size at an early stage after irradiation and then decreased to a size smaller than that of control cells, whereas the size of irradiated M10 cells increased continuously. Apoptosis detected by morphological changes and DNA ladder formation (the cleavage of DNA into oligonucleosomal fragments) occurred in X-irradiated MOLT-4N1 cells but not in M10 cells. Pulsed-field gel electrophoresis showed that the ladder formation involved an intermediate-sized DNA (about 20 kbp). Most of the DNA was detected at the origin in both methods of electrophoresis in the case of M10 cells, though a trace amount of ladder formation was observed. Heat treatment of M10 cells induced apoptosis within 30 min after treatment, in contrast to MOLT-4N1 cells. The results suggest that apoptosis and necrosis are induced by X rays in a manner which is dependent on the cell line irrespective of the capability of the cells to develop apoptosis. DNA fragmentation was the earliest change observed in the development of apoptosis. 27 refs., 8 figs., 1 tab.

  14. Antioxidants induce apoptosis of rat ovarian theca-interstitial cells.

    PubMed

    Rzepczynska, Izabela J; Foyouzi, Nastaran; Piotrowski, Piotr C; Celik-Ozenci, Ciler; Cress, Amanda; Duleba, Antoni J

    2011-01-01

    Regulation of growth of ovarian theca-interstitial tissues is essential for normal ovarian development and function. Reactive oxygen species are involved in modulation of signal transduction pathways, including regulation of tissue growth and apoptosis. Previously, we have demonstrated that antioxidants inhibit proliferation of theca-interstitial cells. This report evaluates the effects of antioxidants on apoptosis of rat theca-interstitial cells. The cells were cultured in chemically defined media without or with vitamin E succinate and ebselen. Apoptosis was evaluated by cytochemical assessment of nuclear morphology, activity of executioner caspases 3 and 7, and determination of staining with annexin V in combination with propidium iodide. Both tested antioxidants induced significant morphological changes consistent with apoptosis, including chromatin condensation, nuclear shrinkage, and pyknosis. Antioxidants also induced other hallmarks of apoptosis including increased activity of caspases 3/7 as well as increased staining with annexin V. The present findings demonstrate that antioxidants with distinctly different mechanisms of action induce a series of events consistent with the process of apoptosis in ovarian mesenchyme. These observations may be of translational-clinical relevance, providing mechanistic support for the use of antioxidants in the treatment of PCOS, a condition associated with excessive growth and activity of theca-interstitial cells. PMID:20844276

  15. Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells

    PubMed Central

    SHI, MIAO-QIAN; SU, FEI-FEI; XU, XUAN; LIU, XIONG-TAO; WANG, HONG-TAO; ZHANG, WEI; LI, XUE; LIAN, CHENG; ZHENG, QIANG-SUN; FENG, ZHI-CHUN

    2016-01-01

    Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase-3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII-induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII-induced p-Akt downregulation and cleaved caspase-3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII-induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII-induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway. PMID:26862035

  16. Csk regulates angiotensin II-induced podocyte apoptosis.

    PubMed

    Zhang, Lu; Ren, Zhilong; Yang, Qian; Ding, Guohua

    2016-07-01

    Increasing data have shown that angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. The mechanism underlying Ang II-induced podocyte apoptosis has not been established. C-terminal Src kinase (Csk) is a cytoplasmic kinase that interacts with scaffolding proteins involved in cell growth, adhesion, and polarization, and the role of Csk in regulating cellular apoptosis has gradually attracted attention. This study evaluates the role of Csk in Ang II-induced podocyte apoptosis. In vivo, Wistar rats were randomly subjected to a normal saline or Ang II infusion. In vitro, we exposed differentiated mouse podocytes to Ang II. Ang II increased Csk expression and induced podocyte apoptosis, stimulated Csk translocation and binding to Caveolin-1, and stimulated decreased Fyn pY416, increased Fyn pY529, and nephrin dephosphorylation. Csk knockdown prevented Ang II-induced podocyte apoptosis, reduced Fyn kinase inactivation, and increased the interaction between nephrin and the activated form of Fyn, accompanied by a reduced interaction between Csk and Caveolin-1. These findings indicate that Ang II induces podocyte injury via a Csk-dependent pathway. PMID:27225249

  17. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    PubMed

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

  18. Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT-116 cells.

    PubMed

    Chen, Tzu-Hsuan; Pan, Shiow-Lin; Guh, Jih-Hwa; Chen, Chien-Chih; Huang, Yao-Ting; Pai, Hui-Chen; Teng, Che-Ming

    2008-11-01

    Denbinobin is a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla. We showed that denbinobin induces apoptosis in human colorectal cancer cells (HCT-116) in a concentration-dependent manner. The addition of a pan-caspase inhibitor (zVAD-fmk) did not suppress the denbinobin-induced apoptotic effect, and denbinobin-induced apoptosis was not accompanied by processing of procaspase-3, -6, -7, -9, and -8. However, denbinobin triggered the translocation of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. Small interfering RNA targeting of AIF effectively protected HCT-116 cells against denbinobin-induced apoptosis. Denbinobin treatment also caused DNA damage, activation of the p53 tumor suppressor gene, and upregulation of numerous downstream effectors (p21WAF1/CIP1, Bax, PUMA, and NOXA). A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of denbinobin. Taken together, our findings suggest that denbinobin induces apoptosis of human colorectal cancer HCT-116 cells via DNA damage and an AIF-mediated pathway. These results indicate that denbinobin has potential as a novel anticancer agent. PMID:18607570

  19. Calpain Inhibitor PD150606 Attenuates Glutamate Induced Spiral Ganglion Neuron Apoptosis through Apoptosis Inducing Factor Pathway In Vitro

    PubMed Central

    Song, Yong-Li; Chen, Xiao-Dong; Mi, Wen-Juan; Wang, Jian; Lin, Ying; Chen, Fu-Quan; Qiu, Jian-Hua

    2015-01-01

    Objective This research aimed to investigate whether glutamate induced spiral ganglion neurons (SGNs) apoptosis through apoptosis inducing factor (AIF) pathway. And verify whether PD150606, a calpain inhibitor could prevent apoptosis by inhibiting cleaving and releasing AIF in mitochondrion. Methods SGNs of postnatal days 0-3 were harvested and cultured in dishes. 20 mM Glu, the caspase inhibitor Z-VAD-FMK and calpain inhibitor PD150606 were added into cultured dishes separately. We used optical microscope and immunofluoresence staining to observe cell morphology and AIF distribution, RT-PCR and Westernblot to analyse AIF and calpain expression in SGNs. TUNEL assay was used to test cell apoptosis. Results Cell morphology and nuclear translocation of AIF were altered in SGNs by 20 mM Glu treated in vitro. The axon of SGN shortened, more apoptosis SGN were observed and the expression of AIF and calpain were up-regulated in Glu-treated group than the normal one (P<0.05). The same experiments were conducted in 20 mM+PD150606 treated group and 20 mM+Z-VAD-FMK group. Obviously AIF were located from cytoplasm to the nuclear and the expressions of AIF and calpain were down-regulated by PD150606 (P<0.05). Positive cells in TUNEL staining decreased after PD150606 treating. However, Z-VAD-FMK had no influence on AIF, calpain expression or cell apoptosis. Conclusion The AIF-related apoptosis pathway is involved in the process of Glu-induced SGN injury. Furthermore, the inhibition of calpain can prevent AIF from releasing the nuclear or inducing SGN apoptosis. PMID:25874633

  20. Induction of p53-mediated transcription and apoptosis by exportin-1 (XPO1) inhibition in mantle cell lymphoma.

    PubMed

    Yoshimura, Mariko; Ishizawa, Jo; Ruvolo, Vivian; Dilip, Archana; Quintás-Cardama, Alfonso; McDonnell, Timothy J; Neelapu, Sattva S; Kwak, Larry W; Shacham, Sharon; Kauffman, Michael; Tabe, Yoko; Yokoo, Masako; Kimura, Shinya; Andreeff, Michael; Kojima, Kensuke

    2014-07-01

    The nuclear transporter exportin-1 (XPO1) is highly expressed in mantle cell lymphoma (MCL) cells, and is believed to be associated with the pathogenesis of this disease. XPO1-selective inhibitors of nuclear export (SINE) compounds have been shown to induce apoptosis in MCL cells. Given that p53 is a cargo protein of XPO1, we sought to determine the significance of p53 activation through XPO1 inhibition in SINE-induced apoptosis of MCL cells. We investigated the prognostic impact of XPO1 expression in MCL cells using Oncomine analysis. The significance of p53 mutational/functional status on sensitivity to XPO1 inhibition in cell models and primary MCL samples, and the functional role of p53-mediated apoptosis signaling, were also examined. Increased XPO1 expression was associated with poor prognosis in MCL patients. The XPO1 inhibitor KPT-185 induced apoptosis in MCL cells through p53-dependent and -independent mechanisms, and p53 status was a critical determinant of its apoptosis induction. The KPT-185-induced, p53-mediated apoptosis in the MCL cells occurred in a transcription-dependent manner. Exportin-1 appears to influence patient survival in MCL, and the SINE XPO1 antagonist KPT-185 effectively activates p53-mediated transcription and apoptosis, which would provide a novel strategy for the therapy of MCL. PMID:24766216

  1. Rabies virus infects mouse and human lymphocytes and induces apoptosis.

    PubMed Central

    Thoulouze, M I; Lafage, M; Montano-Hirose, J A; Lafon, M

    1997-01-01

    Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response. PMID:9311815

  2. 6-Gingerol induces autophagy to protect HUVECs survival from apoptosis.

    PubMed

    Wang, Shaopeng; Sun, Xiance; Jiang, Liping; Liu, Xiaofang; Chen, Min; Yao, Xiaofeng; Sun, Qinghua; Yang, Guang

    2016-08-25

    6-Gingerol, the major pharmacologically-active component of ginger, has the potential to prevent heart disease. However, the mechanisms are not well understood. In this study, the protective effect of 6-gingerol against hydrogen peroxide-induced apoptosis in human umbilical vein endothelial cells (HUVECs) was investigated. Apoptosis was detected by Hoechst 33342 and Flow cytometry analysis. To further elucidate the crosstalk between apoptosis and autophagy, we tested the expression of autophagy related proteins, LC3B, Bcl-2, Beclin1, AKT, p-AKT, mechanistic target of rapamycin (mTOR), and p-mTOR. Furthermore, mitochondrial membrane potential and the intracellular generation of reactive oxygen species (ROS) were also investigated. Our data revealed that 6-gingerol significantly reduced apoptosis by inducing autophagy. It has been demonstrated that 6-gingerol suppressed the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway, increased the expression of Beclin1 to promote autophagy, and increased Bcl-2 expression to inhibit apoptosis. In addition, the damage of mitochondrial was protected, and ROS level was decreased by 6-gingerol. These firmly indicate 6-gingerol has a strong protective ability against the apoptosis caused by oxidative stress in HUVECs, and the mechanism may relate to the induction of autophagy. Our data suggest 6-gingerol may be beneficial in the prevention of atherosclerosis. PMID:27451028

  3. The retinoblastoma protein induces apoptosis directly at the mitochondria

    PubMed Central

    Hilgendorf, Keren I.; Leshchiner, Elizaveta S.; Nedelcu, Simona; Maynard, Mindy A.; Calo, Eliezer; Ianari, Alessandra; Walensky, Loren D.; Lees, Jacqueline A.

    2013-01-01

    The retinoblastoma protein gene RB-1 is mutated in one-third of human tumors. Its protein product, pRB (retinoblastoma protein), functions as a transcriptional coregulator in many fundamental cellular processes. Here, we report a nonnuclear role for pRB in apoptosis induction via pRB's direct participation in mitochondrial apoptosis. We uncovered this activity by finding that pRB potentiated TNFα-induced apoptosis even when translation was blocked. This proapoptotic function was highly BAX-dependent, suggesting a role in mitochondrial apoptosis, and accordingly, a fraction of endogenous pRB constitutively associated with mitochondria. Remarkably, we found that recombinant pRB was sufficient to trigger the BAX-dependent permeabilization of mitochondria or liposomes in vitro. Moreover, pRB interacted with BAX in vivo and could directly bind and conformationally activate BAX in vitro. Finally, by targeting pRB specifically to mitochondria, we generated a mutant that lacked pRB's classic nuclear roles. This mito-tagged pRB retained the ability to promote apoptosis in response to TNFα and also additional apoptotic stimuli. Most importantly, induced expression of mito-tagged pRB in Rb−/−;p53−/− tumors was sufficient to block further tumor development. Together, these data establish a nontranscriptional role for pRB in direct activation of BAX and mitochondrial apoptosis in response to diverse stimuli, which is profoundly tumor-suppressive. PMID:23618872

  4. Autophagy Regulates Colistin-Induced Apoptosis in PC-12 Cells

    PubMed Central

    Zhang, Ling; Zhao, Yonghao; Ding, Wenjian; Jiang, Guozheng; Lu, Ziyin; Li, Li; Wang, Jinli

    2015-01-01

    Colistin is a cyclic cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. Our recent study demonstrated that colistin induces apoptosis in primary chick cortex neurons and PC-12 cells. Although apoptosis and autophagy have different impacts on cell fate, there is a complex interaction between them. Autophagy plays an important role as a homeostasis regulator by removing excessive or unnecessary proteins and damaged organelles. The aim of the present study was to investigate the modulation of autophagy and apoptosis regulation in PC-12 cells in response to colistin treatment. PC-12 cells were exposed to colistin (125 to 250 μg/ml), and autophagy was detected by visualization of monodansylcadaverine (MDC)-labeled vacuoles, LC3 (microtubule-associated protein 1 light chain 3) immunofluorescence microscopic examination, and Western blotting. Apoptosis was measured by flow cytometry, Hoechst 33258 staining, and Western blotting. Autophagosomes were observed after treatment with colistin for 12 h, and the levels of LC3-II gene expression were determined; observation and protein levels both indicated that colistin induced a high level of autophagy. Colistin treatment also led to apoptosis in PC-12 cells, and the level of caspase-3 expression increased over the 24-h period. Pretreatment of cells with 3-methyladenine (3-MA) increased colistin toxicity in PC-12 cells remarkably. However, rapamycin treatment significantly increased the expression levels of LC3-II and beclin 1 and decreased the rate of apoptosis of PC-12 cells. Our results demonstrate that colistin induced autophagy and apoptosis in PC-12 cells and that the latter was affected by the regulation of autophagy. It is very likely that autophagy plays a protective role in the reduction of colistin-induced cytotoxicity in neurons. PMID:25645826

  5. An Aqueous Extract of Fagonia cretica Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Breast Cancer Cells via FOXO3a and p53 Expression

    PubMed Central

    Lam, Matt; Carmichael, Amtul R.; Griffiths, Helen R.

    2012-01-01

    Background Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as γ-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53

  6. Hydrogen peroxide induces apoptosis via a mitochondrial pathway in chondrocytes

    NASA Astrophysics Data System (ADS)

    Zhuang, Cai-ping; Liang, Qian; Wang, Xiao-ping; Chen, Tong-sheng

    2012-03-01

    The degenerative joint disease such as osteoarthritis (OA) is closely associated with the death of chondrocytes in apoptosis fashion. Hydrogen peroxide (H2O2), higher expression following acute damage in OA patients, has been shown to be up-regulated during apoptosis in a bulk of experimental models. This study was aimed to explore the mechanism of H2O2-induced rabbit chondrocytes apoptosis. Articular cartilage was biopsied from the joints of 6 weeks old New Zealand rabbits. Cell Counting Kit (CCK-8) assay was used to assess the inhibitory effect of H2O2 on cell viability. H2O2 treatment induced a remarkable reduction of cell viability. We used flow cytometry to assess the form of cell death with Annexin-V/PI double staining, and found that H2O2 treatment induced apoptosis in a dose-and time-dependent manner. Exposure of chondrocytes to 1.5 mM of H2O2 for 2 h induced a burst apoptosis that can be alleviated by N-acetyl cysteine (NAC) pretreatment, an anti-oxidant amino-acid derivative. Loss of mitochondria membrane potential (▵Ψm) was evaluated using confocal microscopy imaging and flow cytometry (FCM). H2O2 treatment induced a marked reduction of ▵Ψm, and the abrupt disappearance of ▵Ψm occurred within 5 minutes. These results indicate that H2O2 induces a rapid apoptosis via a mitochondrial pathway in rabbit chondrocytes.

  7. Iron dysregulation combined with aging prevents sepsis-induced apoptosis

    PubMed Central

    Javadi, Pardis; Buchman, Timothy G.; Stromberg, Paul E.; Turnbull, Isaiah R.; Vyas, Dinesh; Hotchkiss, Richard S.; Karl, Irene E.; Coopersmith, Craig M.

    2005-01-01

    Background Sepsis, iron loading and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels. Methods Hfe−/− mice (a murine homolog of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24–26 months) or mature (16–18 months) Hfe−/− mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 hours later and assessed for apoptosis and cytokine levels. Results Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe−/− mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe−/− mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe−/− mice than septic mature Hfe−/− animals. Interleukin-6 was elevated in septic aged Hfe−/− mice compared to sham mice. Conclusions Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe−/− mice are able to mount an inflammatory response following CLP and mature Hfe−/− mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation. PMID:15921699

  8. Cellular Oxidative Stress and the Control of Apoptosis by Wild-Type p53, Cytotoxic Compounds, and Cytokines

    NASA Astrophysics Data System (ADS)

    Lotem, Joseph; Peled-Kamar, Mira; Groner, Yoram; Sachs, Leo

    1996-08-01

    Apoptosis induced by wild-type p53 or cytotoxic compounds in myeloid leukemic cells can be inhibited by the cytokines interleukin 6, interleukin 3, granulocyte-macrophage colony-stimulating factor, and interferon γ and by antioxidants. The antioxidants and cytokines showed a cooperative protective effect against induction of apoptosis. Cells with a higher sensitivity to induction of apoptosis and required a higher cytokine concentration to inhibit apoptosis. Decreasing the intrinsic oxidative stress in cells by antioxidants thus inhibited apoptosis, whereas increasing this intrinsic stress by adding H2O2 enhanced apoptosis. Induction of apoptosis by wild-type p53 was not preceded by increased peroxide production or lipid peroxidation and the protective effect of cytokines was not associated with a decrease in these properties. The results indicate that the intrinsic degree of oxidative stress can regulate cell susceptibility to wild-type p53-dependent and p53-independent induction of apoptosis and the ability of cytokines to protect cells against apoptosis.

  9. An increase of granulosa cell apoptosis mediates aqueous neem (Azadirachta indica) leaf extract-induced oocyte apoptosis in rat

    PubMed Central

    Tripathi, Anima; Shrivastav, Tulsidas G; Chaube, Shail K

    2013-01-01

    Objective: Neem plant (Azadirachta indica) has been extensively used in Ayurvedic system of medicine for female fertility regulation for a long time, but its mechanism of action remains poorly understood. Hence, the present study was aimed to determine whether an increase of granulosa cell apoptosis is associated with aqueous neem leaf extract (NLE)-induced oocyte apoptosis. Materials and Methods: Sexually immature female rats of 20 days old were fed NLE (50 mg/day) for 10 days and then subjected to superovulation induction protocol. The morphological changes in cumulus oocyte complexes (COCs), rate of oocyte apoptosis, hydrogen peroxide (H2O2), total nitrite, and cytochrome c concentrations, inducible nitric oxide synthase (iNOS), cytochrome c, p53, Bcl2 and Bax expressions, deoxyribonucleic acid (DNA) fragmentation, and estradiol 17β level in granulosa cells collected from preovulatory COCs were analyzed. Results: Aqueous NLE increased H2O2 concentration and decreased catalase activity, increased iNOS expression and total nitrite concentration, increased p53, Bax, and p53 expressions but decreased Bcl2 expression, increased cytochrome c concentration and induced DNA fragmentation in granulosa cells. An increased granulosa cell apoptosis resulted in reduced estradiol 17β concentration and induced apoptosis in ovulated oocytes. Conclusion: We conclude that aqueous NLE-induced granulosa cell apoptosis through the mitochondria-mediated pathway, reduced estradiol 17β concentration and induced apoptosis in ovulated oocytes. Thus, granulosa cell apoptosis mediates NLE-induced oocyte apoptosis during female fertility regulation in rat. PMID:23776837

  10. Cystamine induces AIF-mediated apoptosis through glutathione depletion.

    PubMed

    Cho, Sung-Yup; Lee, Jin-Haeng; Ju, Mi-kyeong; Jeong, Eui Man; Kim, Hyo-Jun; Lim, Jisun; Lee, Seungun; Cho, Nam-Hyuk; Park, Hyun Ho; Choi, Kihang; Jeon, Ju-Hong; Kim, In-Gyu

    2015-03-01

    Cystamine and its reduced form cysteamine showed protective effects in various models of neurodegenerative disease, including Huntington's disease and Parkinson's disease. Other lines of evidence demonstrated the cytotoxic effect of cysteamine on duodenal mucosa leading to ulcer development. However, the mechanism for cystamine cytotoxicity remains poorly understood. Here, we report a new pathway in which cystamine induces apoptosis by targeting apoptosis-inducing factor (AIF). By screening of various cell lines, we observed that cystamine and cysteamine induce cell death in a cell type-specific manner. Comparison between cystamine-sensitive and cystamine-resistant cell lines revealed that cystamine cytotoxicity is not associated with unfolded protein response, reactive oxygen species generation and transglutaminase or caspase activity; rather, it is associated with the ability of cystamine to trigger AIF nuclear translocation. In cystamine-sensitive cells, cystamine suppresses the levels of intracellular glutathione by inhibiting γ-glutamylcysteine synthetase expression that triggers AIF translocation. Conversely, glutathione supplementation completely prevents cystamine-induced AIF translocation and apoptosis. In rats, cysteamine administration induces glutathione depletion and AIF translocation leading to apoptosis of duodenal epithelium. These results indicate that AIF translocation through glutathione depletion is the molecular mechanism of cystamine toxicity, and provide important implications for cystamine in the neurodegenerative disease therapeutics as well as in the regulation of AIF-mediated cell death. PMID:25549939

  11. (+)-Catechin protects dermal fibroblasts against oxidative stress-induced apoptosis

    PubMed Central

    2014-01-01

    Background Oxidative stress has been suggested as a mechanism underlying skin aging, as it triggers apoptosis in various cell types, including fibroblasts, which play important roles in the preservation of healthy, youthful skin. Catechins, which are antioxidants contained in green tea, exert various actions such as anti-inflammatory, anti-bacterial, and anti-cancer actions. In this study, we investigated the effect of (+)-catechin on apoptosis induced by oxidative stress in fibroblasts. Methods Fibroblasts (NIH3T3) under oxidative stress induced by hydrogen peroxide (0.1 mM) were treated with either vehicle or (+)-catechin (0–100 μM). The effect of (+)-catechin on cell viability, apoptosis, phosphorylation of c-Jun terminal kinases (JNK) and p38, and activation of caspase-3 in fibroblasts under oxidative stress were evaluated. Results Hydrogen peroxide induced apoptotic cell death in fibroblasts, accompanied by induction of phosphorylation of JNK and p38 and activation of caspase-3. Pretreatment of the fibroblasts with (+)-catechin inhibited hydrogen peroxide-induced apoptosis and reduced phosphorylation of JNK and p38 and activation of caspase-3. Conclusion (+)-Catechin protects against oxidative stress-induced cell death in fibroblasts, possibly by inhibiting phosphorylation of p38 and JNK. These results suggest that (+)-catechin has potential as a therapeutic agent for the prevention of skin aging. PMID:24712558

  12. Lysophosphatidic acid induces necrosis and apoptosis in hippocampal neurons.

    PubMed

    Holtsberg, F W; Steiner, M R; Keller, J N; Mark, R J; Mattson, M P; Steiner, S M

    1998-01-01

    A diverse body of evidence indicates a role for the lipid biomediator lysophosphatidic acid (LPA) in the CNS. This study identifies and characterizes the induction of neuronal death by LPA. Treatment of cultured hippocampal neurons from embryonic rat brains with 50 microM LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10 microM led to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 microM LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 microM LPA was characterized as apoptotic on the basis of terminal dUTP nick end-labeling (TUNEL) staining, externalization of phosphatidylserine, and protection against chromatin condensation, TUNEL staining, and phosphatidylserine externalization by treatment with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum inhibitor of caspases, i.e., members of the interleukin-1beta converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 microM LPA. LPA (1 microM) also induced a decrease in mitochondrial membrane potential. Moreover, pretreatment of neurons with cyclosporin A protected against the LPA-induced decrease in mitochondrial membrane potential and neuronal apoptosis. Thus, LPA, at pathophysiological levels, can induce neuronal apoptosis and could thereby participate in neurodegenerative disorders. PMID:9422348

  13. Hydrogen peroxide-induced apoptosis in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Guadarrama-Solís, Adriana; Muñoz-Seca, Carmen; Arreguín-Cano, Juan Antonio

    2015-01-01

    In the process of bleaching vital, discolored teeth, low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2. However, interest has been expressed in the assessment of pathological effects of long-term exposure to bleaching agents such as irritation and ulceration of the gingival or other soft tissues. The aim of the present study was to determine the effect of hydrogen peroxide on apoptosis in human gingival fibroblasts (HGF). Cytochrome c, Bcl-2, Bax, Bid and caspase-3 protein expression were detected by Western blotting. HGF cell apoptosis induced by H2O2 was both dose and time dependent. The addition of H2O2 resulted in the release of cytochrome c to the cytosol, and an increase of Caspase-3 cleavage. Data suggest that oxidative stress-induced apoptosis in HGF is intrinsic pathway involved the release of apoptotic signal from mitochondria. PMID:26884825

  14. Hydrogen peroxide-induced apoptosis in human gingival fibroblasts

    PubMed Central

    Gutiérrez-Venegas, Gloria; Guadarrama-Solís, Adriana; Muñoz-Seca, Carmen; Arreguín-Cano, Juan Antonio

    2015-01-01

    In the process of bleaching vital, discolored teeth, low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2. However, interest has been expressed in the assessment of pathological effects of long-term exposure to bleaching agents such as irritation and ulceration of the gingival or other soft tissues. The aim of the present study was to determine the effect of hydrogen peroxide on apoptosis in human gingival fibroblasts (HGF). Cytochrome c, Bcl-2, Bax, Bid and caspase-3 protein expression were detected by Western blotting. HGF cell apoptosis induced by H2O2 was both dose and time dependent. The addition of H2O2 resulted in the release of cytochrome c to the cytosol, and an increase of Caspase-3 cleavage. Data suggest that oxidative stress-induced apoptosis in HGF is intrinsic pathway involved the release of apoptotic signal from mitochondria. PMID:26884825

  15. Glutathione peroxidase-1 protects from CD95-induced apoptosis.

    PubMed

    Gouaze, Valerie; Andrieu-Abadie, Nathalie; Cuvillier, Olivier; Malagarie-Cazenave, Sophie; Frisach, Marie-Francoise; Mirault, Marc-Edouard; Levade, Thierry

    2002-11-01

    Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis. PMID:12221075

  16. Taurine induces the apoptosis of breast cancer cells by regulating apoptosis-related proteins of mitochondria.

    PubMed

    Zhang, Xiali; Lu, Hongfei; Wang, Yibing; Liu, Chunju; Zhu, Weifeng; Zheng, Shuangyan; Wan, Fusheng

    2015-01-01

    Taurine (Tau), the most abundant free amino acid in humans has numerous potential health benefits through its antioxidant and anti-inflammatory properties. However, limited studies have assessed its effect on tumors and the antitumor mechanism remains unknown. The present study investigated the cellular and molecular changes induced by Tau, leading to the induction of apoptosis in human breast cancer cell lines MCF-7 and MDA-MB-231. MCF-7 is p53 proficient (p53+/+) and MDA-MB-231 is a p53 null mutant (p53-/-). Cell proliferation and viability were assessed by MTT. Flow cytometry and hoechst33342 fluorescent staining were employed to detect apoptosis. Spectrophotometry was used to detect caspase-3 activity. Reverse transcription-polymerase chain reaction and western blot analysis were used to detect the levels of mRNA and proteins of p53-upregulated modulator of apoptosis (PUMA), Bax and Bcl-2. Finally, the affect of Tau on the growth of MDA-MB-231-cell-nude mice xenografts was examined. In the study, Tau inhibited growth and induced apoptosis of the two cell lines in a concentration- and time-dependent manner. Notably, the inhibitory effect of Tau on p53-/- cancer cells was clearly significant compared to the p53+/+ cancer cells. Further studies showed that Tau promoted apoptosis in human breast cancer cells and inhibited the growth of tumor in nude mice by inducing the expression of PUMA, which further up- and downregulated the expression of Bax and Bcl-2 protein, giving rise to increased activation of caspase-3. Collectively, these results indicate that Tau is a potent candidate for the chemotherapy of breast cancer through increasing the PUMA expression independent of p53 status. PMID:25395275

  17. MG132, a proteasome inhibitor, induces apoptosis in tumor cells.

    PubMed

    Guo, Na; Peng, Zhilan

    2013-03-01

    The balance between cell proliferation and apoptosis is critical for normal development and for the maintenance of homeostasis in adult organisms. Disruption of this balance has been implicated in a large number of disease processes, ranging from autoimmunity and neurodegenerative disorders to cancer. The ubiquitin-proteasome pathway, responsible for mediating the majority of intracellular proteolysis, plays a crucial role in the regulation of many normal cellular processes, including the cell cycle, differentiation and apoptosis. Apoptosis in cancer cells is closely connected with the activity of ubiquitin-proteasome pathway. The peptide-aldehyde proteasome inhibitor MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucine) induces the apoptosis of cells by a different intermediary pathway. Although the pathway of induction of apoptosis is different, it plays a crucial role in anti-tumor treatment. There are many cancer-related molecules in which the protein levels present in cells are regulated by a proteasomal pathway; for example, tumor inhibitors (P53, E2A, c-Myc, c-Jun, c-Fos), transcription factors (transcription factor nuclear factor-kappa B, IκBα, HIFI, YYI, ICER), cell cycle proteins (cyclin A and B, P27, P21, IAP1/3), MG132 induces cell apoptosis through formation of reactive oxygen species or the upregulation and downregulation of these factors, which is ultimately dependent upon the activation of the caspase family of cysteine proteases. In this article we review the mechanism of the induction of apoptosis in order to provide information required for research. PMID:22897979

  18. Salmonella typhimurium Invasion Induces Apoptosis in Infected Macrophages

    NASA Astrophysics Data System (ADS)

    Monack, Denise M.; Raupach, Barbel; Hromockyj, Alexander E.; Falkow, Stanley

    1996-09-01

    Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.

  19. Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury

    PubMed Central

    Sakhatskyy, Pavlo; Newton, Julie; Shamirian, Paul; Hsiao, Vivian; Curren, Sean; Gabino Miranda, Gustavo Andres; Pedroza, Mesias; Blackburn, Michael R.; Rounds, Sharon

    2013-01-01

    Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases. PMID:23316066

  20. Chemotherapeutic-Induced Apoptosis – A Phenotype for Pharmacogenomics Studies

    PubMed Central

    Wen, Yujia; Gorsic, Lidija K.; Wheeler, Heather E.; Ziliak, Dana M.; Huang, R. Stephanie; Dolan, M. Eileen

    2011-01-01

    Lymphoblastoid cell lines have been used as a model system to identify genetic determinants of chemotherapeutic-induced cytotoxicity, a phenotype thought to represent cellular sensitivity to drug. However, cytotoxicity is a broad measurement encompassing cell cycle inhibition as well as cell death (apoptotic and non-apoptotic). We evaluated caspase 3/7 mediated cellular apoptosis with six chemotherapeutic agents: 5′-deoxy-fluorouridine, pemetrexed, cytarabine, paclitaxel, carboplatin and cisplatin. Using monozygotic twin pair and sibling pair lymphoblastoid cell lines, we identified conditions for measurement of caspase activity. Although treatment with 5′-deoxy-fluorouridine and pemetrexed for up to 24 h did not result in significant apoptosis or inter-individual variation in caspase dependent cell death; paclitaxel, cisplatin, carboplain and cytarabine treatment for 24 h resulted in 9.4, 9.1, 7.0 and 6.0 fold increases in apoptosis relative to control, respectively. There was a weak correlation between caspase activity and cytotoxicity (r2=0.03 to 0.29) demonstrating that cytotoxicity and apoptosis are two distinct phenotypes that may produce independent genetic associations. Estimated heritability (h2) for apoptosis was 0.57 and 0.29 for cytarabine (5 μM and 40 μM respectively), 0.22 for paclitaxel (12.5 nM), and 0.34 for cisplatin (5 μM). The HapMap CEU panel of lymphoblastoid cell lines (n = 77) were evaluated for sensitivity to cisplatin followed by genome wide association studies with over 2 million SNPs at p < 0.001. We identified a significant enrichment of cisplatin-induced apoptosis SNPs within the significant cisplatin induced cytotoxicity SNPs and an enrichment of expression quantitative trait loci. PMID:21642893

  1. Capsaicin induces apoptosis in PC12 cells through ER stress.

    PubMed

    Krizanova, Olga; Steliarova, Iveta; Csaderova, Lucia; Pastorek, Michal; Hudecova, Sona

    2014-02-01

    Capsaicin, the pungent agent in chili peppers, has been shown to act as a tumor-suppressor in cancer. In our previous study, capsaicin was shown to induce apoptosis in the rat pheochromocytoma cell line (PC12 cells). Thus, the aim of the present study was to determine the potential mechanism by which capsaicin induces apoptosis. We treated PC12 cells with 50, 100 and 500 µM capsaicin and measured the reticular calcium content and expression of the reticular calcium transport systems. These results were correlated with endoplasmic reticulum (ER) stress markers CHOP, ATF4 and X-box binding protein 1 (XBP1), as well as with apoptosis induction. We observed that capsaicin decreased reticular calcium in a concentration-dependent manner. Simultaneously, expression levels of the sarco/endoplasmic reticulum pump and ryanodin receptor of type 2 were modified. These changes were accompanied by increased ER stress, as documented by increased stress markers. Thus, from these results we propose that in PC12 cells capsaicin induces apoptosis through increased ER stress. PMID:24337105

  2. Lipopolysaccharide-Induced Apoptosis of Astrocytes: Therapeutic Intervention by Minocycline.

    PubMed

    Sharma, Arpita; Patro, Nisha; Patro, Ishan K

    2016-05-01

    Astrocytes are most abundant glial cell type in the brain and play a main defensive role in central nervous system against glutamate-induced toxicity by virtue of numerous transporters residing in their membranes and an astrocyte-specific enzyme glutamine synthetase (GS). In view of that, a dysregulation in the astrocytic activity following an insult may result in glutamate-mediated toxicity accompanied with astrocyte and microglial activation. The present study suggests that the lipopolysaccharide (LPS)-induced inflammation results in significant astrocytic apoptosis compared to other cell types in hippocampus and minocycline could not efficiently restrict the glutamate-mediated toxicity and apoptosis of astrocytes. Upon LPS exposure 76 % astrocytes undergo degeneration followed by 44 % oligodendrocytes, 26 % neurons and 10 % microglia. The pronounced astrocytic apoptosis resulted from the LPS-induced glutamate excitotoxicity leading to their hyperactivation as evident from their hypertrophied morphology, glutamate transporter 1 upregulation and downregulation of GS. Therapeutic minocycline treatment to LPS-infused rats efficiently restricted the inflammatory response and degeneration of other cell types but could not significantly combat with the apoptosis of astrocytes. Our study demonstrates a novel finding on cellular degeneration in the hippocampus revealing more of astrocytic death and suggests a more careful consideration on the protective efficacy of minocycline. PMID:26188416

  3. Phytoconstituents as apoptosis inducing agents: strategy to combat cancer.

    PubMed

    Kumar, Manish; Kaur, Varinder; Kumar, Subodh; Kaur, Satwinderjeet

    2016-08-01

    Advancement in the field of cancer molecular biology has aided researchers to develop various new chemopreventive agents which can target cancer cells exclusively. Cancer chemopreventive agents have proficiency to inhibit, reverse and delay process of carcinogenesis during its early and later course. Chemopreventive agents can act as antioxidative, antimutagenic/antigenotoxic, anti-inflammatory agents or via aiming various molecular targets in a cell to induce cell death. Apoptosis is a kind of cell death which shows various cellular morphological alterations such as cell shrinkage, blebbing of membrane, chromatin condensation, DNA fragmentation, formation of apoptotic bodies etc. Nowadays, apoptosis is being one of the new approaches for the identification and development of novel anticancer therapies. For centuries, plants are known to play part in daily routine from providing food to management of human health. In the last two decades, diverse phytochemicals and various botanical formulations have been characterized as agents that possess potential to execute cancer cells via inducing apoptosis. Data obtained from the research carried out globally pointed out that natural products are the potential candidates which have capability to combat cancer. In the present review, we surveyed literature on natural products which throws light on the mechanism through which these phytochemicals induce apoptosis in cancer cells. PMID:26239338

  4. Determinants of PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Kessel, David; Luo, Yu; Kim, Hyeong-Reh C.

    2000-03-01

    Photodynamic therapy can initiate cell death by apoptosis or necrosis. Using agents with known patterns of sub-cellular localization, we examined the correlation between sites of photodamage and the mode of cell death, using murine leukemia cells in vitro. Mitochondrial or mitochondrial/lysosomal photodamage caused the rapid release of cytochrome c. This effect was not temperature sensitive, and could be demonstrated immediately after irradiation of photosensitized cells at 10 degrees C. Subsequent warming to 37 degrees C led to a rapid apoptotic response, consistent with the known ability of cytochrome c to trigger the activation of caspase-3. In contrast, lysosomal or lysosomal/membrane photodamage resulted in the release of cathepsins and other proteolytic enzymes. A subsequent incubation at 37 degrees C resulted in mitochondrial degradation, leading to loss of cytochrome c within 30 min. The apoptotic response was both delayed and incomplete, with many dead cells not exhibiting an apoptotic morphology. The latter outcome was traced to photodamage to procaspase-3, an effect not observed with sensitizers that caused mainly mitochondrial photodamage. Studies in a cell-free system demonstrated that agents with lysosomal and/or membrane targets could bring about photoinactivation of caspase-3. These result are consistent with the proposal that photodynamic therapy can both activate and inactivate components of the apoptotic process.

  5. Plasma-activated medium induced apoptosis on tumor cells

    NASA Astrophysics Data System (ADS)

    Hori, Masaru; Tanaka, Hiromasa; Mizuno, Masaaki; Nakamura, Kae; Kajiyama, Hiroaki; Takeda, Keigo; Ishikawa, Kenji; Kano, Hiroyuki; Kikkawa, Fumitaka

    2013-09-01

    The non-equilibrium atmospheric pressure plasma (NEAPP) has attracted attention in cancer therapy. In this study, the fresh medium was treated with our developed NEAPP, ultra-high electron density (approximately 2 × 1016 cm-3). The medium called the plasma-activated medium (PAM) killed not normal cells but tumor cells through induction of apoptosis. Cell proliferation assays showed that the tumor cells were selectively killed by the PAM. Those cells induced apoptosis using an apoptotic molecular marker, cleaved Caspase3/7. The molecular mechanisms of PAM-mediated apoptosis in the tumor cells were also found that the PAM downregulated the expression of AKT kinase, a marker molecule in a survival signal transduction pathway. These results suggest that PAM may be a promising tool for tumor therapy by downregulating the survival signals in cancers.

  6. p73-induced apoptosis: A question of compartments and cooperation

    SciTech Connect

    Dobbelstein, Matthias; Strano, Sabrina; Roth, Judith; Blandino, Giovanni . E-mail: blandino@ifo.it

    2005-06-10

    The transcriptionally active forms of p73 are capable of inducing apoptosis, and the isoforms termed TAp73 are important players when E2F and its oncogenic activators induce programmed cell death. However, the conditions under that TAp73 can kill a cell remain to be clarified. Recently, it has been found that p73 proteins are not merely floating in the nucleoplasm but rather can associate with specific compartments in the cell. Examples of intranuclear compartments associated with p73 proteins include the PML oncogenic domains and the nuclear matrix. In addition, p73 is found in the cytoplasm. It remains to be seen whether p73 might also associate with mitochondria, in analogy with p53. The relocalization of p73 is expected to be mediated by specific binding partners, mostly other proteins. Here, we discuss the possibility that the compartmentalization of p73, and the cooperation with the corresponding binding partners, might decide about its apoptosis-inducing activity.

  7. Molecular Mechanisms of Particle Ration Induced Apoptosis in Lymphocyte

    NASA Astrophysics Data System (ADS)

    Shi, Yufang

    Space radiation, composed of high-energy charged nuclei (HZE particles) and protons, has been previously shown to severely impact immune homeostasis in mice. To determine the molecular mechanisms that mediate acute lymphocyte depletion following exposure to HZE particle radiation mice were exposed to particle radiation beams at Brookhaven National Laboratory. We found that mice given whole body 5 6Fe particle irradiation (1GeV /n) had dose-dependent losses in total lymphocyte numbers in the spleen and thymus (using 200, 100 and 50 cGy), with thymocytes being more sensitive than splenocytes. All phenotypic subsets were reduced in number. In general, T cells and B cells were equally sensitive, while CD8+ T cells were more senstive than CD4+ T cells. In the thymus, immature CD4+CD8+ double-positive thymocytes were exquisitely sensitive to radiation-induced losses, single-positive CD4 or CD8 cells were less sensitive, and the least mature double negative cells were resistant. Irradiation of mice deficient in genes encoding essential apoptosis-inducing proteins revealed that the mechanism of lymphocyte depletion is independent of Fas ligand and TRAIL (TNF-ralated apoptosis-inducing ligand), in contrast to γ-radiation-induced lymphocyte losses which require the Fas-FasL pathway. Using inhibitors in vitro, lymphocyte apoptosis induced by HZE particle radiation was found to be caspase dependent, and not involve nitric oxide or oxygen free radicals.

  8. Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53

    PubMed Central

    Bishopric, Nanette H.; Discher, Daryl J.; Kaiser, Shari; Hernandez, Olga; Sato, Barbara; Zang, Jie; Webster, Keith A.

    1999-01-01

    Ischemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH]o). Apoptosis did not occur when [pH]o was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis–mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25–30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53. J. Clin. Invest. 104:239–252 (1999). PMID:10430605

  9. Herbal Medicine as Inducers of Apoptosis in Cancer Treatment

    PubMed Central

    Safarzadeh, Elham; Sandoghchian Shotorbani, Siamak; Baradaran, Behzad

    2014-01-01

    Cancer is uncontrolled growth of abnormal cells in the body. Nowadays, cancer is considered as a human tragedy and one of the most prevalent diseases in the wide, and its mortality resulting from cancer is being increased. It seems necessary to identify new strategies to prevent and treat such a deadly disease. Control survival and death of cancerous cell are important strategies in the management and therapy of cancer. Anticancer agents should kill the cancerous cell with the minimal side effect on normal cells that is possible through the induction of apoptosis. Apoptosis is known as programmed cell death in both normal and damaged tissues. This process includes some morphologically changes in cells such as rapid condensation and budding of the cell, formation of membrane-enclosed apoptotic bodies with well-preserved organelles. Induction of apoptosis is one of the most important markers of cytotoxic antitumor agents. Some natural compounds including plants induce apoptotic pathways that are blocked in cancer cells through various mechanisms in cancer cells. Multiple surveys reported that people with cancer commonly use herbs or herbal products. Vinca Alkaloids, Texans, podo phyllotoxin, Camptothecins have been clinically used as Plant derived anticancer agents. The present review summarizes the literature published so far regarding herbal medicine used as inducers of apoptosis in cancer. PMID:25364657

  10. Herbal medicine as inducers of apoptosis in cancer treatment.

    PubMed

    Safarzadeh, Elham; Sandoghchian Shotorbani, Siamak; Baradaran, Behzad

    2014-10-01

    Cancer is uncontrolled growth of abnormal cells in the body. Nowadays, cancer is considered as a human tragedy and one of the most prevalent diseases in the wide, and its mortality resulting from cancer is being increased. It seems necessary to identify new strategies to prevent and treat such a deadly disease. Control survival and death of cancerous cell are important strategies in the management and therapy of cancer. Anticancer agents should kill the cancerous cell with the minimal side effect on normal cells that is possible through the induction of apoptosis. Apoptosis is known as programmed cell death in both normal and damaged tissues. This process includes some morphologically changes in cells such as rapid condensation and budding of the cell, formation of membrane-enclosed apoptotic bodies with well-preserved organelles. Induction of apoptosis is one of the most important markers of cytotoxic antitumor agents. Some natural compounds including plants induce apoptotic pathways that are blocked in cancer cells through various mechanisms in cancer cells. Multiple surveys reported that people with cancer commonly use herbs or herbal products. Vinca Alkaloids, Texans, podo phyllotoxin, Camptothecins have been clinically used as Plant derived anticancer agents. The present review summarizes the literature published so far regarding herbal medicine used as inducers of apoptosis in cancer. PMID:25364657

  11. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.

    PubMed

    Rachlin, Kenneth; Moore, Dan H; Yount, Garret

    2013-11-01

    The development of nontoxic agents that can selectively enhance the cytotoxicity of chemotherapy is an important aim in oncology. This study evaluates the ability of infrasound exposure to sensitize glioblastoma cells to cisplatin-induced apoptosis. The infrasound was delivered using a device designed to replicate the unique infrasound emissions measured during external Qigong treatments. Human glioblastoma cell lines harboring wild-type p53 (U87) or mutant p53 (U251, SF210, and SF188) were treated in culture with cisplatin, infrasound emissions, or the combination of the 2 agents. Induction of apoptosis was quantified after 24 hours by flow cytometry following annexin V/propidium iodide staining. Infrasound emissions alone, delivered at moderate levels (~10 mPa) with dynamic frequency content (7-13 Hz), did not induce apoptosis, yet combining infrasound with cisplatin augmented the induction of apoptosis by cisplatin in all the 4 cell lines (P < .05). Increased cellular uptake of the fluorophore calcein associated with infrasound exposure was quantified by fluorescence microscopy as well as flow cytometry, demonstrating increased cell membrane permeability. The 4 cell lines differed in the degree to which infrasound exposure increased calcein uptake, and these differences were predictive of the extent to which infrasound enhanced cisplatin-induced apoptosis. When exposed to specific frequencies, membrane permeabilization also appeared to be differentially responsive for each cell line, suggesting the potential for selective targeting of tissue types using isolated infrasonic frequencies. Additionally, the pressure amplitudes used in this study were several orders of magnitude less than those used in similar studies involving ultrasound and shock waves. The results of this study provide support for using infrasound to enhance the chemotherapeutic effects of cisplatin in a clinical setting. PMID:23165942

  12. Minerval induces apoptosis in Jurkat and other cancer cells

    PubMed Central

    Llado, Victoria; Gutierrez, Antonio; Martínez, Jordi; Casas, Jesús; Terés, Silvia; Higuera, Mónica; Galmés, Antonio; Saus, Carles; Besalduch, Joan; Busquets, Xavier; Escribá, Pablo V

    2010-01-01

    Abstract Minerval is an oleic acid synthetic analogue that impairs lung cancer (A549) cell proliferation upon modulation of the plasma membrane lipid structure and subsequent regulation of protein kinase C localization and activity. However, this mechanism does not fully explain the regression of tumours induced by this drug in animal models of cancer. Here we show that Minerval also induced apoptosis in Jurkat T-lymphoblastic leukaemia and other cancer cells. Minerval inhibited proliferation of Jurkat cells, concomitant with a decrease of cyclin D3 and cdk2 (cyclin-dependent kinase2). In addition, the changes that induced on Jurkat cell membrane organization caused clustering (capping) of the death receptor Fas (CD95), caspase-8 activation and initiation of the extrinsic apoptosis pathway, which finally resulted in programmed cell death. The present results suggest that the intrinsic pathway (associated with caspase-9 function) was activated downstream by caspase-8. In a xenograft model of human leukaemia, Minerval also inhibited tumour progression and induced tumour cell death. Studies carried out in a wide variety of cancer cell types demonstrated that apoptosis was the main molecular mechanism triggered by Minerval. This is the first report on the pro-apoptotic activity of Minerval, and in part explains the effectiveness of this non-toxic anticancer drug and its wide spectrum against different types of cancer. PMID:19413889

  13. Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

    PubMed Central

    Park, Choon-Ho; Choi, Bo-Hwa; Jeong, Min-Woo; Kim, Sangjune; Kim, Wanil; Song, Yun Seon; Kim, Kyong-Tai

    2011-01-01

    Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage–induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner. PMID:21346188

  14. Selenite suppression of cadmium-induced testicular apoptosis.

    PubMed

    Jones, M M; Xu, C; Ladd, P A

    1997-01-15

    The characteristic apoptotic ladder-like patterns of rat testicular DNA on agarose gel electrophoresis which results from treatment with CdCl2 are suppressed by the administration of Na2SeO3. The examination of testicular tissue using an ELISA programmed cell death detection procedure confirmed this selenite suppression of cadmium-induced apoptosis. The administration of the Na2SeO3 at either 0.5, 1, 2 h prior to or 0.5, 1, 2 h after the administration of the CdCl2 appear to be almost equally effective at suppressing the apoptotic response. These results are in accord with previous studies on the Na2SeO3 suppression of cadmium induced necrotic changes in tissues and suggest that Na2SeO3 interferes with both necrosis and apoptosis. PMID:9020518

  15. Pulse mode of laser photodynamic treatment induced cell apoptosis.

    PubMed

    Klimenko, Vladimir V; Knyazev, Nickolay A; Moiseenko, Fedor V; Rusanov, Anatoliy A; Bogdanov, Alexey A; Dubina, Michael V

    2016-03-01

    One of the factors limiting photodynamic therapy (PDT) is hypoxia in tumor cells during photodynamic action. PDT with pulse mode irradiation and appropriate irradiation parameters could be more effective in the singlet oxygen generation and tissue re-oxygenation than continuous wave (CW) mode. We theoretically demonstrate differences between the cumulative singlet oxygen concentration in PDT using pulse mode and CW mode of laser irradiation. In vitro experimental results show that photodynamic treatment with pulse mode irradiation has similar cytotoxicity to CW mode and induces mainly cell apoptosis, whereas CW mode induces necrotic cell death. We assume that the cumulative singlet oxygen concentration and the temporal distribution of singlet oxygen are important in photodynamic cytotoxicity and apoptosis initiation. We expect our research may improve irradiation protocols and photodynamic therapy efficiency. PMID:26790610

  16. Alpha Particles Induce Apoptosis through the Sphingomyelin Pathway

    PubMed Central

    Seideman, Jonathan H.; Stancevic, Branka; Rotolo, Jimmy A.; McDevitt, Michael R.; Howell, Roger W.; Kolesnick, Richard N.; Scheinberg, David A.

    2011-01-01

    The sphingomyelin pathway involves the enzymatic cleavage of sphingomyelin to produce ceramide, a second messenger that serves as a key mediator in the rapid apoptotic response to various cell stressors. Low-linear energy transfer (LET) γ radiation can initiate this pathway, independent of DNA damage, via the cell membrane. Whether short-ranged, high-LET a particles, which are of interest as potent environmental carcinogens, radiotherapies and potential components of dirty bombs, can act through this mechanism to signal apoptosis is unknown. Here we show that irradiation of Jurkat cells with a particles emitted by the 225Ac-DOTA-anti-CD3 IgG antibody construct results in dose-dependent apoptosis. This apoptosis was significantly reduced by pretreating cells with cholesterol-depleting nystatin, a reagent known to inhibit ceramide signaling by interfering with membrane raft coalescence and ceramide-rich platform generation. The effects of nystatin on α-particle-induced apoptosis were related to disruption of the ceramide pathway and not to microdosimetry alterations, because similar results were obtained after external irradiation of the cells with a broad beam of collimated a particles using a planar 241Am source. External irradiation allowed for more precise control of the dosimetry and geometry of the irradiation, independent of antibody binding or cell internalization kinetics. Mechanistically consistent with these findings, Jurkat cells rapidly increased membrane concentrations of ceramide after external irradiation with an average of five α-particle traversals per cell. These data indicate that a particles can activate the sphingomyelin pathway to induce apoptosis. PMID:21631289

  17. Alpha particles induce apoptosis through the sphingomyelin pathway.

    PubMed

    Seideman, Jonathan H; Stancevic, Branka; Rotolo, Jimmy A; McDevitt, Michael R; Howell, Roger W; Kolesnick, Richard N; Scheinberg, David A

    2011-10-01

    The sphingomyelin pathway involves the enzymatic cleavage of sphingomyelin to produce ceramide, a second messenger that serves as a key mediator in the rapid apoptotic response to various cell stressors. Low-linear energy transfer (LET) γ radiation can initiate this pathway, independent of DNA damage, via the cell membrane. Whether short-ranged, high-LET α particles, which are of interest as potent environmental carcinogens, radiotherapies and potential components of dirty bombs, can act through this mechanism to signal apoptosis is unknown. Here we show that irradiation of Jurkat cells with α particles emitted by the ²²⁵Ac-DOTA-anti-CD3 IgG antibody construct results in dose-dependent apoptosis. This apoptosis was significantly reduced by pretreating cells with cholesterol-depleting nystatin, a reagent known to inhibit ceramide signaling by interfering with membrane raft coalescence and ceramide-rich platform generation. The effects of nystatin on α-particle-induced apoptosis were related to disruption of the ceramide pathway and not to microdosimetry alterations, because similar results were obtained after external irradiation of the cells with a broad beam of collimated α particles using a planar ²⁴¹Am source. External irradiation allowed for more precise control of the dosimetry and geometry of the irradiation, independent of antibody binding or cell internalization kinetics. Mechanistically consistent with these findings, Jurkat cells rapidly increased membrane concentrations of ceramide after external irradiation with an average of five α-particle traversals per cell. These data indicate that α particles can activate the sphingomyelin pathway to induce apoptosis. PMID:21631289

  18. Tumor Cell Selective Cytotoxicity and Apoptosis Induction by an Herbal Preparation from Brucea javanica

    PubMed Central

    Gao, Hua; Lamusta, Julie; Zhang, Wei-Fang; Salmonsen, Rebecca; Liu, Yingwang; O’Connell, Edward; Evans, James E.; Burstein, Sumner; Chen, Jason J.

    2011-01-01

    The plant Brucea javanica has shown impressive efficacy for treating various diseases including cancer. However, the mechanism by which B. javanica acts is poorly understood. We have established tissue culture assays to study the effects of B. javanica on cervical and several other cancer cells. Our results demonstrated that the aqueous extract from B. javanica is selectively toxic to cancer cells. Induction of apoptosis by B. javanica appears to be a possible mechanism by which it kills cancer cells. Interestingly, a significant increase of p53 protein level was observed in these apoptotic cells. Our studies indicated that both p53-dependent and p53-independent activities contributed to herb-induced cell death. These results imply that further studies with B. javanica may lead to the development of novel anti-cancer drugs. PMID:21654932

  19. CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

    EPA Science Inventory

    Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

    Abstract
    HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

  20. Oxidative Stress Mediates Radiation Lung Injury by Inducing Apoptosis

    SciTech Connect

    Zhang Yu; Zhang Xiuwu; Rabbani, Zahid N.; Jackson, Isabel L.; Vujaskovic, Zeljko

    2012-06-01

    Purpose: Apoptosis in irradiated normal lung tissue has been observed several weeks after radiation. However, the signaling pathway propagating cell death after radiation remains unknown. Methods and Materials: C57BL/6J mice were irradiated with 15 Gy to the whole thorax. Pro-apoptotic signaling was evaluated 6 weeks after radiation with or without administration of AEOL10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Results: Apoptosis was observed primarily in type I and type II pneumocytes and endothelium. Apoptosis correlated with increased PTEN expression, inhibition of downstream PI3K/AKT signaling, and increased p53 and Bax protein levels. Transforming growth factor-{beta}1, Nox4, and oxidative stress were also increased 6 weeks after radiation. Therapeutic administration of AEOL10150 suppressed pro-apoptotic signaling and dramatically reduced the number of apoptotic cells. Conclusion: Increased PTEN signaling after radiation results in apoptosis of lung parenchymal cells. We hypothesize that upregulation of PTEN is influenced by Nox4-derived oxidative stress. To our knowledge, this is the first study to highlight the role of PTEN in radiation-induced pulmonary toxicity.

  1. Methanol extract of wheatgrass induces G1 cell cycle arrest in a p53-dependent manner and down regulates the expression of cyclin D1 in human laryngeal cancer cells-an in vitro and in silico approach

    PubMed Central

    Shakya, Garima; Balasubramanian, Sangeetha; Rajagopalan, Rukkumani

    2015-01-01

    Background: Deregs been implicated in the malignancy of cancer. Since many years investigation on the traditional herbs has been the focus to develop novel and effective drug for cancer remedies. Wheatgrass is a medicinal plant, used in folk medicine to cure various diseases. The present study was undertaken to gain insights into antiproliferative effect of methanol extract of wheatgrass. Materials Methods: Cell viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Lactate Dehydrogenase assays. Cell cycle was analyzed by flow cytometry. Western blot was performed to determine the p53 and cyclin D1 levels. In silico docking interaction of the 14 active components (identified by high-performance liquid chromatography/gas chromatography-mass spectroscopy) of the methanol extract was tested with cyclin D1 (Protein Data Bank ID: 2W96) and compared with the reference cyclin D1/Cdk4 inhibitor. Results: Methanol extract of wheatgrass effectively reduced the cell viability. The cell cycle analysis showed that the extract treatment caused G1 arrest. The level of cyclin D1 was decreased, whereas p53 level was increased. Molecular docking studies revealed interaction of seven active compounds of the extract with the vital residues (Lys112/Glu141) of cyclin D1. Conclusion: These findings indicate that the methanol extract of wheatgrass inhibits human laryngeal cancer cell proliferation via cell cycle G1 arrest and p53 induction. The seven active compounds of the extract were also found to be directly involved in the inhibition of cyclin D1/Cdk4 binding, thus inhibiting the cell proliferation. PMID:26109759

  2. Dracorhodin perchlorate induces the apoptosis of glioma cells.

    PubMed

    Chen, Xin; Luo, Junjie; Meng, Linghu; Pan, Taifeng; Zhao, Binjie; Tang, Zhen-Gang; Dai, Yongjian

    2016-04-01

    Dracorhodin perchlorate (Dp), a synthetic analogue of the antimicrobial anthocyanin red pigment, has recently been shown to induce apoptotic cell death in various types of cancer cells. Yet, the inhibitory effect of Dp on human glioma cells remains uninvestigated. Therefore, in the present study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to detect cell viability and cell cycle progression in glioma U87MG and T98G cells, respectively. Annexin V-FITC/propidium iodide double staining and JC-1 staining were separately applied to determine cellular apoptosis and mitochondrial membrane potential damage in the cells. The expression levels of associated proteins involved in cell cycle progression and apoptosis were measured by western blotting. The activities of caspase‑9/-3 were determined by Caspase-Glo-9/3 assay. The results indicated that Dp treatment significantly inhibited cell proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at the G1/S phase in the U87MG and T98G cells via the upregulation of p53 and p21 protein expression, and simultaneous downregulation of Cdc25A, Cdc2 and P-Cdc2 protein expression. Additionally, Dp treatment led to the loss of cellular mitochondrial membrane potential, and the release of cytochrome c, and strongly induced the occurence of apoptosis. Increased expression levels of Bim and Bax protein and the downregulated expression of Bcl-2 protein were observed. Caspase-9/-3 were activated and their activities were elevated after Dp treatment. These findings indicate that Dp inhibits cell proliferation, induces cell cycle arrest and apoptosis in glioma cells, and is a possible candidate for glioma treatment. PMID:26846469

  3. Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

    PubMed Central

    Vucic, Domagoj; Kaiser, William J.; Miller, Lois K.

    1998-01-01

    Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins. PMID:9584170

  4. Select forms of tumor cell apoptosis induce dendritic cell maturation.

    PubMed

    Demaria, Sandra; Santori, Fabio R; Ng, Bruce; Liebes, Leonard; Formenti, Silvia C; Vukmanovic, Stanislav

    2005-03-01

    Dendritic cells (DC) play a crucial role in initiating immune responses to tumors. DC can efficiently present antigens from apoptotic tumor cells, but apoptotic cells are thought to lack the inflammatory signals required to induce DC maturation. Here, we show that apoptosis of 67NR mouse carcinoma cells via the Fas (CD95) pathway or induced by the anticancer drug bortezomib (PS-341) but not by ultraviolet irradiation is associated with the production of maturation signals for DC. These data have important implications for the effects of chemotherapy on antitumor immunity in solid and hematologic malignancies. PMID:15569694

  5. Inhibition of TLR8 mediated signaling promotes BCG induced apoptosis in THP-1 cells.

    PubMed

    Tang, Jun; Zhan, Lingjun; Qin, Chuan

    2016-04-01

    Apoptosis was considered as one of the important host defense mechanisms against mycobacteria infection. In macrophage, the main target cell of Mycobacterium tuberculosis, apoptosis after infection could help kill the bacillus inside and process the antigens for further presentation and proper immune response. Here, we identified a role of TLR8 during the apoptosis induced by Bacillus Calmette Guérin (BCG) infection in THP-1 cells. Knockdown TLR8 further increased the apoptosis induced by BCG infection, and this enhanced apoptosis was caspase-dependent. During this process, Erk1/2, JNK and NFκB pathways were negatively affected and contributed to the enhanced apoptosis. PMID:26657720

  6. Thyroid hormone and anti-apoptosis in tumor cells.

    PubMed

    Lin, Hung-Yun; Glinsky, Gennadi V; Mousa, Shaker A; Davis, Paul J

    2015-06-20

    The principal secretory product of the thyroid gland, L-thyroxine (T4), is anti-apoptotic at physiological concentrations in a number of cancer cell lines. Among the mechanisms of anti-apoptosis activated by the hormone are interference with the Ser-15 phosphorylation (activation) of p53 and with TNFα/Fas-induced apoptosis. The hormone also decreases cellular abundance and activation of proteolytic caspases and of BAX and causes increased expression of X-linked inhibitor of apoptosis (XIAP). The anti-apoptotic effects of thyroid hormone largely are initiated at a cell surface thyroid hormone receptor on the extracellular domain of integrin αvβ3 that is amply expressed and activated in cancer cells. Tetraiodothyroacetic acid (tetrac) is a T4 derivative that, in a model of resveratrol-induced p53-dependent apoptosis in glioma cells, blocks the anti-apoptotic action of thyroid hormone, permitting specific serine phosphorylation of p53 and apoptosis to proceed. In a nanoparticulate formulation limiting its action to αvβ3, tetrac modulates integrin-dependent effects on gene expression in human cancer cell lines that include increased expression of a panel of pro-apoptotic genes and decreased transcription of defensive anti-apoptotic XIAP and MCL1 genes. By a variety of mechanisms, thyroid hormone (T4) is an endogenous anti-apoptotic factor that may oppose chemotherapy-induced apoptosis in αvβ3-expressing cancer cells. It is possible to decrease this anti-apoptotic activity pharmacologically by reducing circulating levels of T4 or by blocking effects of T4 that are initiated at αvβ3. PMID:26041883

  7. Thyroid hormone and anti-apoptosis in tumor cells

    PubMed Central

    Lin, Hung-Yun; Glinsky, Gennadi V.; Mousa, Shaker A.; Davis, Paul J.

    2015-01-01

    The principal secretory product of the thyroid gland, L-thyroxine (T4), is anti-apoptotic at physiological concentrations in a number of cancer cell lines. Among the mechanisms of anti-apoptosis activated by the hormone are interference with the Ser-15 phosphorylation (activation) of p53 and with TNFα/Fas-induced apoptosis. The hormone also decreases cellular abundance and activation of proteolytic caspases and of BAX and causes increased expression of X-linked inhibitor of apoptosis (XIAP). The anti-apoptotic effects of thyroid hormone largely are initiated at a cell surface thyroid hormone receptor on the extracellular domain of integrin αvβ3 that is amply expressed and activated in cancer cells. Tetraiodothyroacetic acid (tetrac) is a T4 derivative that, in a model of resveratrol-induced p53-dependent apoptosis in glioma cells, blocks the anti-apoptotic action of thyroid hormone, permitting specific serine phosphorylation of p53 and apoptosis to proceed. In a nanoparticulate formulation limiting its action to αvβ3, tetrac modulates integrin-dependent effects on gene expression in human cancer cell lines that include increased expression of a panel of pro-apoptotic genes and decreased transcription of defensive anti-apoptotic XIAP and MCL1 genes. By a variety of mechanisms, thyroid hormone (T4) is an endogenous anti-apoptotic factor that may oppose chemotherapy-induced apoptosis in αvβ3-expressing cancer cells. It is possible to decrease this anti-apoptotic activity pharmacologically by reducing circulating levels of T4 or by blocking effects of T4 that are initiated at αvβ3. PMID:26041883

  8. Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Daosen; Feng, Yanyan; Zhang, Qinnan; Su, Xin; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

    2015-04-01

    Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

  9. Erythropoietin protects cardiac myocytes against anthracycline-induced apoptosis

    SciTech Connect

    Fu Ping; Arcasoy, Murat O. . E-mail: arcas001@mc.duke.edu

    2007-03-09

    The cardiotoxic adverse effects of anthracycline antibiotics limit their therapeutic utility as essential components of chemotherapy regimens for hematologic and solid malignancies. Here we show that the hematopoietic cytokine erythropoietin attenuates doxorubicin-induced apoptosis of primary neonatal rat ventricular cardiomyocytes in a dose-dependent manner. Erythropoietin treatment induced rapid, time-dependent phosphorylation of MAP kinases (MAPK) Erk1/2 and the phosphatidylinositol 3-kinase substrate Akt. Treatment of cardiomyocytes with inhibitors of phosphatidylinositol 3-kinase (LY294002) or Akt (Akti-1/2) abolished the protective effect of erythropoietin, whereas treatment with MAPK kinase (MEK1) inhibitor U0126 did not. Erythropoietin also induced the phosphorylation of GSK-3{beta}, a downstream target of PI3K-Akt. Because phosphorylation is known to inactivate GSK-3{beta}, we investigated whether GSK-3{beta} inhibition is cardioprotective. We found that GSK-3{beta} inhibitors SB216763 or lithium chloride blocked doxorubicin-induced cardiomyocyte apoptosis in a manner similar to erythropoietin, suggesting that GSK-3{beta} inhibition is involved in erythropoietin-mediated cardioprotection. Erythropoietin may serve as a novel cardioprotective agent against anthracycline-induced cardiotoxicity.

  10. Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

    PubMed Central

    Doroodgar, Masoud; Delavari, Mahdi; Doroodgar, Moein; Abbasi, Ali; Taherian, Ali Akbar; Doroodgar, Abbas

    2016-01-01

    Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 μg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC50. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC50) of tamoxifen on promastigotes was 2.6 μg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed. PMID:26951973

  11. Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo.

    PubMed

    Galvin, J P; Spaeny-Dekking, L H; Wang, B; Seth, P; Hack, C E; Froelich, C J

    1999-05-01

    Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro. PMID:10228010

  12. Roscovitine ameliorates endotoxin-induced uveitis through neutrophil apoptosis

    PubMed Central

    Jiang, Zhao-Xin; Qiu, Suo; Lou, Bing-Sheng; Yang, Yao; Wang, Wen-Cong; Lin, Xiao-Feng

    2016-01-01

    Neutrophils have been recognized as critical response cells during the pathogenesis of endotoxin-induced uveitis (EIU). Apoptosis of neutrophils induced by roscovitine has previously been demonstrated to ameliorate inflammation in several in vivo models. The present study aimed to assess whether roscovitine ameliorates EIU. EIU was induced in female C57BL/6 mice by a single intravitreal injection of lipopolysaccharide (LPS; 250 ng). The mice were divided into three groups as follows: LPS alone, LPS plus vehicle, LPS plus roscovitine (50 mg/kg). The mice were euthanized 12, 24, 48 and 72 h after LPS-induced uveitis. Accumulation of inflammatory cells in the vitreous body was confirmed by immunohistochemistry, and quantified following hematoxylin and eosin staining. Terminal deoxynucleotidyl transferase dUTP nick-end labeling was performed to detect of apoptotic cells. The mRNA levels of inflammatory cytokines were analyzed by reverse transcription-quantitative polymerase chain reaction and the changes in protein levels were analyzed by western blotting. Inflammatory cells accumulated in the vitreous near the optic nerve head and the quantity peaked at 24 h after LPS injection. Immunohistochemistry revealed that the majority of the inflammatory cells were neutrophils. The number of infiltrating cells was similar in the LPS and LPS plus vehicle groups, while there were significantly less in the roscovitine group at 24 h. Apoptosis of neutrophils was observed between 12 and 48 h after roscovitine injection, while no apoptosis was observed in the other groups. The mRNA expression levels of GMCSF, CINC-1 and ICAM-1 peaked at 12 h after LPS injection, and decreased to normal levels at 72 h. This trend in mRNA expression was similar in the LPS and LPS plus vehicle groups; however, the expression levels decreased more quickly in the roscovitine group at 24 and 48 h. Following roscovitine administration, upregulated cleaved caspase 3 expression levels and downregulated Mcl-1

  13. Roscovitine ameliorates endotoxin-induced uveitis through neutrophil apoptosis.

    PubMed

    Jiang, Zhao-Xin; Qiu, Suo; Lou, Bing-Sheng; Yang, Yao; Wang, Wen-Cong; Lin, Xiao-Feng

    2016-08-01

    Neutrophils have been recognized as critical response cells during the pathogenesis of endotoxin‑induced uveitis (EIU). Apoptosis of neutrophils induced by roscovitine has previously been demonstrated to ameliorate inflammation in several in vivo models. The present study aimed to assess whether roscovitine ameliorates EIU. EIU was induced in female C57BL/6 mice by a single intravitreal injection of lipopolysaccharide (LPS; 250 ng). The mice were divided into three groups as follows: LPS alone, LPS plus vehicle, LPS plus roscovitine (50 mg/kg). The mice were euthanized 12, 24, 48 and 72 h after LPS‑induced uveitis. Accumulation of inflammatory cells in the vitreous body was confirmed by immunohistochemistry, and quantified following hematoxylin and eosin staining. Terminal deoxynucleotidyl transferase dUTP nick‑end labeling was performed to detect of apoptotic cells. The mRNA levels of inflammatory cytokines were analyzed by reverse transcription‑quantitative polymerase chain reaction and the changes in protein levels were analyzed by western blotting. Inflammatory cells accumulated in the vitreous near the optic nerve head and the quantity peaked at 24 h after LPS injection. Immunohistochemistry revealed that the majority of the inflammatory cells were neutrophils. The number of infiltrating cells was similar in the LPS and LPS plus vehicle groups, while there were significantly less in the roscovitine group at 24 h. Apoptosis of neutrophils was observed between 12 and 48 h after roscovitine injection, while no apoptosis was observed in the other groups. The mRNA expression levels of GMCSF, CINC‑1 and ICAM‑1 peaked at 12 h after LPS injection, and decreased to normal levels at 72 h. This trend in mRNA expression was similar in the LPS and LPS plus vehicle groups; however, the expression levels decreased more quickly in the roscovitine group at 24 and 48 h. Following roscovitine administration, upregulated cleaved caspase 3 expression levels

  14. Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation.

    PubMed

    Boren, J; Brindle, K M

    2012-09-01

    A characteristic of apoptosis is the rapid accumulation of cytoplasmic lipid droplets, which are composed largely of neutral lipids. The proton signals from these lipids have been used for the non-invasive detection of cell death using magnetic resonance spectroscopy. We show here that despite an apoptosis-induced decrease in the levels and activities of enzymes involved in lipogenesis, which occurs downstream of p53 activation and inhibition of the mTOR signaling pathway, the increase in lipid accumulation is due to increased de novo lipid synthesis. This results from inhibition of mitochondrial fatty acid β-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverts fatty acids away from oxidation and into lipid synthesis. The inhibition of fatty acid oxidation can be explained by a rapid rise in mitochondrial membrane potential and an attendant increase in the levels of reactive oxygen species. PMID:22460322

  15. Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

    PubMed Central

    Ueda, Norishi

    2015-01-01

    Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

  16. Cimetidine induces apoptosis of human salivary gland tumor cells.

    PubMed

    Fukuda, Masakatsu; Tanaka, Shin; Suzuki, Seiji; Kusama, Kaoru; Kaneko, Tadayoshi; Sakashita, Hideaki

    2007-03-01

    It has been reported that cimetidine, a histamine type-2 receptor (H2R) antagonist, inhibits the growth of glandular tumors such as colorectal cancer. However, its effects against salivary gland tumors are still unknown. We demonstrated previously that human salivary gland tumor (HSG) cells spontaneously express the neural cell adhesion molecule (NCAM) and also that HSG cell proliferation could be controlled via a homophilic (NCAM-NCAM) binding mechanism and that NCAM may be associated with perineural invasion by malignant salivary gland tumors. In the present study, we investigated the effects of cimetidine via the expression of NCAM on tumor growth and perineural/neural invasion in salivary gland tumor cells. Expression of both NCAM mRNA and protein was found to decrease in a dose-dependent manner upon treatment with cimetidine for 24 h. The MTT assay and confocal laser microscopy clearly showed that HSG cells underwent apoptosis after treatment with cimetidine. Activation of caspases 3, 7, 8 and 9 was observed in HSG cells after cimetidine treatment, thus confirming that the apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with cimetidine. We also found that the cimetidine-mediated down-regulation of NCAM expression in HSG cells did not occur via blocking of the histamine receptor, even though H2R expression was observed on HSG cells, as two other H2R antagonists, famotidine and ranitidine, did not show similar effects. We demonstrated for the first time that cimetidine can induce significant apoptosis of salivary gland tumor cells, which express NCAM, at least in part by down-regulation of NCAM expression on the cells. These findings suggest that the growth, development and perineural/neural invasion of salivary gland tumor cells can be blocked by cimetidine administration through down-regulation of NCAM expression, as well as induction of apoptosis. PMID:17273750

  17. AIRE-induced apoptosis is associated with nuclear translocation of stress sensor protein GAPDH

    SciTech Connect

    Liiv, Ingrid; Haljasorg, Uku; Kisand, Kai; Maslovskaja, Julia; Laan, Martti; Peterson, Paert

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer AIRE induces apoptosis in epithelial cells. Black-Right-Pointing-Pointer CARD domain of AIRE is sufficient for apoptosis induction. Black-Right-Pointing-Pointer AIRE induced apoptosis involves GAPDH translocation to the nuclei. Black-Right-Pointing-Pointer Deprenyl inhibits AIRE induced apoptosis. -- Abstract: AIRE (Autoimmune Regulator) has a central role in the transcriptional regulation of self-antigens in medullary thymic epithelial cells, which is necessary for negative selection of autoreactive T cells. Recent data have shown that AIRE can also induce apoptosis, which may be linked to cross-presentation of these self-antigens. Here we studied AIRE-induced apoptosis using AIRE over-expression in a thymic epithelial cell line as well as doxycycline-inducible HEK293 cells. We show that the HSR/CARD domain in AIRE together with a nuclear localization signal is sufficient to induce apoptosis. In the nuclei of AIRE-positive cells, we also found an increased accumulation of a glycolytic enzyme, glyceraldehyde-3-phosphate (GAPDH) reflecting cellular stress and apoptosis. Additionally, AIRE-induced apoptosis was inhibited with an anti-apoptotic agent deprenyl that blocks GAPDH nitrosylation and nuclear translocation. We propose that the AIRE-induced apoptosis pathway is associated with GAPDH nuclear translocation and induction of NO-induced cellular stress in AIRE-expressing cells.

  18. Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus

    PubMed Central

    Craciunescu, Corneliu N.; Wu, Renan; Zeisel, Steven H.

    2006-01-01

    Diethanolamine (DEA) is present in many consumer products such as shampoo. Dermal administration of DEA diminishes hepatic stores of the essential nutrient choline, and we previously reported that dietary choline deficiency during pregnancy reduces neurogenesis and increases apoptosis in the hippocampus of fetal rats and mice. Therefore, DEA could also alter brain development. Timed-pregnant C57BL/6 mice were dosed dermally from gestation day 7 through 17 with DEA at 0, 20, 80, 160, 320, and 640 mg/kg body/day. At doses of DEA > 80 mg/kg body/day, we observed decreased litter size. In fetuses (embryonic day 17) collected from dams treated dermally with 80 mg/kg body/day DEA, we observed decreased neural progenitor cell mitosis at the ventricular surface of the ventricular zone of the hippocampus [to 56±14% (SE) histone 3 (H3) phosphorylation as compared to controls; P < 0.01]. We also observed increased apoptosis in fetal hippocampus (to 170±10% of control measured using TUNEL and to 178±7% of control measured using activated caspase 3; P < 0.01). Thus, maternal exposure to DEA reduces the number of neural progenitor cells in hippocampus by two mechanisms, and this could permanently alter memory function in offspring of mothers exposed to this common ingredient of shampoos and soaps.—Craciunescu, C. N., Wu, R., Zeisel, S. H. Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus. PMID:16873886

  19. Tetranectin gene deletion induces Parkinson's disease by enhancing neuronal apoptosis.

    PubMed

    Chen, Zhifeng; Wang, Ersong; Hu, Rong; Sun, Yu; Zhang, Lei; Jiang, Jue; Zhang, Ying; Jiang, Hong

    Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We previously identified tetranectin (TET) as a potential biomarker for PD whose expression is downregulated in the cerebrospinal fluid of PD patients. In the present study, we investigate the role of TET in neurodegeneration in vitro and in vivo. Our results showed that siRNA knockdown of TET decreased cell viability and the number of tyrosine hydroxylase (TH) positive cells, whereas it increased caspase-3 activity and the Bax/Bcl-2 ratio in cultured primary dopaminergic neurons. Overexpression of TET protected dopaminergic neurons against neuronal apoptosis in 1-methyl-4-phenylpyridinium cell culture model in vitro. In TET knockdown mouse model of PD, TET gene deletion decreased the number of TH positive cells in the SNpc, induced apoptosis via the p53/Bax pathway, and significantly impaired the motor behavior of transgenic mice. The findings suggest that TET plays a neuroprotective role via reducing neuron apoptosis and could be a valuable biomarker or potential therapeutic target for the treatment of patients with PD. PMID:26597345

  20. Dihydrolipoic acid induces cytotoxicity in mouse blastocysts through apoptosis processes.

    PubMed

    Houng, Wei-Li; Lin, Cheng-An J; Shen, Ji-Lin; Yeh, Hung-I; Wang, Hsueh-Hsiao; Chang, Walter H; Chan, Wen-Hsiung

    2012-01-01

    α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet, taken up by cells and tissues, and subsequently reduced to dihydrolipoic acid (DHLA). In view of the recent application of DHLA as a hydrophilic nanomaterial preparation, determination of its biosafety profile is essential. In the current study, we examined the cytotoxic effects of DHLA on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, in vivo implantation by embryo transfer, and early embryonic development in an animal model. Blastocysts treated with 50 μM DHLA exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with DHLA were lower than that of their control counterparts. Moreover, in vitro treatment with 50 μM DHLA was associated with increased resorption of post-implantation embryos and decreased fetal weight. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 100 μM DHLA led to decreased early embryo development, specifically, inhibition of development to the blastocyst stage. However, it appears that concentrations of DHLA lower than 50 μM do not exert a hazardous effect on embryonic development. Our results collectively indicate that in vitro and in vivo exposure to concentrations of DHLA higher than 50 μM DHLA induces apoptosis and retards early pre- and post-implantation development, and support the potential of DHLA to induce embryonic cytotoxicity. PMID:22489194

  1. Vanadate induces apoptosis in epidermal JB6 P+ cells via hydrogen peroxide-mediated reactions.

    PubMed

    Ye, J; Ding, M; Leonard, S S; Robinson, V A; Millecchia, L; Zhang, X; Castranova, V; Vallyathan, V; Shi, X

    1999-12-01

    Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P+ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 microM vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H2O2 as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H2O2 production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H2O2 induced by vanadate plays a major role in this process. PMID:10705990

  2. Fangchinoline inhibits breast adenocarcinoma proliferation by inducing apoptosis.

    PubMed

    Xing, Zhi-Bo; Yao, Lei; Zhang, Guo-Qiang; Zhang, Xian-Yu; Zhang, You-Xue; Pang, Da

    2011-01-01

    Radix Stephaniae tetrandrae, which contains tetrandrine (Tet) and fangchinoline, is traditionally used as an analgesic, antirheumatic, and antihypertensive drug in China. In this study, we investigated its effect on breast cancer cell proliferation and its potential mechanism of action in vitro. Treatment of cells with fangchinoline significantly inhibited MDA-MB-231 cell proliferation in a concentration- and time-dependent manner. To define the mechanism underlying the antiproliferative effects of fangchinoline, we studied its effects on critical molecular events known to regulate the apoptotic machinery. Specifically, we addressed the potential of fangchinoline to induce apoptosis of breast cancer cells. Fangchinoline induced internucleosomal DNA fragmentation, chromatin condensation, activation of caspases-3, -8, and -9, and cleavage of poly(ADP ribose) polymerase, as well as enhanced mitochondrial cytochrome c release. Furthermore, fangchinoline increased the expression of the proapoptotic protein B cell lymphoma-2 associated X (Bax) and decreased the expression of the antiapoptotic protein B cell lymphoma-2 (Bcl-2). In addition, the proliferation-inhibitory effect of fangchinoline was associated with decreased levels of phosphorylated Akt. Our results indicate that fangchinoline can inhibit breast cancer cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and decreasing phosphorylated Akt. Thus fangchinoline may be a novel agent that can potentially be developed clinically to target human malignancies. PMID:22130369

  3. Cupressus lusitanica (Cupressaceae) leaf extract induces apoptosis in cancer cells.

    PubMed

    Lopéz, L; Villavicencio, M A; Albores, A; Martínez, M; de la Garza, J; Meléndez-Zajgla, J; Maldonado, V

    2002-05-01

    A crude ethanolic extract of Cupressus lusitanica Mill. leaves demonstrate cytotoxicity in a panel of cancer cell lines. Cell death was due to apoptosis, as assessed by morphologic features (chromatin condensation and apoptotic bodies formation) and specific DNA fragmentation detected by in situ end-labeling of DNA breaks (TUNEL). The apoptotic cell death was induced timely in a dose-dependent manner. Despite the absence of changes in the expression levels of antiapoptotic protein Bcl-2, proapoptotic Bax protein variants omega and delta were increased. These results warrant further research of possible antitumor compounds in this plant. PMID:12007700

  4. Nitroxoline induces apoptosis and slows glioma growth in vivo

    PubMed Central

    Lazovic, Jelena; Guo, Lea; Nakashima, Jonathan; Mirsadraei, Leili; Yong, William; Kim, Hyun J.; Ellingson, Benjamin; Wu, Hong; Pope, Whitney B.

    2015-01-01

    Background Nitroxoline is an FDA-approved antibiotic with potential antitumor activity. Here we evaluated whether nitroxoline has antiproliferative properties on glioma cell growth in vitro and in vivo using glioma cell lines and a genetically engineered PTEN/KRAS mouse glioma model. Methods The effect of nitroxoline treatment on U87 and/or U251 glioma cell proliferation, cell-cycle arrest, invasion, and ability to induce an apoptotic cascade was determined in vitro. Magnetic resonance imaging was used to measure glioma volumes in genetically engineered PTEN/KRAS mice prior to and after nitroxoline therapy. Induction of apoptosis by nitroxoline was evaluated at the end of treatment using terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL). Results Nitroxoline inhibited the proliferation and invasion of glioblastoma cells in a time- and dose-dependent manner in vitro. Growth inhibition was associated with cell-cycle arrest in G1/G0 phase and induction of apoptosis via caspase 3 and cleaved poly(ADP-ribose) polymerase. In vivo, nitroxoline-treated mice had no increase in tumor volume after 14 days of treatment, whereas tumor volumes doubled in control mice. Histological examination revealed 15%–20% TUNEL-positive cells in nitroxoline-treated mice, compared with ∼5% in the control group. Conclusion Nitroxoline induces apoptosis and inhibits glioma growth in vivo and in vitro. As an already FDA-approved treatment for urinary tract infections with a known safety profile, nitroxoline could move quickly into clinical trials pending confirmatory studies. PMID:25074541

  5. Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion.

    PubMed

    Morlé, A; Garrido, C; Micheau, O

    2015-01-01

    TRAIL is involved in immune tumor surveillance and is considered a promising anti-cancer agent owing to its limited side effects on healthy cells. However, some cancer cells display resistance, or become resistant to TRAIL-induced cell death. Hyperthermia can enhance sensitivity to TRAIL-induced cell death in various resistant cancer cell lines, including lung, breast, colon or prostate carcinomas. Mild heat shock treatment has been proposed to restore Fas ligand or TRAIL-induced apoptosis through c-FLIP degradation or the mitochondrial pathway. We demonstrate here that neither the mitochondria nor c-FLIP degradation are required for TRAIL-induced cell death restoration during hyperthermia. Our data provide evidence that insolubilization of c-FLIP, alone, is sufficient to enhance apoptosis induced by death receptors. Hyperthermia induced c-FLIP depletion from the cytosolic fraction, without apparent degradation, thereby preventing c-FLIP recruitment to the TRAIL DISC and allowing efficient caspase-8 cleavage and apoptosis. Hyperthermia-induced c-FLIP depletion was independent of c-FLIP DED2 FL chain assembly motif or ubiquitination-mediated c-FLIP degradation, as assessed using c-FLIP point mutants on lysine 167 and 195 or threonine 166, a phosphorylation site known to regulate ubiquitination of c-FLIP. Rather, c-FLIP depletion was associated with aggregation, because addition of glycerol not only prevented the loss of c-FLIP from the cytosol but also enabled c-FLIP recruitment within the TRAIL DISC, thus inhibiting TRAIL-induced apoptosis during hyperthermia. Altogether our results demonstrate that c-FLIP is a thermosensitive protein whose targeting by hyperthermia allows restoration of apoptosis induced by TNF ligands, including TRAIL. Our findings suggest that combining TRAIL agonists with whole-body or localized hyperthermia may be an interesting approach in cancer therapy. PMID:25675293

  6. Ursodeoxycholic acid induces apoptosis in hepatocellular carcinoma xenografts in mice

    PubMed Central

    Liu, Hui; Xu, Hong-Wei; Zhang, Yu-Zhen; Huang, Ya; Han, Guo-Qing; Liang, Tie-Jun; Wei, Li-Li; Qin, Cheng-Yong; Qin, Cheng-Kun

    2015-01-01

    AIM: To evaluate the efficacy of ursodeoxycholic acid (UDCA) as a chemotherapeutic agent for the treatment of hepatocellular carcinoma (HCC). METHODS: BALB/c nude mice were randomized into four groups 24 h before subcutaneous injection of hepatocarcinoma BEL7402 cells suspended in phosphate buffered saline (PBS) into the right flank. The control group (n = 10) was fed a standard diet while treatment groups (n = 10 each) were fed a standard daily diet supplemented with different concentrations of UDCA (30, 50 and 70 mg/kg per day) for 21 d. Tumor growth was measured once each week, and tumor volume (V) was calculated with the following equation: V = (L × W2) × 0.52, where L is the length and W is the width of the xenograft. After 21 d, mice were killed under ether anesthesia, and tumors were excised and weighed. Apoptosis was evaluated through detection of DNA fragmentation with gel electrophoresis and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Western blot analysis was performed to determine the expression of apoptosis-related proteins BAX, BCL2, APAF1, cleaved caspase-9, and cleaved caspase-3. RESULTS: UDCA suppressed tumor growth relative to controls. The mean tumor volumes were the following: control, 1090 ± 89 mm3; 30 mg/kg per day, 612 ± 46 mm3; 50 mg/kg per day, 563 ± 38 mm3; and 70 mg/kg per day, 221 ± 26 mm3. Decreased tumor volumes reached statistical significance relative to control xenografts (30 mg/kg per day, P < 0.05; 50 mg/kg per day, P < 0.05; 70 mg/kg per day, P < 0.01). Increasing concentrations of UDCA led to increased DNA fragmentation observed on gel electrophoresis and in the TUNEL assay (control, 1.6% ± 0.3%; 30 mg/kg per day, 2.9% ± 0.5%; 50 mg/kg per day, 3.15% ± 0.7%, and 70 mg/kg per day, 4.86% ± 0.9%). Western blot analysis revealed increased expression of BAX, APAF1, cleaved-caspase-9 and cleaved-caspase-3 proteins, which induce apoptosis, but decreased expression of BCL2

  7. Lead Induces Apoptosis and Histone Hyperacetylation in Rat Cardiovascular Tissues

    PubMed Central

    Xu, Li-Hui; Mu, Fang-Fang; Zhao, Jian-Hong; He, Qiang; Cao, Cui-Li; Yang, Hui; Liu, Qi; Liu, Xue-Hui; Sun, Su-Ju

    2015-01-01

    Acute and chronic lead (Pb) exposure might cause hypertension and cardiovascular diseases. The purpose of this study was to evaluate the effects of early acute exposure to Pb on the cellular morphology, apoptosis, and proliferation in rats and to elucidate the early mechanisms involved in the development of Pb-induced hypertension. Very young Sprague-Dawley rats were allowed to drink 1% Pb acetate for 12 and 40 days. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA) decreased in the tissues of the abdominal and thoracic aortas and increased in the cardiac tissue after 12 and 40 days of Pb exposure, respectively. Bax was upregulated and Bcl-2 was downregulated in vascular and cardiac tissues after 40 days of Pb exposure. In addition, an increase in caspase-3 activity was observed after 40 days of exposure to Pb. In terms of morphology, we found that the internal elastic lamina (IEL) of aorta lost the original curve and the diameter of cardiac cell was enlarged after 40 days. Furthermore, the exposure led to a marked increase in acetylated histone H3 levels in the aortas and cardiac tissue after 12 and 40 days, than that in the control group. These findings indicate that Pb might increase the level of histone acetylation and induce apoptosis in vascular and cardiac tissues. However, the mechanism involved need to be further investigated. PMID:26075388

  8. Vanadium induced ultrastructural changes and apoptosis in male germ cells.

    PubMed

    Aragón, M A; Ayala, M E; Fortoul, T I; Bizarro, P; Altamirano-Lozano, M

    2005-01-01

    Vanadium is a transition metal that is emitted to the atmosphere during combustion of fossil fuels. In the environment, vanadium occurs in the (V) oxidized form, but in the body it is found exclusively in the (IV) oxidized form. Vanadium tetraoxide is an inorganic chemical species in the (IV) oxidized form that has been shown to induce toxic effects in vitro and in vivo. The reproductive toxicity of vanadium in males was studied through monitoring germ cell apoptosis during spermatogenesis. We analyzed ultrastructural damage, and testosterone and progesterone concentrations following vanadium tetraoxide administered to male mice for 60 days. Spermatogenesis stages I-III and X-XII frequently showed apoptotic germ cells in control and treated animals; vanadium tetraoxide treatment induced an increase in the number of germ cell apoptosis in stages I-III and XII at 9.4 and 18.8 mg/kg, respectively. Although spermatogenesis is regulated by testosterone, in our study this hormone level was not modified by vanadium administration; thus, germ cell death was not related with testosterone concentration. At the ultrastructural level, we observed inclusion structures that varied as to location and content in the Sertoli and germ cells. PMID:15808796

  9. Aggregated Myocilin Induces Russell Bodies and Causes Apoptosis

    PubMed Central

    Yam, Gary Hin-Fai; Gaplovska-Kysela, Katarina; Zuber, Christian; Roth, Jürgen

    2007-01-01

    Primary open-angle glaucoma with elevated intraocular pressure is a leading cause of blindness worldwide. Mutations of myocilin are known to play a critical role in the manifestation of the disease. Misfolded mutant myocilin forms secretion-incompetent intracellular aggregates. The block of myocilin secretion was proposed to alter the extracellular matrix environment of the trabecular meshwork, with subsequent impediment of aqueous humor outflow leading to elevated intraocular pressure. However, the molecular pathogenesis of myocilin-caused glaucoma is poorly defined. In this study, we show that heteromeric complexes composed of wild-type and mutant myocilin were retained in the rough endoplasmic reticulum, aggregating to form inclusion bodies typical of Russell bodies. The presence of myocilin aggregates induced the unfolded protein response proteins BiP and phosphorylated endoplasmic reticulum-localized eukaryotic initiation factor-2α kinase (PERK) with the subsequent activation of caspases 12 and 3 and expression of C/EBP homologous protein (CHOP)/GADD153, leading to apoptosis. Our findings identify endoplasmic reticulum stress-induced apoptosis as a pathway to explain the reduction of trabecular meshwork cells in patients with myocilin-caused glaucoma. As a consequence, the phagocytotic capacity of the remaining trabecular meshwork cell population would be insufficient for effective cleaning of aqueous humor, constituting a major pathogenetic factor for the development of increased intraocular pressure in primary open-angle glaucoma. PMID:17200186

  10. Involvement of FAN in TNF-induced apoptosis.

    PubMed

    Ségui, B; Cuvillier, O; Adam-Klages, S; Garcia, V; Malagarie-Cazenave, S; Lévêque, S; Caspar-Bauguil, S; Coudert, J; Salvayre, R; Krönke, M; Levade, T

    2001-07-01

    TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF. PMID:11435466

  11. Aloe-emodin-induced apoptosis in human gastric carcinoma cells.

    PubMed

    Chen, Sheng-Hsuan; Lin, Kai-Yuan; Chang, Chun-Chao; Fang, Chia-Lang; Lin, Chih-Ping

    2007-11-01

    The purpose of this study was to investigate the anticancer effect of aloe-emodin, an anthraquinone compound present in the leaves of Aloe vera, on two distinct human gastric carcinoma cell lines, AGS and NCI-N87. We demonstrate that aloe-emodin induced cell death in a dose- and time-dependent manner. Noteworthy is that the AGS cells were generally more sensitive than the NCI-N87 cells. Aloe-emodin caused the release of apoptosis-inducing factor and cytochrome c from mitochondria, followed by the activation of caspase-3, leading to nuclear shrinkage and apoptosis. In addition, exposure to aloe-emodin suppressed the casein kinase II activity in a time-dependent manner and was accompanied by a reduced phosphorylation of Bid, a downstream substrate of casein kinase II and a pro-apoptotic molecule. These preclinical studies suggest that aloe-emodin represents a suitable and novel chemotherapeutic drug candidate for the treatment of human gastric carcinoma. PMID:17637488

  12. Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes.

    PubMed

    Fernández-Presas, Ana María; Tato, Patricia; Becker, Ingeborg; Solano, Sandra; Copitin, Natalia; Kopitin, Natalia; Berzunza, Miriam; Willms, Kaethe; Hernández, Joselin; Molinari, José Luis

    2010-05-01

    The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated. PMID:20237802

  13. Antiplatelet drugs induce apoptosis in cultured cancer cells.

    PubMed

    Chen, W H; Yin, H L; Chang, Y Y; Lan, M Y; Hsu, H Y; Liu, J S

    1997-10-01

    In order to understand if antiplatelet drugs possess direct antineoplastic property, we tested the apoptotic effect of 5 popularly marketed antiplatelet drugs in Taiwan in 6 cultured cancer cell lines (Hep 3B hepatocarcinoma, U87-MG malignant glioma, PC-3 prostate adenocarcinoma, HeLa cervical adenocarcinoma, HL-60 preleukemia and K-562 chronic myelogenous leukemia). While acetylsalicylate and flunarizine exerted no effect on these cancer cells, pentoxifyline (PTX), dipyridamole (DYA) and ticlopidine hydrochloride (T. HCl) displayed a time and dose-dependent apoptotic effect on them except for HL-60 and K-562 cells. PTX induced apoptosis in U87-MG, Hep 3B and HeLa cells, DYA in HeLa cells, while T. HCl in U87-MG, Hep 3B, PC-3 and HeLa cells. Adriamycin also provoked apoptotic effect in all 6 cell lines but neither PTX, DYA nor T. HCl acted synergy with adriamycin to HeLa cells, implicating that they may share a similar pathway for inducing apoptosis. Therefore, our results show that the antiplatelet drugs do possess antineoplastic property in vitro. A co-administration of antiplatelet drugs is noteworthy for an alternative adjunctive therapy in cancer patients. PMID:9385774

  14. Targeting Glutamine Induces Apoptosis: A Cancer Therapy Approach

    PubMed Central

    Chen, Lian; Cui, Hengmin

    2015-01-01

    Glutamine metabolism has been proved to be dysregulated in many cancer cells, and is essential for proliferation of most cancer cells, which makes glutamine an appealing target for cancer therapy. In order to be well used by cells, glutamine must be transported to cells by specific transporters and converted to glutamate by glutaminase. There are currently several drugs that target glutaminase under development or clinical trials. Also, glutamine metabolism restriction has been proved to be effective in inhibiting tumor growth both in vivo and vitro through inducing apoptosis, growth arrest and/or autophagy. Here, we review recent researches about glutamine metabolism in cancer, and cell death induced by targeting glutamine, and their potential roles in cancer therapy. PMID:26402672

  15. Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells

    SciTech Connect

    Ota, Kimiko; Nakamura, Jiro; Li, Weiguo; Kozakae, Mika; Watarai, Atsuko; Nakamura, Nobuhisa; Yasuda, Yutaka; Nakashima, Eirtaro; Naruse, Keiko; Watabe, Kazuhiko; Kato, Koichi; Oiso, Yutaka; Hamada, Yoji . E-mail: yhama@med.nagoya-u.ac.jp

    2007-05-25

    Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS.

  16. Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

    PubMed Central

    Chen, Xin; Gu, Ying; Singh, Karnika; Shang, Chaowei; Barzegar, Mansoureh; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death. PMID:25531367

  17. Maduramicin inhibits proliferation and induces apoptosis in myoblast cells.

    PubMed

    Chen, Xin; Gu, Ying; Singh, Karnika; Shang, Chaowei; Barzegar, Mansoureh; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death. PMID:25531367

  18. Host Immune Defense Peptide LL-37 Activates Caspase-Independent Apoptosis and Suppresses Colon Cancer

    PubMed Central

    Ren, Shun X.; Cheng, Alfred S.L.; To, Ka F.; Tong, Joanna H.M.; Li, May S.; Shen, Jin; Wong, Clover C.M.; Zhang, Lin; Chan, Ruby L.Y.; Wang, Xiao J.; Ng, Simon S.M.; Chiu, Lawrence C.M.; Marquez, Victor E.; Gallo, Richard L.; Chan, Francis K.L.; Yu, Jun; Sung, Joseph J.Y.; Wu, William K.K.; Cho, Chi H.

    2014-01-01

    Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin–sensitive G-protein–coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG–mediated apoptosis in colon cancer cells. PMID:23100468

  19. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner.

    PubMed

    Pellegrini, Gretel G; Morales, Cynthya C; Wallace, Taylor C; Plotkin, Lilian I; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  20. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    PubMed Central

    Pellegrini, Gretel G.; Morales, Cynthya C.; Wallace, Taylor C.; Plotkin, Lilian I.; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  1. β-Arrestin1 inhibits chemotherapy-induced intestinal stem cell apoptosis and mucositis.

    PubMed

    Zhan, Y; Xu, C; Liu, Z; Yang, Y; Tan, S; Yang, Y; Jiang, J; Liu, H; Chen, J; Wu, B

    2016-01-01

    The mechanism of chemotherapy-induced gastrointestinal (GI) syndrome (CIGIS) is still controversial, and it is unclear whether chemotherapy induces intestinal stem cell (ISC) apoptosis. β-Arrestins are regulators and mediators of G protein-coupled receptor signaling in cell apoptosis, division and growth. In this study, we aimed to investigate whether chemotherapy induces ISC apoptosis to contribute to mucositis in CIGIS and whether β-arrestin1 (β-arr1) is involved in this apoptosis. Different chemotherapeutic agents were used to generate a CIGIS model. Lgr5-EGFP-IRES-creERT2(+/-) knock-in mice were used as a CIGIS model to investigate ISC apoptosis. β-arr1 knockout mice were used to determine whether β-arr1 is involved in the apoptosis in CIGIS. Intestinal histology was performed, the ISC apoptosis was analyzed and the mucosal barrier was examined. The effects of β-arr1 in apoptosis were investigated in the samples from humans and mice as well as in cell lines. Here, we demonstrate that chemotherapy induced intestinal mucositis by promoting crypt cell apoptosis, especially in Lgr5+ stem cells and Paneth cells but not in goblet cells, epithelial cells or vascular endothelial cells. Furthermore, β-arr1 deficiency exacerbated the Lgr5+ stem cell apoptosis, but not Paneth cell apoptosis, in CIGIS. In addition, the data showed that β-arr1 reduced the chemotherapy-induced Lgr5+ stem cell apoptosis by inhibiting endoplasmic reticulum stress-mediated mitochondrial apoptotic signaling. Our study indicates that β-arr1 inhibits chemotherapy-induced ISC apoptosis to alleviate intestinal mucositis in CIGIS. PMID:27195676

  2. β-Arrestin1 inhibits chemotherapy-induced intestinal stem cell apoptosis and mucositis

    PubMed Central

    Zhan, Y; Xu, C; Liu, Z; Yang, Y; Tan, S; Yang, Y; Jiang, J; Liu, H; Chen, J; Wu, B

    2016-01-01

    The mechanism of chemotherapy-induced gastrointestinal (GI) syndrome (CIGIS) is still controversial, and it is unclear whether chemotherapy induces intestinal stem cell (ISC) apoptosis. β-Arrestins are regulators and mediators of G protein-coupled receptor signaling in cell apoptosis, division and growth. In this study, we aimed to investigate whether chemotherapy induces ISC apoptosis to contribute to mucositis in CIGIS and whether β-arrestin1 (β-arr1) is involved in this apoptosis. Different chemotherapeutic agents were used to generate a CIGIS model. Lgr5-EGFP-IRES-creERT2+/− knock-in mice were used as a CIGIS model to investigate ISC apoptosis. β-arr1 knockout mice were used to determine whether β-arr1 is involved in the apoptosis in CIGIS. Intestinal histology was performed, the ISC apoptosis was analyzed and the mucosal barrier was examined. The effects of β-arr1 in apoptosis were investigated in the samples from humans and mice as well as in cell lines. Here, we demonstrate that chemotherapy induced intestinal mucositis by promoting crypt cell apoptosis, especially in Lgr5+ stem cells and Paneth cells but not in goblet cells, epithelial cells or vascular endothelial cells. Furthermore, β-arr1 deficiency exacerbated the Lgr5+ stem cell apoptosis, but not Paneth cell apoptosis, in CIGIS. In addition, the data showed that β-arr1 reduced the chemotherapy-induced Lgr5+ stem cell apoptosis by inhibiting endoplasmic reticulum stress-mediated mitochondrial apoptotic signaling. Our study indicates that β-arr1 inhibits chemotherapy-induced ISC apoptosis to alleviate intestinal mucositis in CIGIS. PMID:27195676

  3. Statins induce apoptosis in rat and human myotube cultures by inhibiting protein geranylgeranylation but not ubiquinone.

    PubMed

    Johnson, Timothy E; Zhang, Xiaohua; Bleicher, Kimberly B; Dysart, Gary; Loughlin, Amy F; Schaefer, William H; Umbenhauer, Diane R

    2004-11-01

    Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential

  4. Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

    SciTech Connect

    Geel, Tessa M.; Meiss, Gregor; Gun, Bernardina T. van der; Kroesen, Bart Jan; Leij, Lou F. de; Zaremba, Mindaugas; Silanskas, Arunas; Kokkinidis, Michael; Ruiters, Marcel H.; McLaughlin, Pamela M.; Rots, Marianne G.

    2009-09-10

    TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18{sup Registered-Sign }:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

  5. Fumonisin and beauvericin induce apoptosis in turkey peripheral blood lymphocytes.

    PubMed

    Dombrink-Kurtzman, Mary Ann

    2003-01-01

    Fumonisins, a family of mycotoxins produced by Fusarium verticillioides (synonym Fusarium moniliforme Sheldon) and F. proliferatum, have been associated with various deleterious effects in different animal species. Serological, hematological and pathological effects and mortality have previously been observed in broiler chicks fed F. proliferatum culture material containing known concentrations of fumonisin, moniliformin and beauvericin. Turkey peripheral blood lymphocytes were exposed in vitro for 72 hours to fumonisin B1 (FB1), fumonisin B2 (FB2), hydrolyzed fumonisin B1 (HFB1), moniliformin and tricarballylic acid (TCA) (0.01-25 microg/ml). A decrease in cell proliferation, as determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] bioassay, occurred in the order: FB2 > FB1 > HFB1, with IC50 = 0.6 microM, 1 microM and 10 microM, respectively. Internucleosomal DNA fragmentation and morphological features characteristic of apoptosis were observed following exposure to fumonisin B1 and beauvericin; cytoplasmic condensation and membrane blebbing were seen by light microscopy. Tricarballylic acid and moniliformin did not interfere with cell proliferation. Results suggested that fumonisin B1 and beauvericin may affect immune functions by suppressing proliferation and inducing apoptosis of lymphocytes. PMID:14682463

  6. Apoptosis-Inducing Factor: Structure, Function, and Redox Regulation

    PubMed Central

    2011-01-01

    Abstract Apoptosis-inducing factor (AIF) is a flavin adenine dinucleotide-containing, NADH-dependent oxidoreductase residing in the mitochondrial intermembrane space whose specific enzymatic activity remains unknown. Upon an apoptotic insult, AIF undergoes proteolysis and translocates to the nucleus, where it triggers chromatin condensation and large-scale DNA degradation in a caspase-independent manner. Besides playing a key role in execution of caspase-independent cell death, AIF has emerged as a protein critical for cell survival. Analysis of in vivo phenotypes associated with AIF deficiency and defects, and identification of its mitochondrial, cytoplasmic, and nuclear partners revealed the complexity and multilevel regulation of AIF-mediated signal transduction and suggested an important role of AIF in the maintenance of mitochondrial morphology and energy metabolism. The redox activity of AIF is essential for optimal oxidative phosphorylation. Additionally, the protein is proposed to regulate the respiratory chain indirectly, through assembly and/or stabilization of complexes I and III. This review discusses accumulated data with respect to the AIF structure and outlines evidence that supports the prevalent mechanistic view on the apoptogenic actions of the flavoprotein, as well as the emerging concept of AIF as a redox sensor capable of linking NAD(H)-dependent metabolic pathways to apoptosis. Antioxid. Redox Signal. 14, 2545–2579. PMID:20868295

  7. Biomarkers of breast cancer apoptosis induced by chemotherapy and TRAIL.

    PubMed

    Leong, Sharon; McKay, Matthew J; Christopherson, Richard I; Baxter, Robert C

    2012-02-01

    Treatment of breast cancer is complex and challenging due to the heterogeneity of the disease. To avoid significant toxicity and adverse side-effects of chemotherapy in patients who respond poorly, biomarkers predicting therapeutic response are essential. This study has utilized a proteomic approach integrating 2D-DIGE, LC-MS/MS, and bioinformatics to analyze the proteome of breast cancer (ZR-75-1 and MDA-MB-231) and breast epithelial (MCF-10A) cell lines induced to undergo apoptosis using a combination of doxorubicin and TRAIL administered in sequence (Dox-TRAIL). Apoptosis induction was confirmed using a caspase-3 activity assay. Comparative proteomic analysis between whole cell lysates of Dox-TRAIL and control samples revealed 56 differentially expressed spots (≥2-fold change and p < 0.05) common to at least two cell lines. Of these, 19 proteins were identified yielding 11 unique protein identities: CFL1, EIF5A, HNRNPK, KRT8, KRT18, LMNA, MYH9, NACA, RPLP0, RPLP2, and RAD23B. A subset of the identified proteins was validated by selected reaction monitoring (SRM) and Western blotting. Pathway analysis revealed that the differentially abundant proteins were associated with cell death, cellular organization, integrin-linked kinase signaling, and actin cytoskeleton signaling pathways. The 2D-DIGE analysis has yielded candidate biomarkers of response to treatment in breast cancer cell models. Their clinical utility will depend on validation using patient breast biopsies pre- and post-treatment with anticancer drugs. PMID:22133146

  8. Porcine JAB1 significantly enhances apoptosis induced by staurosporine

    PubMed Central

    Jiang, P; Wang, J; Kang, Z; Li, D; Zhang, D

    2013-01-01

    c-Jun activation domain-binding protein-1 (JAB1), also known as the subunit 5 of the COP9 signalosome, is a multifunctional protein that regulates cell proliferation, apoptosis and oncogenesis by interacting with and subsequently degrading a large number of proteins. Although human JAB1 (hJAB1) has been studied for a long time, studies on porcine JAB1 (pJAB1) have never been reported. In the present study, we cloned and characterized the pJAB1 gene. The genomic structure of the pJAB1 gene was determined. The open-reading frame of pJAB1 encoded 334 amino acids. The deduced amino acid sequence was highly similar to homologs in other species. Furthermore, the tertiary structure analysis and phylogenetic analysis indicated that JAB1 was highly conservative among species. pJAB1 may interact with several proteins according to protein–protein interactions analysis. In addition, pJAB1 was found to be universally expressed in porcine tissues. Subcellular localization analysis showed that GFP–pJAB1 fusion protein distributed specifically in the cytoplasm. Flow cytometric analysis proved that pJAB1 significantly enhanced apoptosis induced by staurosporine, which at least partially depended on the activation of caspase-9 and caspase-3. This study is useful for understanding the function of pJAB1 and offers a potential molecular model for the investigation of diseases related to hJAB1. PMID:24091666

  9. PDT-induced apoptosis in arterial smooth muscles cells

    NASA Astrophysics Data System (ADS)

    Nyamekye, Isaac; Renick, R.; Gilbert, C.; McEwan, Jean R.; Evan, G.; Bishop, Christopher C. R.; Bown, Stephen G.

    1995-03-01

    PDT kills smooth muscle cells (SMC) in vivo and thus prevents intimal hyperplasia after angioplasty. It causes little inflammation and structural integrity of the artery is not compromised. We have studied the process of the SMC death in vitro. Cultured rat SMC (cell line sv40 ATCC) were sensitized with aluminum disulphonated phthalocyanine (AlS2Pc), and then irradiated with 675 nm laser light (2.5 J/cm2). Controls were studied using only sensitizer or laser for treatment. The cells were incubated and the dying process observed with a time lapse video and microscope system. PDT caused a characteristic pattern of death. Cells lost contact with neighbors, shrank, and showed hyperactivity and membrane ruffling. The cells imploded into active and condensed membrane bound vesicles which were terminally reduced to residual bodies. These are the morphological changes of apoptosis. The control cells which were given AlS2Pc alone or laser alone showed no death. PDT induced cultured arterial SMC death by apoptosis rather than necrosis. An apoptotic mechanism of cell death in vivo would explain the relative lack of inflammation and local tissue destruction in the face of massive death.

  10. N-Acetylcholinesterase-Induced Apoptosis in Alzheimer's Disease

    PubMed Central

    Toiber, Debra; Berson, Amit; Greenberg, David; Melamed-Book, Naomi; Diamant, Sophia; Soreq, Hermona

    2008-01-01

    Background Alzheimer's disease (AD) involves loss of cholinergic neurons and Tau protein hyper-phosphorylation. Here, we report that overexpression of an N-terminally extended “synaptic” acetylcholinesterase variant, N-AChE-S is causally involved in both these phenomena. Methodology and Principal Findings In transfected primary brain cultures, N-AChE-S induced cell death, morphological impairments and caspase 3 activation. Rapid internalization of fluorescently labeled fasciculin-2 to N-AChE-S transfected cells indicated membranal localization. In cultured cell lines, N-AChE-S transfection activated the Tau kinase GSK3, induced Tau hyper-phosphorylation and caused apoptosis. N-AChE-S-induced cell death was suppressible by inhibiting GSK3 or caspases, by enforced overexpression of the anti-apoptotic Bcl2 proteins, or by AChE inhibition or silencing. Moreover, inherent N-AChE-S was upregulated by stressors inducing protein misfolding and calcium imbalances, both characteristic of AD; and in cortical tissues from AD patients, N-AChE-S overexpression coincides with Tau hyper-phosphorylation. Conclusions Together, these findings attribute an apoptogenic role to N-AChE-S and outline a potential value to AChE inhibitor therapeutics in early AD. PMID:18769671

  11. Apoptosis induced by weisiensin B isolated from Rabdosia weisiensis C.Y. Wu in K562.

    PubMed

    Liu, Guo-An; Chang, Jin-Chun; Feng, Xiao-Lu; Ding, Lan

    2015-04-01

    The ent-kaurane diterpenoid weisinensis B shows significant cytotoxicity to human chronic myeloid leukemia K562 cells. It inhibits cell growth at low concentration and kills cells at high concentration. The compound induced cell apoptosis and necrosis mainly associated with G2/M phase cell cycle arrest and the ROS generation is the early event in weisiensin B induced cell apoptosis. PMID:26012257

  12. Zinc protects human kidney cells from depleted uranium-induced apoptosis.

    PubMed

    Hao, Yuhui; Ren, Jiong; Liu, Cong; Li, Hong; Liu, Jing; Yang, Zhangyou; Li, Rong; Su, Yongping

    2014-03-01

    Depleted uranium (DU) is a weak radioactive heavy metal, and zinc (Zn) is an effective antidote to heavy metal poisoning. However, the effect of Zn on DU-induced cytotoxicity and apoptosis is not completely understood. The purpose of this study was to evaluate the effect of Zn on DU-induced cell apoptosis in human kidney cells (HK-2) and explore its molecular mechanism. Pre-treatment with Zn significantly inhibited DU-induced apoptosis. It reduced the formation of reactive oxygen species in the cells, increased the catalase (CAT) and glutathione (GSH) concentrations, suppressed the DU-induced soluble Fas receptor (sFasR) and soluble Fas ligand (sFasL) overexpression, suppressed the release of cytochrome c and apoptosis inhibitor factor (AIF) from mitochondria to cytoplasm, inhibited the activation of caspase-9, caspase-8 and caspase-3, and induced metallothionein (MT) expression. Furthermore, exogenous MT effectively inhibited DU-induced cell apoptosis. In conclusion, mitochondrial and FasR-mediated apoptosis pathways contribute to DU-induced apoptosis in HK-2 cells. Through independent mechanisms, such as indirect antioxidant effects, inhibition of the activation of caspase-9, caspase-8 and caspase-3, and induction of MT expression, Zn inhibits DU-induced apoptosis. PMID:24330236

  13. Modulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by chemotherapy in thyroid cancer cell lines.

    PubMed

    Park, Jin-Woo; Wong, Mariwil G; Lobo, Margaret; Hyun, William C; Duh, Quan-Yang; Clark, Orlo H

    2003-12-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many human cancer cells but not in normal cells. Thyroid cancer cells, however, appear to be relatively resistant to TRAIL-induced apoptosis. We therefore investigated the effect of chemotherapy on TRAIL-induced apoptosis in thyroid cancer cells. We used six thyroid cancer cell lines: TPC-1, FTC-133, FTC-236, FTC-238, XTC-1, and ARO82-1. We used flow cytometry to measure apoptosis, dimethyl-thiazol-diphenyltetrazolium bromide (MTT) assay to measure antiproliferation effects and Western blot to determine the expression of Bcl family proteins. Troglitazone, paclitaxel, geldanamycin, and cycloheximide were used for pretreatment. We used the Student's t test and analysis of variance (ANOVA) for statistical analysis. All thyroid cancer cell lines, except the TPC-1 cell line, were resistant to TRAIL, and growth inhibition was less than 20% at concentration of 800 ng/mL of TRAIL. In both TPC-1 (TRAIL-sensitive) and FTC-133 (TRAIL-resistant) thyroid cancer cell lines, pretreatment with troglitazone, cycloheximide, and paclitaxel enhanced TRAIL-induced cell death significantly but pretreatment with geldanamycin did not. There were no significant changes in Bcl-2, Bcl-xl, and Bax protein expression after troglitazone treatment. In conclusion, TRAIL in combination with troglitazone, paclitaxel, and cycloheximide induces apoptosis in thyroid cancer cells at suboptimal concentrations that cannot be achieved using TRAIL alone. PMID:14751030

  14. Evidence that tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits angiogenesis by inducing vascular endothelial cell apoptosis

    SciTech Connect

    Chen, Pei-Lin; Easton, Alexander S.

    2010-01-01

    Tumor necrosis factor (TNF) and its related ligands TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL) play roles in the regulation of vascular responses, but their effect on the formation of new blood vessels (angiogenesis) is unclear. Therefore, we have examined the effects of these ligands on angiogenesis modeled with primary cultures of human umbilical vein endothelial cells (HUVEC). To examine angiogenesis in the context of the central nervous system, we have also modeled cerebral angiogenesis with the human brain endothelial cell line hCMEC/D3. Parameters studied were bromodeoxyuridine (BrdU) incorporation and cell number (MTT) assay (to assess endothelial proliferation), scratch assay (migration) and networks on Matrigel (tube formation). In our hands, neither TRAIL nor FasL (1, 10, and 100 ng/ml) had an effect on parameters of angiogenesis in the HUVEC model. In hCMEC/D3 cells by contrast, TRAIL inhibited all parameters (10-100 ng/ml, 24 h). This was due to apoptosis, since its action was blocked by the pan-caspase inhibitor zVADfmk (5 x 10{sup -5} mol/l) and TRAIL increased caspase-3 activity 1 h after application. However FasL (100 ng/ml) increased BrdU uptake without other effects. We conclude that TRAIL has different effects on in vitro angiogenesis depending on which model is used, but that FasL is generally ineffective when applied in vitro. The data suggest that TRAIL primarily influences angiogenesis by the induction of vascular endothelial apoptosis, leading to vessel regression.

  15. Geniposide prevents rotenone-induced apoptosis in primary cultured neurons

    PubMed Central

    Li, Lin; Zhao, Juan; Liu, Ke; Li, Guang-lai; Han, Yan-qing; Liu, Yue-ze

    2015-01-01

    Geniposide, a monomer extracted from gardenia and widely used in Chinese medicine, is a novel agonist at the glucagon-like peptide-1 receptor. This receptor is involved in neuroprotection. In the present study, we sought to identify an anti-apoptotic mechanism for the treatment of neurodegenerative diseases. Primary cultured neurons were treated with different concentrations of rotenone for 48 hours. Morphological observation, cell counting kit-8 assay, lactate dehydrogenase detection and western blot assay demonstrated that 0.5 nM rotenone increased lactate dehydrogenase release, decreased the expression of procaspase-3 and Bcl-2, and increased cleaved caspase-3 expression in normal neurons. All these effects were prevented by geniposide. Our results indicate that geniposide diminished rotenone-induced injury in primary neurons by suppressing apoptosis. This may be one of the molecular mechanisms underlying the efficacy of geniposide in the treatment of neurodegenerative diseases. PMID:26692859

  16. 5-Ene-4-thiazolidinones induce apoptosis in mammalian leukemia cells.

    PubMed

    Senkiv, Julia; Finiuk, Nataliya; Kaminskyy, Danylo; Havrylyuk, Dmytro; Wojtyra, Magdalena; Kril, Iryna; Gzella, Andrzej; Stoika, Rostyslav; Lesyk, Roman

    2016-07-19

    The article presents the synthesis of 5-ene-4-thiazolidinone derivatives with pyrazole core linked by enamine group. The structure and purity of compounds were confirmed by analytical and spectral data including X-ray analysis. Target compounds were screened for their anticancer activity and selective antileukemic action was confirmed. 5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one (compound 1) was selected as most active agent against HL-60 and HL-60/ADR cell lines; IC50 = 118 nM/HL-60 with low toxicity towards pseudonormal cells. The mitochondria-depended apoptosis was identified as the main mode of 1 action. Moreover compound's effect induces G0/G1 arrest of the treated cells and causes inhibition of cell division and is related with activation of ROS production. PMID:27089210

  17. Docosahexaenoic Acid Induces Apoptosis in Primary Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Gyan, Emmanuel; Tournilhac, Olivier; Halty, Christelle; Veyrat-Masson, Richard; Akil, Saïda; Berger, Marc; Hérault, Olivier; Callanan, Mary; Bay, Jacques-Olivier

    2015-01-01

    Chronic lymphocytic leukemia is an indolent disorder with an increased infectious risk remaining one of the main causes of death. Development of therapies with higher safety profile is thus a challenging issue. Docosahexaenoic acid (DHA, 22:6) is an omega-3 fatty acid, a natural compound of normal cells, and has been shown to display antitumor potency in cancer. We evaluated the potential in vitro effect of DHA in primary CLL cells. DHA induces high level of in vitro apoptosis compared to oleic acid in a dose-dependent and time-dependent manner. Estimation of IC50 was only of 4.813 µM, which appears lower than those reported in solid cancers. DHA is highly active on CLL cells in vitro. This observation provides a rationale for further studies aiming to understand its mechanisms of action and its potent in vivo activity. PMID:26734128

  18. Transcriptional inhibition of p21{sup WAF1/CIP1} gene (CDKN1) expression by survivin is at least partially p53-dependent: Evidence for survivin acting as a transcription factor or co-factor

    SciTech Connect

    Tang, Lei; Ling, Xiang; Liu, Wensheng; Das, Gokul M.; Li, Fengzhi

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Survivin inhibits the expression of p21 protein, mRNA and promoter activity. Black-Right-Pointing-Pointer Survivin neutralizes p53-induced p21 expression and promoter activity. Black-Right-Pointing-Pointer Survivin physically interacts with p53 in cancer cells. Black-Right-Pointing-Pointer Genetic silencing of endogenous survivin upregulates p21 in p53 wild type cancer cells. Black-Right-Pointing-Pointer Both p53 and survivin interacts on the two p53-binding sites in the p21 promoter. -- Abstract: Growing evidence suggests a role for the antiapoptotic protein survivin in promotion of cancer cell G1/S transition and proliferation. However, the underlying mechanism is unclear. Further, although upregulation of p21{sup WAF1/CIP1} by p53 plays an important role in p53-mediated cell G1 arrests in response to various distresses, it is unknown whether survivin plays a role in the regulation of p21{sup WAF1/CIP1} expression. Here, we report that exogenous expression of survivin in p53-wild type MCF-7 breast cancer cells inhibits the expression of p21{sup WAF1/CIP1} protein, mRNA and promoter activity, while the survivin C84A mutant and antisense failed to do so. Cotransfection experiments in the p53 mutant H1650 lung cancer cell line showed that survivin neutralizes p53-induced p21{sup WAF1/CIP1} expression and promoter activity. Importantly, genetically silencing of endogenous survivin using lentiviral survivin shRNA also enhances endogenous p21 in p53 wild type cancer cells, suggesting the physiological relevance of the fining. We further demonstrated that both p53 and survivin interacts on the two p53-binding sites in the p21{sup WAF1/CIP1} promoter (-2313 to -2212; -1452 to -1310), and survivin physically interacts with p53 in cancer cells. Together, we propose that survivin may act as a transcription factor or cofactor to interact with p53 on the p21{sup WAF1/CIP1} promoter leading to the inhibition of p21{sup WAF1/CIP1

  19. Toll-like Receptor 9 Can be Activated by Endogenous Mitochondrial DNA to Induce Podocyte Apoptosis

    PubMed Central

    Bao, Wenduona; Xia, Hong; Liang, Yaojun; Ye, Yuting; Lu, Yuqiu; Xu, Xiaodong; Duan, Aiping; He, Jing; Chen, Zhaohong; Wu, Yan; Wang, Xia; Zheng, Chunxia; Liu, Zhihong; Shi, Shaolin

    2016-01-01

    Toll-like receptor 9 (TLR9) senses bacterial DNA characteristic of unmethylated CpG motifs to induce innate immune response. TLR9 is de novo expressed in podocytes of some patients with glomerular diseases, but its role in podocyte injury remains undetermined. Since TLR9 activates p38 MAPK and NFkB that are known to mediate podocyte apoptosis, we hypothesized that TLR9 induces podocyte apoptosis in glomerular diseases. We treated immortalized podocytes with puromycin aminonucleosides (PAN) and observed podocyte apoptosis, accompanied by TLR9 upregulation. Prevention of TLR9 upregulation by siRNA significantly attenuated NFκB p65 or p38 activity and apoptosis, demonstrating that TLR9 mediates podocyte apoptosis. We next showed that endogenous mitochondrial DNA (mtDNA), whose CpG motifs are also unmethylated, is the ligand for TLR9, because PAN induced mtDNA accumulation in endolysosomes where TLR9 is localized, overexpression of endolysosomal DNase 2 attenuated PAN-induced p38 or p65 activity and podocyte apoptosis, and DNase 2 silencing was sufficient to activate p38 or p65 and induce apoptosis. In PAN-treated rats, TLR9 was upregulated in the podocytes, accompanied by increase of apoptosis markers. Thus, de novo expressed TLR9 may utilize endogenous mtDNA as the ligand to facilitate podocyte apoptosis, a novel mechanism underlying podocyte injury in glomerular diseases. PMID:26934958

  20. Apoptosis and proliferation of oligodendrocyte progenitor cells in the irradiated rodent spinal cord

    SciTech Connect

    Atkinson, Shelley L.; Li Yuqing; Wong, C. Shun . E-mail: shun.wong@sw.ca

    2005-06-01

    Purpose: Oligodendrocytes undergo early apoptosis after irradiation. The aim of this study was to determine the relationship between oligodendroglial apoptosis and proliferation of oligodendrocyte progenitor cells (OPC) in the irradiated central nervous system. Methods and Materials: Adult rats and p53 transgenic mice were given single doses of 2 Gy, 8 Gy, or 22 Gy to the cervical spinal cord. Apoptosis was assessed using TUNEL (Tdt-mediated dUTP terminal nick-end labeling) staining or by examining nuclear morphology. Oligodendrocyte progenitor cells were identified with an NG2 antibody or by in situ hybridization for platelet-derived growth factor receptor {alpha}. Proliferation of OPC was assessed by in vivo bromodeoxyuridine (BrdU) labeling and subsequent immunohistochemistry. Because radiation-induced apoptosis of oligodendroglial cells is p53 dependent, p53 transgenic mice were used to study the relationship between apoptosis and cell proliferation. Results: Oligodendrocyte progenitor cells underwent apoptosis within 24 h of irradiation in the rat. That did not result in a change in OPC density at 24 h. Oligodendrocyte progenitor cell density was significantly reduced by 2-4 weeks, but showed recovery by 6 weeks after irradiation. An increase in BrdU-labeled cells was observed at 2 weeks after 8 Gy or 22 Gy, and proliferating cells in the rat spinal cord were immunoreactive for NG2. The mouse spinal cord showed a similar early cell proliferation after irradiation. No difference was observed in the proliferation response in the spinal cord of p53 -/- mice compared with wild type animals. Conclusions: Oligodendroglial cells undergo early apoptosis and OPC undergo early proliferation after ionizing radiation. However, apoptosis is not likely to be the trigger for early proliferation of OPC in the irradiated central nervous system.

  1. Puma cooperates with Bim, the rate-limiting BH3-only protein in cell death during lymphocyte development, in apoptosis induction

    PubMed Central

    Erlacher, Miriam; Labi, Verena; Manzl, Claudia; Böck, Günther; Tzankov, Alexandar; Häcker, Georg; Michalak, Ewa; Strasser, Andreas; Villunger, Andreas

    2006-01-01

    The physiological role of B cell lymphoma 2 (Bcl-2) homology 3–only proteins has been investigated in mice lacking the individual genes identifying rate-limiting roles for Bim (Bcl-2–interacting mediator of cell death) and Puma (p53–up-regulated modulator of apoptosis) in apoptosis induction. The loss of Bim protects lymphocytes from apoptosis induced by cytokine deprivation and deregulated Ca++ flux and interferes with the deletion of autoreactive lymphocytes and the shutdown of immune responses. In contrast, Puma is considered the key mediator of p53-induced apoptosis. To investigate the hypothesis that Bim and Puma have overlapping functions, we generated mice lacking both genes and found that bim−/−/puma−/− animals develop multiple postnatal defects that are not observed in the single knockout mice. Most strikingly, hyperplasia of lymphatic organs is comparable with that observed in mice overexpressing Bcl-2 in all hemopoietic cells exceeding the hyperplasia observed in bim−/− mice. Bim and Puma also have clearly overlapping functions in p53-dependent and -independent apoptosis. Their combined loss promotes spontaneous tumorigenesis, causing the malignancies observed in Bcl-2 transgenic mice, but does not exacerbate the autoimmunity observed in the absence of Bim. PMID:17178918

  2. Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

    SciTech Connect

    Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.; Bilimoria, Shaen L.

    2008-01-20

    Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV{sub XS}; 400 {mu}g/ml), UV-irradiated virus (CIV{sub UV}; 10 {mu}g/ml) and CVPE (CIV protein extract; 10 {mu}g/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 {mu}g/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV{sub UV} or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV{sub UV} particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV{sub UV}, CIV{sub XS} or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae

  3. Tanshinone IIA blocks dexamethasone-induced apoptosis in osteoblasts through inhibiting Nox4-derived ROS production

    PubMed Central

    Li, Jia; He, Chongru; Tong, Wenwen; Zou, Yuming; Li, Dahe; Zhang, Chen; Xu, Weidong

    2015-01-01

    Apoptosis of osteoblasts caused by glucocorticoids has been identified as an important contributor to the development of osteoporosis. Tanshinone IIA (Tan), an active ingredient extracted from the rhizome of the Salvia miltiorrhiza Bunge (Danshen), has been reported to cast positive effects on osteoporosis. However, the precise mechanisms accounting this action remain elusive. In this study, by using osteoblastic MC3T3-E1 cells as a model, we confirmed the protective effects of Tan against dexamethasone (Dex)-induced cell apoptosis and further clarified its molecular mechanism of action. Our results showed that treatment with Dex caused cell injury, increased cytosol cytochrome c level and Nox expression, induced apoptosis in caspase-9-dependent manner, and enhanced reactive oxygen species (ROS) production. Tan attenuated these deleterious consequence triggered by Dex. Moreover, Dex-induced ROS production and cell injury were inhibited by antioxidant, NADPH oxidases inhibitors, Nox4 inhibitor, and Nox4 small interfering RNA (siRNA). Overexpression of Nox4 almost abolished the inhibitory effect of Tan on Dex-induced cell injury and apoptosis. The results also demonstrated significant involvement of Nox4 in the Dex-induced apoptosis. Nox4-derived ROS led to apoptosis through activation of intrinsic mitochondrial pathway. Additionally, we evidenced that Tan reversed Dex-induced apoptosis via inactivation of Nox4. The present findings suggest that inhibition of Nox4 may be a novel therapeutic approach of Tan to prevent against glucocorticoids-induced osteoblasts apoptosis and osteoporosis. PMID:26722597

  4. Cyclin C mediates stress-induced mitochondrial fission and apoptosis

    PubMed Central

    Wang, Kun; Yan, Ruilan; Cooper, Katrina F.; Strich, Randy

    2015-01-01

    Mitochondria are dynamic organelles that undergo constant fission and fusion cycles. In response to cellular damage, this balance is shifted dramatically toward fission. Cyclin C–Cdk8 kinase regulates transcription of diverse gene sets. Using knockout mouse embryonic fibroblasts (MEFs), we demonstrate that cyclin C directs the extensive mitochondrial scission induced by the anticancer drug cisplatin or oxidative stress. This activity is independent of transcriptional regulation, as Cdk8 is not required for this activity. Furthermore, adding purified cyclin C to unstressed permeabilized MEF cultures induced complete mitochondrial fragmentation that was dependent on the fission factors Drp1 and Mff. To regulate fission, a portion of cyclin C translocates from the nucleus to the cytoplasm, where it associates with Drp1 and is required for its enhanced mitochondrial activity in oxidatively stressed cells. In addition, although HeLa cells regulate cyclin C in a manner similar to MEF cells, U2OS osteosarcoma cultures display constitutively cytoplasmic cyclin C and semifragmented mitochondria. Finally, cyclin C, but not Cdk8, is required for loss of mitochondrial outer membrane permeability and apoptosis in cells treated with cisplatin. In conclusion, this study suggests that cyclin C connects stress-induced mitochondrial hyperfission and programmed cell death in mammalian cells. PMID:25609094

  5. A component of the mir-17-92 polycistronic oncomir promotes oncogene-dependent apoptosis

    PubMed Central

    Olive, Virginie; Sabio, Erich; Bennett, Margaux J; De Jong, Caitlin S; Biton, Anne; McGann, James C; Greaney, Samantha K; Sodir, Nicole M; Zhou, Alicia Y; Balakrishnan, Asha; Foth, Mona; Luftig, Micah A; Goga, Andrei; Speed, Terence P; Xuan, Zhenyu; Evan, Gerard I; Wan, Ying; Minella, Alex C; He, Lin

    2013-01-01

    mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eμ-myc Burkitt’s lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk. DOI: http://dx.doi.org/10.7554/eLife.00822.001 PMID:24137534

  6. Valproic Acid and Other HDAC Inhibitors Induce Microglial Apoptosis and Attenuate Lipopolysaccharide- induced Dopaminergic Neurotoxicity

    PubMed Central

    Chen, Po See; Wang, Chao-Chuan; Bortner, Carl D.; Peng, Giia-Sheun; Wu, Xuefei; Pang, Hao; Lu, Ru-Band; Gean, Po-Wu; Chuang, De-Maw; Hong, Jau-Shyong

    2009-01-01

    Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, has been shown to be an inhibitor of histone deacetylase (HDAC). Our previous study has demonstrated that VPA pretreatment reduces lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity through the inhibition of microglia over-activation. The aim of this study was to determine the mechanism underlying VPA-induced attenuation of microglia over-activation. Other HDAC inhibitors (HDACIs) were compared with VPA for their effects on microglial activity. We found that VPA induced apoptosis of microglia cells in a time and concentration-dependent manner. VPA-treated microglial cells showed typical apoptotic hallmarks including phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Further studies revealed that trichostatin A (TSA) and sodium butyrate (SB), two structurally dissimilar HDACIs, also induced microglial apoptosis. The apoptosis of microglia was accompanied by the disruption of mitochondrial membrane potential and the enhancement of acetylation levels of the histone H3 protein. Moreover, pretreatment with SB or TSA caused a robust decrease in LPS-induced pro-inflammatory responses and protected DA neurons from damage in mesencephalic neuron-glia cultures. Taken together, our results shed light on a novel mechanism whereby HDACIs induce neuroprotection and underscore the potential utility of HDACIs in preventing inflammation-related neurodegenerative disorders such as Parkinson’s disease. PMID:17850978

  7. Transcriptional inhibition of p21WAF1/CIP1 gene (CDKN1) expression by survivin is at least partially p53-dependent: evidence for survivin acting as a transcription factor or co-factor.

    PubMed

    Tang, Lei; Ling, Xiang; Liu, Wensheng; Das, Gokul M; Li, Fengzhi

    2012-05-01

    Growing evidence suggests a role for the antiapoptotic protein survivin in promotion of cancer cell G1/S transition and proliferation. However, the underlying mechanism is unclear. Further, although upregulation of p21(WAF1/CIP1) by p53 plays an important role in p53-mediated cell G1 arrests in response to various distresses, it is unknown whether survivin plays a role in the regulation of p21(WAF1/CIP1) expression. Here, we report that exogenous expression of survivin in p53-wild type MCF-7 breast cancer cells inhibits the expression of p21(WAF1/CIP1) protein, mRNA and promoter activity, while the survivin C84A mutant and antisense failed to do so. Cotransfection experiments in the p53 mutant H1650 lung cancer cell line showed that survivin neutralizes p53-induced p21(WAF1/CIP1) expression and promoter activity. Importantly, genetically silencing of endogenous survivin using lentiviral survivin shRNA also enhances endogenous p21 in p53 wild type cancer cells, suggesting the physiological relevance of the fining. We further demonstrated that both p53 and survivin interacts on the two p53-binding sites in the p21(WAF1/CIP1) promoter (-2313 to -2212; -1452 to -1310), and survivin physically interacts with p53 in cancer cells. Together, we propose that survivin may act as a transcription factor or cofactor to interact with p53 on the p21(WAF1/CIP1) promoter leading to the inhibition of p21(WAF1/CIP1) expression at least in part by neutralizing p53-mediated transcriptional activation of the p21 gene. PMID:22503977

  8. Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1

    PubMed Central

    Rahmutulla, Bahityar; Tanaka, Nobuko; Ishige, Takayuki; Satoh, Mamoru; Hoshino, Tyuji; Miyagi, Satoru; Mori, Takeshi; Itoga, Sakae; Shimada, Hideaki; Tomonaga, Takeshi; Kito, Minoru; Nakajima-Takagi, Yaeko; Kubo, Shuji; Nakaseko, Chiaki; Hatano, Masahiko; Miki, Takashi; Matsuo, Masafumi; Fukuyo, Masaki; Kaneda, Atsushi; Iwama, Atsushi; Nomura, Fumio

    2015-01-01

    FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR+/−) C57BL6 mice were generated. FIR complete knockout (FIR−/−) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR+/− mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR+/−TP53−/− generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR+/−TP53−/− compared with that in FIR+/+TP53−/− mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion

  9. Zinc Induces Apoptosis of Human Melanoma Cells, Increasing Reactive Oxygen Species, p53 and FAS Ligand.

    PubMed

    Provinciali, Mauro; Pierpaoli, Elisa; Bartozzi, Beatrice; Bernardini, Giovanni

    2015-10-01

    The aim of this study was to examine the in vitro effect of zinc on the apoptosis of human melanoma cells, by studying the zinc-dependent modulation of intracellular levels of reactive oxygen species (ROS) and of p53 and FAS ligand proteins. We showed that zinc concentrations ranging from 33.7 μM to 75 μM Zn(2+) induced apoptosis in the human melanoma cell line WM 266-4. This apoptosis was associated with an increased production of intracellular ROS, and of p53 and FAS ligand protein. Treatment of tumor cells with the antioxidant N-acetylcysteine was able to prevent Zn(2+)-induced apoptosis, as well as the increase of p53 and FAS ligand protein induced by zinc. Zinc induces apoptosis in melanoma cells by increasing ROS and this effect may be mediated by the ROS-dependent induction of p53 and FAS/FAS ligand. PMID:26408691

  10. Ghrelin Prevents Cisplatin-Induced Testicular Damage by Facilitating Repair of DNA Double Strand Breaks Through Activation of p53 in Mice.

    PubMed

    Garcia, Jose M; Chen, Ji-an; Guillory, Bobby; Donehower, Lawrence A; Smith, Roy G; Lamb, Dolores J

    2015-07-01

    Cisplatin administration induces DNA damage resulting in germ cell apoptosis and subsequent testicular atrophy. Although 50 percent of male cancer patients receiving cisplatin-based chemotherapy develop long-term secondary infertility, medical treatment to prevent spermatogenic failure after chemotherapy is not available. Under normal conditions, testicular p53 promotes cell cycle arrest, which allows time for DNA repair and reshuffling during meiosis. However, its role in the setting of cisplatin-induced infertility has not been studied. Ghrelin administration ameliorates the spermatogenic failure that follows cisplatin administration in mice, but the mechanisms mediating these effects have not been well established. The aim of the current study was to characterize the mechanisms of ghrelin and p53 action in the testis after cisplatin-induced testicular damage. Here we show that cisplatin induces germ cell damage through inhibition of p53-dependent DNA repair mechanisms involving gamma-H2AX and ataxia telangiectasia mutated protein kinase. As a result, testicular weight and sperm count and motility were decreased with an associated increase in sperm DNA damage. Ghrelin administration prevented these sequelae by restoring the normal expression of gamma-H2AX, ataxia telangiectasia mutated, and p53, which in turn allows repair of DNA double stranded breaks. In conclusion, these findings indicate that ghrelin has the potential to prevent or diminish infertility caused by cisplatin and other chemotherapeutic agents by restoring p53-dependent DNA repair mechanisms. PMID:26019260

  11. Ghrelin Prevents Cisplatin-Induced Testicular Damage by Facilitating Repair of DNA Double Strand Breaks Through Activation of p53 in Mice1

    PubMed Central

    Garcia, Jose M.; Chen, Ji-an; Guillory, Bobby; Donehower, Lawrence A.; Smith, Roy G.; Lamb, Dolores J.

    2015-01-01

    Cisplatin administration induces DNA damage resulting in germ cell apoptosis and subsequent testicular atrophy. Although 50 percent of male cancer patients receiving cisplatin-based chemotherapy develop long-term secondary infertility, medical treatment to prevent spermatogenic failure after chemotherapy is not available. Under normal conditions, testicular p53 promotes cell cycle arrest, which allows time for DNA repair and reshuffling during meiosis. However, its role in the setting of cisplatin-induced infertility has not been studied. Ghrelin administration ameliorates the spermatogenic failure that follows cisplatin administration in mice, but the mechanisms mediating these effects have not been well established. The aim of the current study was to characterize the mechanisms of ghrelin and p53 action in the testis after cisplatin-induced testicular damage. Here we show that cisplatin induces germ cell damage through inhibition of p53-dependent DNA repair mechanisms involving gamma-H2AX and ataxia telangiectasia mutated protein kinase. As a result, testicular weight and sperm count and motility were decreased with an associated increase in sperm DNA damage. Ghrelin administration prevented these sequelae by restoring the normal expression of gamma-H2AX, ataxia telangiectasia mutated, and p53, which in turn allows repair of DNA double stranded breaks. In conclusion, these findings indicate that ghrelin has the potential to prevent or diminish infertility caused by cisplatin and other chemotherapeutic agents by restoring p53-dependent DNA repair mechanisms. PMID:26019260

  12. Myc Prevents Apoptosis and Enhances Endoreduplication Induced by Paclitaxel

    PubMed Central

    Gatti, Giuliana; Maresca, Giovanna; Natoli, Manuela; Florenzano, Fulvio; Nicolin, Angelo; Felsani, Armando; D'Agnano, Igea

    2009-01-01

    Background The role of the MYC oncogene in the apoptotic pathways is not fully understood. MYC has been reported to protect cells from apoptosis activation but also to sensitize cells to apoptotic stimuli. We have previously demonstrated that the down-regulation of Myc protein activates apoptosis in melanoma cells and increases the susceptibility of cells to various antitumoral treatments. Beyond the well-known role in the G1→S transition, MYC is also involved in the G2-M cell cycle phases regulation. Methodology/Principal Findings In this study we have investigated how MYC could influence cell survival signalling during G2 and M phases. We used the microtubules damaging agent paclitaxel (PTX), to arrest the cells in the M phase, in a p53 mutated melanoma cell line with modulated Myc level and activity. An overexpression of Myc protein is able to increase endoreduplication favoring the survival of cells exposed to antimitotic poisoning. The PTX-induced endoreduplication is associated in Myc overexpressing cells with a reduced expression of MAD2, essential component of the molecular core of the spindle assembly checkpoint (SAC), indicating an impairment of this checkpoint. In addition, for the first time we have localized Myc protein at the spindle poles (centrosomes) during pro-metaphase in different cell lines. Conclusions The presence of Myc at the poles during the prometaphase could be necessary for the Myc-mediated attenuation of the SAC and the subsequent induction of endoreduplication. In addition, our data strongly suggest that the use of taxane in antitumor therapeutic strategies should be rationally based on the molecular profile of the individual tumor by specifically analyzing Myc expression levels. PMID:19421315

  13. Nanoparticle-Mediated Mitochondrial Damage Induces Apoptosis in Cancer.

    PubMed

    Mallick, Abhik; More, Piyush; Syed, Muhammed Muazzam Kamil; Basu, Sudipta

    2016-06-01

    Detouring of conventional DNA damaging anticancer drugs into mitochondria to damage mitochondrial DNA is evolving as a promising strategy in chemotherapy. Inhibiting single target in mitochondria would eventually lead to the emergence of drug resistance. Moreover, targeting mitochondria selectively in cancer cells, keeping them intact in healthy cells, remains a major challenge. Herein, triphenylphosphine (TPP)-coated positively charged 131.6 nm spherical nanoparticles (NPs) comprised of α-tocopheryl succinate (TOS, inhibitor of complex II in electron transport chain) and obatoclax (Obt, inhibitor of Bcl-2) were engineered. The TOS-TPP-Obt-NPs entered into acidic lysosomes via macropinocytosis, followed by lysosomal escape and finally homed into mitochondria over a period of 24 h. Subsequently, these TOS-TPP-Obt-NPs triggered mitochondrial outer membrane permeabilization (MOMP) by inhibiting antiapoptotic Bcl-2, leading to Cytochrome C release. These TOS-TPP-Obt-NPs mediated mitochondrial damage induced cellular apoptosis through caspase-9 and caspase-3 cleavage to show improved efficacy in HeLa cells. Moreover, TOS-TPP-Obt-NPs induced MOMP in drug-resistant triple negative breast cancer cells (MDA-MB-231), leading to remarkable efficacy, compared to the combination of free drugs in higher drug concentrations. Results presented here clearly stimulate the usage of multiple drugs to perturb simultaneously diverse targets, selectively in mitochondria, as next-generation cancer therapeutics. PMID:27160664

  14. Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis

    PubMed Central

    Bourgine, Paul E.; Scotti, Celeste; Pigeot, Sebastien; Tchang, Laurent A.; Todorov, Atanas; Martin, Ivan

    2014-01-01

    The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard “freeze & thaw” (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue. PMID:25422415

  15. Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis.

    PubMed

    Bourgine, Paul E; Scotti, Celeste; Pigeot, Sebastien; Tchang, Laurent A; Todorov, Atanas; Martin, Ivan

    2014-12-01

    The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard "freeze & thaw" (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue. PMID:25422415

  16. Agarol, an ergosterol derivative from Agaricus blazei, induces caspase-independent apoptosis in human cancer cells.

    PubMed

    Shimizu, Takamitsu; Kawai, Junya; Ouchi, Kenji; Kikuchi, Haruhisa; Osima, Yoshiteru; Hidemi, Rikiishi

    2016-04-01

    Agaricus blazei (A. blazei) is a mushroom with many biological effects and active ingredients. We purified a tumoricidal substance from A. blazei, an ergosterol derivative, and named it 'Agarol'. Cytotoxic effects of Agarol were determined by the MTT assay using A549, MKN45, HSC-3, and HSC-4 human carcinoma cell lines treated with Agarol. Apoptosis was detected by flow cytometry analysis. Reactive oxygen species (ROS) levels and mitochondria membrane potential (∆ψm) were also determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related proteins. Agarol predominantly induced apoptosis in two p53-wild cell lines (A549 and MKN45) compared to the other p53-mutant cell lines (HSC-3 and HSC-4). Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of ROS, reduced ∆ψm, release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol, upregulation of Bax, and downregulation of Bcl-2. Caspase-3 activities did not increase, and z-VAD-fmk, a caspase inhibitor, did not inhibit the Agarol-induced apoptosis. These findings indicate that Agarol induces caspase-independent apoptosis in human carcinoma cells through a mitochondrial pathway. The in vivo anticancer activity of Agarol was confirmed in a xenograft murine model. This study suggests a molecular mechanism by which Agarol induces apoptosis in human carcinoma cells and indicates the potential use of Agarol as an anticancer agent. PMID:26893131

  17. Par-4/NF-κB Mediates the Apoptosis of Islet β Cells Induced by Glucolipotoxicity.

    PubMed

    QiNan, Wu; XiaGuang, Gan; XiaoTian, Lei; WuQuan, Deng; Ling, Zhang; Bing, Chen

    2016-01-01

    Apoptosis of islet β cells is a primary pathogenic feature of type 2 diabetes, and ER stress and mitochondrial dysfunction play important roles in this process. Previous research has shown that prostate apoptosis response-4 (Par-4)/NF-κB induces cancer cell apoptosis through endoplasmic reticulum (ER) stress and mitochondrial dysfunction. However, the mechanism by which Par-4/NF-κB induces islet β cell apoptosis remains unknown. We used a high glucose/palmitate intervention to mimic type 2 diabetes in vitro. We demonstrated that the high glucose/palmitate intervention induced the expression and secretion of Par-4. It also causes increased expression and activation of NF-κB, which induced NIT-1 cell apoptosis and dysfunction. Overexpression of Par-4 potentiates these effects, whereas downregulation of Par-4 attenuates them. Inhibition of NF-κB inhibited the Par-4-induced apoptosis. Furthermore, these effects occurred through the ER stress cell membrane and mitochondrial pathway of apoptosis. Our findings reveal a novel role for Par-4/NF-κB in islet β cell apoptosis and type 2 diabetes. PMID:27340675

  18. Par-4/NF-κB Mediates the Apoptosis of Islet β Cells Induced by Glucolipotoxicity

    PubMed Central

    QiNan, Wu; XiaGuang, Gan; XiaoTian, Lei; WuQuan, Deng; Ling, Zhang; Bing, Chen

    2016-01-01

    Apoptosis of islet β cells is a primary pathogenic feature of type 2 diabetes, and ER stress and mitochondrial dysfunction play important roles in this process. Previous research has shown that prostate apoptosis response-4 (Par-4)/NF-κB induces cancer cell apoptosis through endoplasmic reticulum (ER) stress and mitochondrial dysfunction. However, the mechanism by which Par-4/NF-κB induces islet β cell apoptosis remains unknown. We used a high glucose/palmitate intervention to mimic type 2 diabetes in vitro. We demonstrated that the high glucose/palmitate intervention induced the expression and secretion of Par-4. It also causes increased expression and activation of NF-κB, which induced NIT-1 cell apoptosis and dysfunction. Overexpression of Par-4 potentiates these effects, whereas downregulation of Par-4 attenuates them. Inhibition of NF-κB inhibited the Par-4-induced apoptosis. Furthermore, these effects occurred through the ER stress cell membrane and mitochondrial pathway of apoptosis. Our findings reveal a novel role for Par-4/NF-κB in islet β cell apoptosis and type 2 diabetes. PMID:27340675

  19. Fructose Protects Murine Hepatocytes from Tumor Necrosis Factor-induced Apoptosis by Modulating JNK Signaling*

    PubMed Central

    Speicher, Tobias; Köhler, Ulrike A.; Choukèr, Alexander; Werner, Sabine; Weiland, Timo; Wendel, Albrecht

    2012-01-01

    Fructose-induced hepatic ATP depletion prevents TNF-induced apoptosis, whereas it contrarily enhances CD95-induced hepatocyte apoptosis in vitro and in vivo. By contrast, transformed liver cells are not protected against TNF due to metabolic alterations, allowing selective tumor targeting. We analyzed the molecular mechanisms by which fructose modulates cytokine-induced apoptosis. A release of adenosine after fructose-induced ATP depletion, followed by a cAMP response, was demonstrated. Likewise, cAMP and adenosine mimicked per se the modulation by fructose of CD95- and TNF-induced apoptosis. The effects of fructose on cytokine-induced apoptosis were sensitive to inhibition of protein kinase A. Fructose prevented the pro-apoptotic, sustained phase of TNF-induced JNK signaling and thereby blocked bid-mediated activation of the intrinsic mitochondrial apoptosis pathway in a PKA-dependent manner. We explain the dichotomal effects of fructose on CD95- and TNF-induced cell death by the selective requirement of JNK signaling for the latter. These findings provide a mechanistic rationale for the protection of hepatocytes from TNF-induced cell death by pharmacological doses of fructose. PMID:22086922

  20. The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1

    PubMed Central

    Prikhod’ko, Elena A.; Miller, Lois K.

    1999-01-01

    While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus, pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection. PMID:10400766

  1. Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts.

    PubMed

    Leccia, M T; Richard, M J; Favier, A; Béani, J C

    1999-09-01

    Ultraviolet A1 (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340-450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dose-dependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation. PMID:10468155

  2. MicroRNA-322 protects hypoxia-induced apoptosis in cardiomyocytes via BDNF gene

    PubMed Central

    Yang, Liguo; Song, Shigang; Lv, Hang

    2016-01-01

    Background: Cardiomyocytes apoptosis under hypoxia condition contributes significantly to various cardiovascular diseases. In this study, we investigated the role of microRNA-322 (miR-322) in regulating hypoxia-induced apoptosis in neonatal murine cardiomyocytes in vitro. Method: Cardiomyocytes of C57BL/6J mice were treated with hypoxia condition in vitro. Cardiomyocyte apoptosis was measured by TUNEL assay. Gene expression pattern of miR-322 was measured by qRT-PCR. Stable downregulation of miR-322 in cardiomyocytes were achieved by lentiviral transduction, and the effect of miR-322 downregulation on hypoxia-induced cardiomyocyte apoptosis was investigated. Possible regulation of miR-322 on its downstream target gene, brain derived neurotrophic factor (BDNF) was investigated in cardiomyocytes. BDNF was then genetically silenced by siRNA to evaluate its role in miR-137 mediated cardiomyocyte apoptosis protection under hypoxia condition. Results: Under hypoxia condition, significant apoptosis was induced and miR-322 was significantly upregulated in cardiomyocytes in vitro. Through lentiviral transduction, miR-322 was efficiently knocked down in cardiomyocytes. Downregulation of miR-322 protected hypoxia-induced cardiomyocyte apoptosis. Luciferase assay showed BDNF was the target gene of miR-322. QRT-PCR showed BDNF expression was associated with miR-322 regulation on hypoxia-induced cardiomyocyte apoptosis. Silencing BDNF in cardiomyocyte through siRNA transfection reversed the protective effect of miR-322 downregulation on hypoxia-induced apoptosis. Conclusion: Our study revealed that miR-322, in association with BDNF, played important role in regulating hypoxia-induced apoptosis in cardiomyocyte. PMID:27398164

  3. Cyclophosphamide-induced apoptosis in COV434 human granulosa cells involves oxidative stress and glutathione depletion.

    PubMed

    Tsai-Turton, Miyun; Luong, Brian T; Tan, Youming; Luderer, Ulrike

    2007-07-01

    The anticancer drug cyclophosphamide induces granulosa cell apoptosis and is detoxified by glutathione (GSH) conjugation. We previously showed that both cyclophosphamide treatment and GSH depletion induced granulosa cell apoptosis in rats, but the role of GSH in apoptosis in human ovarian cells has not been studied. Using the COV434 human granulosa cell line, we tested the hypotheses that (1) GSH depletion or treatment with 4-hydroperoxycyclophosphamide (4HC), a preactivated form of cyclophosphamide, induces apoptosis, (2) GSH depletion potentiates 4HC-induced apoptosis, and (3) 4HC-induced apoptosis is mediated by GSH depletion and oxidative stress. Cells were treated with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, with or without follicle stimulating hormone (FSH) or serum. A significant increase in the number of apoptotic cells, assessed by terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick-end labeling (TUNEL) and Hoechst 33342 staining, occurred with BSO treatment. Treatment with 4HC dose-dependently induced apoptosis by TUNEL, Hoechst staining, and caspase 3 activation. Treatment with 4HC caused an increase in reactive oxygen species generation, measured by dichlorofluorescein fluorescence, oxidative DNA damage, measured by 8-hydroxyguanosine immunostaining, and an oxidation of the redox potential for the oxidized glutathione/reduced glutathione couple. Total intracellular GSH declined after 4HC treatment, preceding the onset of cell death. Treatment with antioxidants inhibited 4HC-induced apoptosis. Combined treatment with BSO and 4HC caused greater induction of apoptosis than either treatment alone. These findings are consistent with roles for oxidative stress and GSH depletion in mediating the induction of apoptosis in COV434 cells by cyclophosphamide. PMID:17434952

  4. The Mitochondria-Mediate Apoptosis of Lepidopteran Cells Induced by Azadirachtin

    PubMed Central

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis. PMID:23516491

  5. Involvement of ASK1 activation in apoptosis induced by NPe6-PDT

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Zhang, Zhen-zhen; Zhang, Zhigang

    2010-02-01

    Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate apoptotic pathway. Apoptosis signal-regulating kinase (ASK1) is an important regulator of apoptosis in response to various stresses, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, lipopolysaccharide (LPS) and calcium influx. In this study, we investigated the molecular mechanisms of apoptosis induced by NPe6-PDT in ASTC-a-1 cells. The results showed that the activities of ASK1 increased in response to NPe6-PDT. Over-expression of wild-type or activated mutant of ASK1 could obviously decrease cell viability and increase cell death; while inhibition of ASK1 significantly decreased cell apoptosis. These results suggested that ASK1 plays an important role in apoptosis induced by NPe6-PDT.

  6. Implications of the involvement of the endoplasmic reticulum stress pathway in drug-induced apoptosis.

    PubMed

    Cory, Ann H; Chen, Jianming; Cory, Joseph G

    2008-01-01

    Apoptosis occurs by distinct pathways that involve the cell surface, mitochondria or the endoplasmic reticulum. Previous studies had shown that deoxyadenosine-resistant L1210 cells (Y8) proceeded to apoptosis under conditions in which the parental L1210 cell line (WT) did not undergo an apoptotic response. Combinations of drugs, acting at different molecular targets, markedly potentiated the apoptotic response in the Y8 cells without inducing apoptosis in the WT cells. In the present study, induction of apoptosis by parthenolide and BAY 11-7085, drugs that targeted nuclear factor kappa B activation, was blocked by the presence of N-acetylcysteine (NAC). On the other hand, the levels of apoptosis induced by parthenolide or BAY 11-7085 were increased by pre-treatment of the cells with glutathione lowering L-buthionine-(S,R)-sulfoximine (BSO). Western blot analyses showed that the levels of the stress proteins, Grp 78 and Gadd 153 were reduced in the parthenolide-treated Y8 cells, but not in those co-treated with NAC. Protection of the cells from apoptosis induced by parthenolide or BAY 11-7085 by NAC was relatively specific as the induction of apoptosis in the Y8 cells by MG-132, flavopiridol, Gemcitabine or PRIMA-1 was not decreased by NAC. These data suggest that multiple pathways, one of which is ER-stress induced, may ultimately be involved and interactive in the induction of apoptosis in specific cell lines. PMID:18507007

  7. Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

    PubMed Central

    Aguiló, N; Uranga, S; Marinova, D; Martín, C; Pardo, J

    2014-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis. PMID:25032866

  8. Low temperature protects mammalian cells from apoptosis initiated by various stimuli in vitro

    SciTech Connect

    Sakurai, Toshiharu; Itoh, Katsuhiko; Liu Yu; Higashitsuji, Hiroaki; Sumitomo, Yasuhiko; Sakamaki, Kazuhiro; Fujita, Jun . E-mail: jfujita@virus.kyoto-u.ac.jp

    2005-10-01

    Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we examined the effects of low temperature (32 deg. C) on cells exposed to a variety of stress in vitro. We found that 32 deg. C suppressed induction of apoptosis by cytotoxic stimuli such as adriamycin, etoposide, thapsigargin, NaCl, H{sub 2}O{sub 2}, and anti-Fas antibody. In adriamycin-treated BALB/3T3 cells, the down-shift in temperature from 37 deg. C to 32 deg. C increased the Bcl-xL protein level and decreased the mRNA level of Puma and mitochondrial translocation of Bax, suppressing caspase-9-mediated apoptosis. Furthermore, the protein level and stability of p53 were decreased, and its nuclear export was increased concomitant with Mdm2 mRNA upregulation. The low temperature effect was not observed in p53 {sup -/-}/Mdm2 {sup -/-} mouse embryonic fibroblasts, suggesting that the effect is mediated by suppression of the p53 pathway. In contrast, while thapsigargin-induced apoptosis was suppressed by the low temperature, no effect on the p53 protein level was observed. Furthermore, the survival rate of p53 {sup -/-}/Mdm2 {sup -/-} cells exposed to thapsigargin was increased when cultured at 32 deg. C compared with 37 deg. C. In conclusion, mild hypothermia protects cells from a variety of stress by p53-dependent and p53-independent mechanisms.

  9. Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells

    PubMed Central

    Chang, Haigang; Song, Shanshan; Chen, Zhongcan; Wang, Yaxiao; Yang, Lujun; Du, Mouxuan; Ke, Yiquan; Xu, Ruxiang; Jin, Baozhe; Jiang, Xiaodan

    2014-01-01

    Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells. PMID:25206849

  10. Experimental study on apoptosis induced by semiconductor laser to hair removal and armpit odor treatment

    NASA Astrophysics Data System (ADS)

    Shi, Hongmin; Yan, Min; Zhang, Meijue

    2005-07-01

    Objective: To observe and explore the effects and mechanism of apoptosis on canine induced by Laser. Try to find a new approach to treat of armpit odor with no traumatism. Method: We used different power of semiconductor Laser to irradiate the black hair canine to observe and evaluate the tissue effects with electroscope, flow cytometry and Tunel technique at different period of time after irradiation. Result: The apoptosis has been observed within the hair follicle cells and apocrine gland cells after irradiation. After repeat irradiation in low power level, more apoptosis has been observed. Conclusion: Apoptosis exists in hair follicle cells and apocrine gland cells after Laser irradiation.

  11. Endocannabinoids participate in placental apoptosis induced by hypoxia inducible factor-1.

    PubMed

    Abán, C; Martinez, N; Carou, C; Albamonte, I; Toro, A; Seyahian, A; Franchi, A; Leguizamón, G; Trigubo, D; Damiano, A; Farina, M

    2016-10-01

    During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed towards the relevance of endocannabinoids (ECs) and hypoxia as modulators of trophoblast cell death. However, the relation between these factors is still unknown. In this report, we evaluated the participation of ECs in placental apoptosis induced by cobalt chloride (CoCl2), a hypoxia mimicking agent that stabilizes the expression of hypoxia inducible factor-1 alpha (HIF-1α). We found that HIF-1α stabilization decreased FAAH mRNA and protein levels, suggesting an increase in ECs tone. Additionally, CoCl2 incubation and Met-AEA treatment reduced cell viability and increased TUNEL-positive staining in syncytiotrophoblast layer. Immunohistochemical analysis demonstrated Bax and Bcl-2 protein expression in the cytoplasm of syncytiotrophoblast. Finally, HIF-1α stabilization produced an increase in Bax/Bcl-2 ratio, activation of caspase 3 and PARP cleavage. All these changes in apoptotic parameters were reversed with AM251, a CB1 antagonist. These results demonstrate that HIF-1α may induce apoptosis in human placenta via intrinsic pathway by a mechanism that involves activation of CB1 receptor suggesting a role of the ECs in this process. PMID:27488203

  12. RCAN1 Overexpression Exacerbates Calcium Overloading-Induced Neuronal Apoptosis

    PubMed Central

    Herculano, Bruno; Song, Weihong

    2014-01-01

    Down Syndrome (DS) patients develop characteristic Alzheimer's Disease (AD) neuropathology after their middle age. Prominent neuronal loss has been observed in the cortical regions of AD brains. However, the underlying mechanism leading to this neuronal loss in both DS and AD remains to be elucidated. Calcium overloading and oxidative stress have been implicated in AD pathogenesis. Two major isoforms of regulator of calcineurin 1 (RCAN1), RCAN1.1 and RCAN1.4, are detected in human brains. In this report we defined the transcriptional regulation of RCAN1.1 and RCAN1.4 by two alternative promoters. Calcium overloading upregulated RCAN1.4 expression by activating RCAN1.4 promoter through calcineurin-NFAT signaling pathway, thus forming a negative feedback loop in isoform 4 regulation. Furthermore, RCAN1.4 overexpression exacerbated calcium overloading-induced neuronal apoptosis, which was mediated by caspase-3 apoptotic pathway. Our results suggest that downregulating RCAN1.4 expression in neurons could be beneficial to AD patients. PMID:24751678

  13. Secondhand smoke induces hepatic apoptosis and fibrosis in hamster fetus.

    PubMed

    Huang, Chien-Wei; Horng, Chi-Ting; Huang, Chih-Yang; Cho, Ta-Hsiung; Tsai, Yi-Chang; Chen, Li-Jeng; Hsu, Tsai-Ching; Tzang, Bor-Show

    2016-09-01

    Secondhand smoke (SHS) is an important health issue worldwide. Inhaling SHS during pregnancy could cause abnormalities in the internal tissues of newborns, which may then impair fetal development and even cause severe intrauterine damage and perinatal death. However, the understanding of cytopathic mechanisms of SHS by maternal passive smoking on fetus liver during pregnancy is still limited. This study analyzed the effects of high-dose SHS (SHSH) on fetus liver using a maternal passive smoking animal model. Experiments showed that hepatic matrix metalloproteinase-9 activity and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling-positive cells were significantly increased in livers from fetuses of hamsters treated with SHSH. Similarly, expressions of both extrinsic and intrinsic apoptotic molecules were significantly higher in livers from fetuses of hamsters exposed to SHSH. Additionally, significantly increased inflammatory proteins, including transforming growth factor β, inducible nitric oxide synthase, and interleukin 1β, and fibrotic signaling molecules, including phosphorylated Smad2/3, SP1, and α-smooth muscle actin, were observed in the fetus livers from hamsters treated with SHSH. This study revealed that SHSH not only increased apoptosis through intrinsic and extrinsic pathways in the livers of fetuses from hamsters exposed to SHSH but also augmented hepatic fibrosis via Smad2/3 signaling. PMID:26612555

  14. Novel fluorescence molecular imaging of chemotherapy-induced intestinal apoptosis

    NASA Astrophysics Data System (ADS)

    Levin, Galit; Shirvan, Anat; Grimberg, Hagit; Reshef, Ayelet; Yogev-Falach, Merav; Cohen, Avi; Ziv, Ilan

    2009-09-01

    Chemotherapy-induced enteropathy (CIE) is one of the most serious complications of anticancer therapy, and tools for its early detection and monitoring are highly needed. We report on a novel fluorescence method for detection of CIE, based on molecular imaging of the related apoptotic process. The method comprises systemic intravenous administration of the ApoSense fluorescent biomarker (N,N'-didansyl-L-cystine DDC) in vivo and subsequent fluorescence imaging of the intestinal mucosa. In the reported proof-of-concept studies, mice were treated with either taxol+cyclophosphamide or doxil. DDC was administered in vivo at various time points after drug administration, and tracer uptake by ileum tissue was subsequently evaluated by ex vivo fluorescent microscopy. Chemotherapy caused marked and selective uptake of DDC in ileal epithelial cells, in correlation with other hallmarks of apoptosis (i.e., DNA fragmentation and Annexin-V binding). Induction of DDC uptake occurred early after chemotherapy, and its temporal profile was parallel to that of the apoptotic process, as assessed histologically. DDC may therefore serve as a useful tool for detection of CIE. Future potential integration of this method with fluorescent endoscopic techniques, or development of radio-labeled derivatives of DDC for emission tomography, may advance early diagnosis and monitoring of this severe adverse effect of chemotherapy.

  15. Significance of p53 dynamics in regulating apoptosis in response to ionizing radiation, and polypharmacological strategies

    PubMed Central

    Liu, Bing; Bhatt, Divesh; Oltvai, Zoltán N.; Greenberger, Joel S.; Bahar, Ivet

    2014-01-01

    Developing pharmacological strategies for controlling ionizing radiation (IR)-induced cell death is important for both mitigating radiation damage and alleviating the side effects of anti-cancer radiotherapy manifested in surrounding tissue morbidity. Exposure to IR often triggers the onset of p53-dependent apoptotic pathways. Here we build a stochastic model of p53 induced apoptosis comprised of coupled modules of nuclear p53 activation, mitochondrial cytochrome c release and cytosolic caspase activation that also takes into account cellular heterogeneity. Our simulations show that the strength of p53 transcriptional activity and its coupling (or timing with respect) to mitochondrial pore opening are major determinants of cell fate: for systems where apoptosis is elicited via a p53-transcription-independent mechanism, direct activation of Bax by p53 becomes critical to IR-induced-damage initiation. We further show that immediate administration of PUMA inhibitors following IR exposure effectively suppresses excessive cell death, provided that there is a strong caspase/Bid feedback loop; however, the efficacy of the treatment diminishes with increasing delay in treatment implementation. In contrast, the combined inhibition of Bid and Bax elicits an anti-apoptotic response that is effective over a range of time delays. PMID:25175563

  16. Coxsackievirus A16 Infection Induces Neural Cell and Non-Neural Cell Apoptosis In Vitro

    PubMed Central

    Liu, Li; Wei, Zhenhong; Ehrlich, Elana S.; Liu, Guanchen; Li, Jingliang; Liu, Xin; Wang, Hong; Yu, Xiao-fang; Zhang, Wenyan

    2014-01-01

    Coxsackievirus A16 (CA16) is one of the main causative pathogens of hand, foot and mouth disease (HFMD). Viral replication typically results in host cell apoptosis. Although CA16 infection has been reported to induce apoptosis in the human rhabdomyosarcoma (RD) cell line, it remains unclear whether CA16 induces apoptosis in diverse cell types, especially neural cells which have important clinical significance. In the current study, CA16 infection was found to induce similar apoptotic responses in both neural cells and non-neural cells in vitro, including nuclear fragmentation, DNA fragmentation and phosphatidylserine translocation. CA16 generally is not known to lead to serious neurological symptoms in vivo. In order to further clarify the correlation between clinical symptoms and cell apoptosis, two CA16 strains from patients with different clinical features were investigated. The results showed that both CA16 strains with or without neurological symptoms in infected patients led to neural and muscle cell apoptosis. Furthermore, mechanistic studies showed that CA16 infection induced apoptosis through the same mechanism in both neural and non-neural cells, namely via activation of both the mitochondrial (intrinsic) pathway-related caspase 9 protein and the Fas death receptor (extrinsic) pathway-related caspase 8 protein. Understanding the mechanisms by which CA16 infection induces apoptosis in both neural and non-neural cells will facilitate a better understanding of CA16 pathogenesis. PMID:25350381

  17. Bupivacaine induces apoptosis through caspase-dependent and -independent pathways in canine mammary tumor cells.

    PubMed

    Chiu, Yi-Shu; Cheng, Yeong-Hsiang; Lin, Sui-Wen; Chang, Te-Sheng; Liou, Chian-Jiun; Lai, Yu-Shen

    2015-06-01

    Local anesthetics have been reported to induce apoptosis in various cell lines. In this study, we showed that bupivacaine also induced apoptosis in DTK-SME cells, a vimentin(+)/AE1(+)/CK7(+)/HSP27(+), tumorigenic, immortalized, canine mammary tumor cell line. Bupivacaine induced apoptosis in DTK-SME cells in a time- and concentration-dependent manner. Apoptosis-associated morphological changes, including cell shrinkage and rounding, chromatin condensation, and formation of apoptotic bodies, were observed in the bupivacaine-treated DTK-SME cells. Apoptosis was further confirmed with annexin V staining, TUNEL staining, and DNA laddering assays. At the molecular level, the activation of caspases-3, -8, and -9 corresponded well to the degree of DNA fragmentation triggered by bupivacaine. We also demonstrated that the pan-caspase inhibitor, z-VAD-fmk, only partially inhibited the apoptosis induced by bupivacaine. Moreover, treated cells increased expression of endonuclease G, a death effector that acts independently of caspases. Our data suggested that bupivacaine-induced apoptosis occurs through both caspase-dependent and caspase-independent apoptotic pathways. PMID:25843897

  18. AMID Mediates Adenosine-Induced Caspase-Independent HuH-7 Cell Apoptosis

    PubMed Central

    Yang, Dongqin; Yaguchi, Takahiro; Nagata, Tetsu; Gotoh, Akinobu; Dovat, Sinisa; Song, Chunhua; Nishizaki, Tomoyuki

    2011-01-01

    Background/Aims: The mechanism underlying extracellular adenosine-induced caspase-independent apoptosis in HuH-7 human hepatoma cells is not fully understood. The present study investigated the role for apoptosis-inducing factor (AIF)-homologous mitochondrion-associated inducer of death (AMID) in the pathway. Methods: To see the implication of AMID in adenosine-induced HuH-7 cell apoptosis, real-time reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescent cytochemistry, time-laps GFP monitoring, cell cycle analysis, flow cytometry, Western blotting, cell viability assay, and TUNEL staining were carried out. Results: Adenosine upregulated AMID expression in HuH-7 cells, and translocated AMID from the cytosol into the nucleus. Adenosine induced HuH-7 cell apoptosis, and the effect was further enhanced by overexpressing AMID. Adenosine-induced HuH-7 cell apoptosis, alternatively, was inhibited by knocking-down AMID. Conclusion: The results of the present study provide evidence for AMID as a critical factor for adenosine-induced caspase-independent HuH-7 cell apoptosis. PMID:21325820

  19. GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

    PubMed Central

    Zhang, Bicheng; Huang, Jun; Li, Hong-Liang; Liu, Ting; Wang, Yan-Yi; Waterman, Paul; Mao, Ai-Ping; Xu, Liang-Guo; Zhai, Zhonghe; Liu, Depei; Marrack, Philippa; Shu, Hong-Bing

    2011-01-01

    We report here the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal Ring finger domain, which is mostly conserved with those of the IAP family members, and which is required for its E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases since the caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockade of JNK activation inhibits GIDE-induced release of cytochrome c and Smac and apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE-induced apoptosis. These proapoptotic properties of GIDE require its E3 ligase activity. When somewhat over or underexpressed, GIDE slows or hastens cell growth respectively. These pro-apoptotic or growth rate effects of GIDE may account for its absence from tumor cells. PMID:18591963

  20. Bim mediates mitochondria-regulated particulate matter-induced apoptosis in alveolar epithelial cells

    PubMed Central

    Zhang, J.; Ghio, A.J.; Chang, W.; Kamdar, O.; Rosen, G.D.; Upadhyay, D.

    2007-01-01

    We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 μm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway. PMID:17716672

  1. Ultrasonication processed Panax ginseng berry extract induces apoptosis through an intrinsic apoptosis pathway in HepG2 cells.

    PubMed

    Jung, Hyunwoo; Bae, Jinhyung; Ko, Sung Kwon; Sohn, Uy Dong

    2016-06-01

    Ginseng's major active components, ginsenosides, have been known to show anti-cancer, neuroprotective, and anti-inflammatory activities. Ultrasonication processed Panax ginseng berry extract (UGB) contains various ginsenosides. The components are different from Panax ginseng berry extract (GBE). This study was aimed to investigate the cytotoxic mechanism of UGB in HepG2 cells, human hepatocellular carcinoma cell line. HepG2 cells were treated with UGB (0, 10, 20 μg/ml). Cell growth and cellular apoptosis were evaluated by MTT assay and Annexin V/Pi staining, respectively. Intracellular Reactive oxygen species (ROS) levels were also determined by 2', 7'-dichlorofluorescin diacetate (DCFDA) staining. The expressions of Bax, Bcl-2 and caspase-3, the apoptotic markers, were evaluated by Western Blot. UGB dose-dependently inhibited cell growth and induced apoptotic cell death. Intracellular ROS levels were increased. UGB increased the expression of the cleaved form of caspase-3. Furthermore, UGB induced apoptosis of HepG2 cells through Bax activation and Bcl-2 inhibition. In conclusion, UGB induced apoptosis through an intrinsic pathway in HepG2 cells suggesting that UGB might play a role as a novel substance for anti-cancer effect. PMID:27233905

  2. Portulaca oleracea extracts protect human keratinocytes and fibroblasts from UV-induced apoptosis.

    PubMed

    Lee, Suyeon; Kim, Ki Ho; Park, Changhoon; Lee, Jong-Suk; Kim, Young Heui

    2014-10-01

    Portulaca oleracea extracts, known as Ma Chi Hyun in the traditional Korean medicine, show a variety of biomedical efficacies including those in anti-inflammation and anti-allergy. In this study, we investigate the protective activity of the P. oleracea extracts against UVB-induced damage in human epithelial keratinocytes and fibroblasts by several apoptosis-related tests. The results suggest that P. oleracea extracts have protective effects from UVB-induced apoptosis. PMID:25234830

  3. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    SciTech Connect

    Li, Fengbo; Sun, Xiaolei; Ma, Jianxiong; Ma, Xinlong; Zhao, Bin; Zhang, Yang; Tian, Peng; Li, Yanjun; Han, Zhe

    2014-09-26

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.

  4. TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells.

    PubMed

    Guelen, Lars; Paterson, Hugh; Gäken, Joop; Meyers, Michelle; Farzaneh, Farzin; Tavassoli, Mahvash

    2004-02-01

    Apoptin has been described to induce apoptosis in various human cancer cell lines, but not in normal cells, thus making it an interesting candidate for the development of novel therapeutic strategies. Apoptin was generated and cloned into several mammalian expression vectors. Transfection or microinjection of apoptin cDNA resulted in its expression, initially in the cytoplasm with a filamentous pattern. Subsequently, apoptin entered the nucleus and efficiently induced apoptosis in several cancer cell lines. Nuclear localization was shown to be required for induction of apoptosis. Apoptin expression level was found to be an important determinant of the efficiency of induction of apoptosis. Surprisingly, expression of apoptin or GFP-apoptin cDNA induced apoptosis in some normal cells. When fused to the HIV-TAT protein transduction domain and delivered as a protein, TAT-apoptin was transduced efficiently (>90%) into normal and tumour cells. However, TAT-apoptin remained in the cytoplasm and did not kill normal 6689 and 1BR3 fibroblasts. In contrast TAT-apoptin migrated from the cytoplasm to the nucleus of Saos-2 and HSC-3 cancer cells resulting in apoptosis after 24 h. This study shows that apoptin is a powerful apoptosis-inducing protein with a potential for cancer therapy. PMID:14691460

  5. miRNA143 Induces K562 Cell Apoptosis Through Downregulating BCR-ABL

    PubMed Central

    Liang, Bing; Song, Yanbin; Zheng, Wenling; Ma, Wenli

    2016-01-01

    Background Leukemia seriously threats human health and life. MicroRNA regulates cell growth, proliferation, apoptosis, and cell cycle. Whether microRNA could be treated as a target for leukemia is still unclear and the mechanism by which microRNA143 regulates K562 cells needs further investigation. Material/Methods miRNA143 and its scramble miRNA were synthesized and transfected to K562 cells. MTT assay was used to detect K562 cell proliferation. Flow cytometry and a caspase-3 activity detection kit were used to test K562 cell apoptosis. Western blot analysis was performed to determine breakpoint cluster region-Abelson (BCR-ABL) expression. BCR-ABL overexpression and siRNA were used to change BCR-ABL level, and cell apoptosis was detected again after lipofection transfection. Results miRNA143 transfection inhibited K562 cell growth and induced its apoptosis. miRNA143 transfection decreased BCR-ABL expression. BCR-ABL overexpression suppressed miRNA143-induced K562 cell apoptosis, while its reduction enhanced miRNA143-induced apoptosis. Conclusions miRNA143 induced K562 cell apoptosis through downregulating BCR-ABL. miRNA143 might be a target for a new leukemia therapy. PMID:27492780

  6. A novel role for the apoptosis inhibitor ARC in suppressing TNFα-induced regulated necrosis.

    PubMed

    Kung, G; Dai, P; Deng, L; Kitsis, R N

    2014-04-01

    TNFα signaling can promote apoptosis or a regulated form of necrosis. ARC (apoptosis repressor with CARD (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. We discovered that ARC blocks not only apoptosis but also necrosis. TNFα-induced necrosis was abrogated by overexpression of wild-type ARC but not by a CARD mutant that is also defective for inhibition of apoptosis. Conversely, knockdown of ARC exacerbated TNFα-induced necrosis, an effect that was rescued by reconstitution with wild-type, but not CARD-defective, ARC. Similarly, depletion of ARC in vivo exacerbated necrosis caused by infection with vaccinia virus, which elicits severe tissue damage through this pathway, and sensitized mice to TNFα-induced systemic inflammatory response syndrome. The mechanism underlying these effects is an interaction of ARC with TNF receptor 1 that interferes with recruitment of RIP1, a critical mediator of TNFα-induced regulated necrosis. These findings extend the role of ARC from an apoptosis inhibitor to a regulator of the TNFα pathway and an inhibitor of TNFα-mediated regulated necrosis. PMID:24440909

  7. Hyperthermia Induces Apoptosis of 786-O Cells through Suppressing Ku80 Expression

    PubMed Central

    Qi, Defeng; Hu, Yuan; Li, Jinhui; Peng, Tao; Su, Jialin; He, Yun; Ji, Weidong

    2015-01-01

    Hyperthermia as an anticancer method has been paid increasing attention in recent years. Several studies have shown that hyperthermia can kill tumor cells by inducing apoptosis. However, the underlying molecular mechanisms of hyperthermia-induced apoptosis are largely unknown. To investigate the effects and molecular mechanism of hyperthermia on the apoptosis in renal carcinoma 786-O cells, we firstly examined apoptosis and Ku expression in 786-O cell line treated with heat exposure (42°C for 0-4 h). The results showed that hyperthermia induced apoptosis of 786-O cells, and suppressed significantly Ku80 expression, but not Ku70 expression. Next, we knock-down Ku80 in 786-O cells, generating stable cell line 786-O-shKu80, and detected apoptosis, cell survival and cell cycle distribution. Our data showed higher apoptotic rate and lower surviving fraction in the stable cell line 786-O-shKu80 compared with those in control cells, exposed to the same heat stress (42°C for 0-4 h). Moreover, the results also showed suppression of Ku80 led to G2/M phase arrest in the stable cell line 786-O-shKu80 following heat treatment. Together, these findings indicate that Ku80 may play an important role in hyperthermia-induced apoptosis and heat-sensitivity of renal carcinoma cells through influencing the cell cycle distribution. PMID:25902193

  8. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    SciTech Connect

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

  9. Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction.

    PubMed

    Kuo, Chen-Tzu; Hsu, Ming-Jen; Chen, Bing-Chang; Chen, Chien-Chih; Teng, Che-Ming; Pan, Shiow-Lin; Lin, Chien-Huang

    2008-02-28

    Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis. PMID:18262737

  10. De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis.

    PubMed Central

    Gómez del Pulgar, Teresa; Velasco, Guillermo; Sánchez, Cristina; Haro, Amador; Guzmán, Manuel

    2002-01-01

    Delta(9)-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells. PMID:11903061

  11. Asymmetric dimethylarginine (ADMA) treatment induces apoptosis in cultured rat mesangial cells via endoplasmic reticulum stress activation.

    PubMed

    Park, Min-Jung; Oh, Ki-Seok; Nho, Jong-Hyun; Kim, Gye-Yeop; Kim, Dong-Il

    2016-06-01

    Asymmetric dimethylarginine (ADMA), a high risk factor for endothelial dysfunction and cardiovascular disease (CVD), has been reported to promote cellular dysfunction via endoplasmic reticulum (ER) stress activation in various cells. Additionally, increased serum ADMA levels have been observed in incipient kidney diseases. Previously, we reported that activated ER stress is associated with mesangial cell apoptosis, observed mainly in overt nephropathy or chronic kidney disease (CKD). However, the effect of ADMA on mesangial cell apoptosis is unknown. Thus, we investigated the effects of ADMA on mesangial cell apoptosis and ER stress signaling. ADMA treatment increased caspase-3 activity and activated three branches of ER stress signaling (PERK, IRE1, and ATF6) that induce mesangial cell apoptosis. Pharmacological inhibitors of ER stress (inhibitors of PERK, IRE1, and S1P) attenuated ADMA-induced cleavage of caspase-3 and induced a decrease in the mitochondrial membrane potential. Furthermore, these inhibitors diminished the number of apoptotic cells induced by ADMA treatment. Taken together, our results indicated that ADMA treatment induces mesangial cell apoptosis via ER stress signaling. These results suggest that ADMA-induced mesangial cell apoptosis could contribute to the progression of overt nephropathy and CKD. PMID:26992443

  12. Identification of genes regulating TRAIL-induced apoptosis in rheumatoid arthritis fibroblasts-like synoviocytes.

    PubMed

    Audo, R; Hegglin, A; Severac, D; Dantec, C; Combe, B; Hahne, M; Morel, J

    2015-10-01

    We previously described that sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis varied in rheumatoid arthritis fibroblasts-like synoviocytes (RAFLS) from one patient to another and was correlated with disease severity. Therefore, we screened for genes differentially expressed in RAFLS sensitive and resistant to TRAIL-induced apoptosis. The sensitivity of RAFLS was defined based on the percentage of TRAIL-induced apoptosis: 0-10% for resistant cells and >25% for sensitive RAFLS. We performed transcriptomic comparison between RAFLS-S (n=6) and RAFLS-R (n=6) and then examined the implication of identified candidates in the regulation of apoptosis using small interference RNA (siRNA). Microarray analysis revealed 10 functional genes differentially expressed according to TRAIL sensitivity. These factors are implicated in different functions, such as the respiratory chain (ND3), the transport of lipids (OSBP2, PLTP), the regulation of signaling linked to extracellular factors (SULF2, GALNT1, SIAE) or the regulation of gene expression (TET2 and LARP6). We confirmed differential expression for GALNT1 and LARP6 by quantitative reverse transcriptase-PCR. Using siRNA extinction, we demonstrated the implication of GALNT1, SULF2 and LARP6 in the control of TRAIL-induced responses. These results are of particular interest as GALNT1 and LARP6 have been implicated in the regulation of cell death and may represent interesting targets to induce apoptosis of RAFLS. PMID:26247836

  13. Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection

    PubMed Central

    Hanauske-Abel, Hartmut M.; Saxena, Deepti; Palumbo, Paul E.; Hanauske, Axel-Rainer; Luchessi, Augusto D.; Cambiaghi, Tavane D.; Hoque, Mainul; Spino, Michael; Gandolfi, Darlene D'Alliessi; Heller, Debra S.; Singh, Sukhwinder; Park, Myung Hee; Cracchiolo, Bernadette M.; Tricta, Fernando; Connelly, John; Popowicz, Anthony M.; Cone, Richard A.; Holland, Bart; Pe’ery, Tsafi; Mathews, Michael B.

    2013-01-01

    HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal

  14. Glycogen synthase kinase-3β regulates tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis via the NF-κB pathway in hepatocellular carcinoma

    PubMed Central

    FU, KAI; PAN, HUAZHENG; LIU, SHIHAI; LV, JING; WAN, ZHAOJUN; LI, JIAO; SUN, QING; LIANG, JUN

    2015-01-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for its ability to selectively induce apoptosis in malignant cells. However, human hepatocellular carcinoma (HCC) cells display resistance to TRAIL-induced cell death. The present study investigated whether TRAIL-induced apoptosis in HCC cells was enhanced by the administration of an inhibitor of glycogen synthase kinase-3β (GSK-3β) or by short hairpin RNA-mediated inhibition of GSK-3β. The results of the current study demonstrated that inhibition of GSK-3β significantly impairs the expression of the nuclear factor-κB (NF-κB) target genes Bcl-xL and clAP2 in HCC cells (P<0.05). This indicates that GSK-3β may regulate NF-κB target genes involved in cell survival. Furthermore, knockdown of Bcl-xL significantly enhanced the sensitizing effect of GSK-3β inhibitor on TRAIL-induced apoptosis (P<0.05). Overall, the present study provides a rationale for further exploration of GSK-3β inhibition combined with TRAIL as a novel treatment for HCC. PMID:26788169

  15. Bim contributes to phenethyl isothiocyanate-induced apoptosis in breast cancer cells.

    PubMed

    Hahm, Eun-Ryeong; Singh, Shivendra V

    2012-06-01

    Phenethyl isothiocyanate (PEITC) is a highly promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress) with in vivo efficacy in experimental rodent cancer models. Research thus far implicates apoptosis induction in cancer chemopreventive response to PEITC, but the mechanism of proapoptotic effect is not fully understood. The present study demonstrates that p53 upregulated modulator of apoptosis (PUMA)-independent apoptosis by PEITC is mediated by B-cell lymphoma 2 interacting mediator of cell death (Bim). Exposure of a cell line (BRI-JM04) derived from spontaneously developing mammary tumor of a MMTV-neu transgenic mouse to pharmacological concentrations of PEITC resulted in decreased cell viability coupled with apoptosis induction, characterized by release of histone-associated DNA fragments into the cytosol and cleavage of poly-(ADP-ribose)-polymerase and procaspase-3. The PEITC-induced apoptosis in BRI-JM04 cells was associated with up-regulation of Bak, PUMA, and Bim (long and short forms of Bim), increased S65 phosphorylation of BimEL (extra-long form), and down-regulation of Bcl-xL and Bcl-2. On the other hand, a non-tumorigenic human mammary epithelial cell line (MCF-10A) was significantly more resistant to PEITC-induced apoptosis compared with BRI-JM04 despite induction of Bax and PUMA due to concomitant overexpression of anti-apoptotic proteins, including Bcl-xL, Bcl-2, and Mcl-1. Wild-type HCT-116 cells and its isogenic PUMA knockout variant exhibited comparable sensitivity to PEITC-induced apoptosis. On the other hand, small interfering RNA knockdown of Bim protein imparted partial but statistically significant protection against PEITC-induced apoptosis in BRI-JM04, MCF-7, and MDA-MB-231 cells. In conclusion, the present study provides novel insight into the mechanism of PEITC-induced apoptosis involving Bim. PMID:21739479

  16. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    SciTech Connect

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-03-07

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.

  17. Apoptosis and inhibition of proliferation of cancer cells induced by cordycepin

    PubMed Central

    TIAN, XUEWEN; LI, YUJIAN; SHEN, YINYU; LI, QIAOQIAO; WANG, QINGLU; FENG, LIANSHI

    2015-01-01

    Cordycepin, a 3-deoxyadenosine, is the predominant functional component of the fungus Cordyceps militaris, a traditional Chinese medicine. Previous studies investigating the inhibition of cancer cells by cordycepin identified that it not only promotes cell apoptosis, but also controls cell proliferation. Furthermore, studies have elucidated the molecular mechanisms of inhibiting cell proliferation by cordycepin binding the A3 adenosine receptor, activating G protein, inhibiting cAMP formation, decreasing glycogen synthase kinase-3β/β-catenin activation and suppressing cyclin D1 and c-myc expression. The most significant signaling pathway in which cell apoptosis is induced by cordycepin is the caspase pathway. Cordycepin induces cell apoptosis via binding the DR3 receptor and consequently activating caspase-8/-3. Taken together, these studies demonstrate that cordycepin may be used as a natural medicine, as it can not only control tumor cell proliferation, but also induce cancer cell apoptosis. PMID:26622539

  18. Convallatoxin, a Dual Inducer of Autophagy and Apoptosis, Inhibits Angiogenesis In Vitro and In Vivo

    PubMed Central

    Yang, Seung Ya; Kim, Nam Hee; Cho, Yoon Sun; Lee, Hukeun; Kwon, Ho Jeong

    2014-01-01

    Autophagy and apoptosis are important processes that control cellular homeostasis and have been highlighted as promising targets for novel cancer therapies. Here, we identified convallatoxin (CNT), isolated from Antiaris toxicaria, as a dual inducer of autophagy and apoptosis. CNT exerts cytotoxic effects on a number of cancer and normal cell lines and induces apoptosis by increasing caspase-3 and poly ADP ribose polymerase (PARP) cleavage. Moreover, dose- and time-dependent autophagic activity was detected in CNT-treated cells, and mammalian target of rapamycin (mTOR)/p70S6K signal pathway inhibition was observed. Notably, CNT inhibits human umbilical vein endothelial cell (HUVEC) growth and exerts anti-angiogenic activity in vitro and in vivo. Collectively, these results demonstrate that the naturally occurring compound, CNT, is a novel anti-angiogenic compound via dual inducing of autophagy and apoptosis. PMID:24663328

  19. Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells

    PubMed Central

    Yuneva, Mariia; Zamboni, Nicola; Oefner, Peter; Sachidanandam, Ravi; Lazebnik, Yuri

    2007-01-01

    The idea that conversion of glucose to ATP is an attractive target for cancer therapy has been supported in part by the observation that glucose deprivation induces apoptosis in rodent cells transduced with the proto-oncogene MYC, but not in the parental line. Here, we found that depletion of glucose killed normal human cells irrespective of induced MYC activity and by a mechanism different from apoptosis. However, depletion of glutamine, another major nutrient consumed by cancer cells, induced apoptosis depending on MYC activity. This apoptosis was preceded by depletion of the Krebs cycle intermediates, was prevented by two Krebs cycle substrates, but was unrelated to ATP synthesis or several other reported consequences of glutamine starvation. Our results suggest that the fate of normal human cells should be considered in evaluating nutrient deprivation as a strategy for cancer therapy, and that understanding how glutamine metabolism is linked to cell viability might provide new approaches for treatment of cancer. PMID:17606868

  20. The extracellular matrix protein Del1 induces apoptosis via its epidermal growth factor motif.

    PubMed

    Kitano, Hisataka; Kokubun, Shinichiro; Hidai, Chiaki

    2010-03-19

    Mouse Del1 is an extracellular matrix protein mainly expressed in the developing embryo. Del1 has three EGF motifs and two discoidin domains. The second EGF motif reportedly contains an RGD sequence that binds to integrin receptors. Here, we provide evidence that Del1 protein induces cell death in vitro. Chromatin condensation and DNA laddering were observed, suggestive of apoptosis. The results of analysis using the TUNEL method and annexin V staining were also consistent with apoptosis. The apoptosis-inducing activity of Del1 could be mapped to the third EGF motif, which fitted the consensus sequence CX(D/N)XXXX(F/Y)XCXC, wherein the aspartic acid residue (D) could be beta-hydroxylated. As little as twenty-five picomolar of recombinant E3 could induce apoptosis. PMID:20171188

  1. Prostaglandin D2 induces apoptosis of human osteoclasts by activating the CRTH2 receptor and the intrinsic apoptosis pathway.

    PubMed

    Yue, Li; Durand, Marianne; Lebeau Jacob, M Christian; Hogan, Philippe; McManus, Stephen; Roux, Sophie; de Brum-Fernandes, Artur J

    2012-09-01

    Prostaglandin D(2) (PGD(2)) is a lipid mediator synthesized from arachidonic acid that directly activates two specific receptors, the D-type prostanoid (DP) receptor and chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). PGD(2) can affect bone metabolism by influencing both osteoblast and osteoclast (OC) functions, both cells involved in bone remodeling and in in vivo fracture repair as well. The objective of the present study was to determine the effects of PGD(2), acting through its two specific receptors, on human OC apoptosis. Human OCs were differentiated in vitro from peripheral blood mononuclear cells in the presence of receptor activator for nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and treated with PGD(2), its specific agonists and antagonists. Treatment with PGD(2) for 24hours in the presence of naproxen (10μM) to inhibit endogenous prostaglandin production increased the percentage of apoptotic OCs in a dose-dependent manner, as did the specific CRTH2 agonist compound DK-PGD(2) but not the DP agonist compound BW 245C. In the absence of naproxen, the CRTH2 antagonist compound CAY 10471 reduced OC apoptosis rate but the DP antagonist BW A868C had no effect. The induction of PGD(2)-CRTH2 dependent apoptosis was associated with the activation of caspase-9, but not caspase-8, leading to caspase-3 cleavage. These data show that PGD(2) induces human OC apoptosis through activation of CRTH2 and the apoptosis intrinsic pathway. PMID:22705147

  2. Angiopoietin 2 induces pericyte apoptosis via α3β1 integrin signaling in diabetic retinopathy.

    PubMed

    Park, Sung Wook; Yun, Jang-Hyuk; Kim, Jin Hyoung; Kim, Kyu-Won; Cho, Chung-Hyun; Kim, Jeong Hun

    2014-09-01

    Pericyte loss is an early characteristic change in diabetic retinopathy (DR). Despite accumulating evidence that hyperglycemia-induced angiopoietin 2 (Ang2) has a central role in pericyte loss, the precise molecular mechanism has not been elucidated. This study investigated the role of Ang2 in pericyte loss in DR. We demonstrated that pericyte loss occurred with Ang2 increase in the diabetic mouse retina and that the source of Ang2 could be the endothelial cell. Ang2 induced pericyte apoptosis via the p53 pathway under high glucose, whereas Ang2 alone did not induce apoptosis. Integrin, not Tie-2 receptor, was involved for Ang2-induced pericyte apoptosis under high glucose as an Ang2 receptor. High glucose changed the integrin expression pattern, which increased integrin α3 and β1 in the pericyte. Furthermore, Ang2-induced pericyte apoptosis in vitro was effectively attenuated via p53 suppression by blocking integrin α3 and β1. Although intravitreal injection of Ang2 induced pericyte loss in C57BL/6J mice retina in vivo, intravitreal injection of anti-integrin α3 and β1 antibodies attenuated Ang2-induced pericyte loss. Taken together, Ang2 induced pericyte apoptosis under high glucose via α3β1 integrin. Glycemic control or blocking Ang2/integrin signaling could be a potential therapeutic target to prevent pericyte loss in early DR. PMID:24722242

  3. Ischemia-induced mitochondrial apoptosis is significantly attenuated by ischemic preconditioning.

    PubMed

    Racay, Peter; Chomova, Maria; Tatarkova, Zuzana; Kaplan, Peter; Hatok, Jozef; Dobrota, Dusan

    2009-09-01

    Ischemic preconditioning (IPC) represents an important adaptation of CNS to sub-lethal ischemia, which results in increased tolerance of CNS to the lethal ischemia. Ischemia-induced mitochondrial apoptosis is considered to be an important event leading to neuronal cell death after cerebral blood flow arrest. In presented study, we have determined the effect of IPC on ischemia/reperfusion-induced mitochondrial apoptosis. Global brain ischemia was induced by permanent occlusion of vertebral arteries and temporal occlusion of carotid arteries for 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later 15 min of lethal ischemia was induced. With respect to mitochondrial apoptosis initiation, translocation of p53 to mitochondria was observed in hippocampus but not in cerebral cortex. However, level of both apoptotic bax and anti-apoptotic bcl-xl in both hippocampal and cortical mitochondria was unchanged after global brain ischemia. Detection of genomic DNA fragmentation as well as Fluoro-Jade C staining showed that ischemia induces apoptosis in vulnerable CA1 layer of rat hippocampus. IPC abolished completely ischemia-induced translocation of p53 to mitochondria and had significant protective effect on ischemia-induced DNA fragmentation. In addition, significant decrease of Fluoro-Jade C positive cells was observed as well. Our results indicate that IPC abolished almost completely both initiation and execution of mitochondrial apoptosis induced by global brain ischemia. PMID:19283470

  4. Emodin induces apoptosis of human breast cancer cells by modulating the expression of apoptosis-related genes

    PubMed Central

    ZU, CONG; ZHANG, MINGDI; XUE, HUI; CAI, XIAOPENG; ZHAO, LEI; HE, ANNING; QIN, GUANGYUAN; YANG, CHUNSHU; ZHENG, XINYU

    2015-01-01

    The aim of this study was to investigate the effects of emodin on the proliferation of human breast cancer cells Bcap-37 and ZR-75-30. Cell viability following emodin treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of emodin on apoptosis were determined by flow cytometry using Annexin V-fluorescein isothiocyanate and propidium iodide staining. Quantitative polymerase chain reaction and western blot analysis were used to determine changes in the expression of apoptotic genes and protein, respectively. The effect of emodin on the invasiveness of breast cancer cells was evaluated by Matrigel invasion assay. Treatment of breast cancer cells Bcap-37 and ZR-75-30 with emodin was observed to inhibit the growth and induced apoptosis in a time- and dose-dependent manner. Emodin reduced the level of Bcl-2 and increased levels of cleaved caspase-3, PARP, p53 and Bax. These findings indicate that emodin induces growth inhibition and apoptosis in human breast cancer cells. Emodin may be a potential therapeutic agent for the treatment of breast cancer. PMID:26722264

  5. Low intensity-pulsed ultrasound induced apoptosis of human hepatocellular carcinoma cells in vitro.

    PubMed

    Shi, Mingfang; Liu, Bangzhong; Liu, Guanghua; Wang, Ping; Yang, Mingzhen; Li, Yun; Zhou, Jian

    2016-01-01

    The present study was conducted to determine whether low intensity-pulsed ultrasound (LIPUS) could induce apoptosis of human hepatocellular carcinoma cells, SMMC-7721, and to define the mechanism of ultrasound-induced apoptosis, in vitro. MTT assay was used to measure cell proliferation. Apoptosis was investigated by multiple methods such as flow cytometry, DNA fragmentation, Ca(2+) mobilizations, pro- and anti-apoptotic protein expression, and light as well as ultramicroscopic morphology. The results provide evidence that LIPUS induced a dose-dependent effect on cell viability and apoptosis of SMMC-7721 cells. Specifically, exposure of cells to >0.5 W/cm(2) intensity significantly increased cell apoptosis, caused shifts in cell cycle phase, and induced structural changes. Ultrasound significantly increased intracellular Ca(2+) concentrations and modulated expression of caspase-3, Bcl-2 and Bax. The findings suggest that this novel technology can be used to induce SMMC-7721 apoptosis via the Ca(2+)/mitochondrial pathway and could potentially be of clinical use for the treatment of hepatocellular carcinoma (SMMC-7721 cell line) and other cancers. PMID:26231998

  6. Heat stress impairs mice granulosa cell function by diminishing steroids production and inducing apoptosis.

    PubMed

    Luo, Man; Li, Lian; Xiao, Cheng; Sun, Yu; Wang, Gen-Lin

    2016-01-01

    Ovarian injury can be induced by heat stress. Mice granulosa cells (GCs) are critical for normal ovarian function and they synthesize a variety of growth factors and steroids for the follicle. Furthermore, the growth, differentiation, and maturate of theca cells and oocyte are dependent upon the synthesis of GCs. Due to the critical biological functions of GCs, we hypothesized that the apoptosis and dysfunction of GCs could also be induced by heat stress. We analyzed GCs apoptosis and evaluated the expression of apoptosis-related genes (caspase-3, Bax, Bcl-2) after heat treatment. Radio immunity assay was used to measure the secretion of 17β-estradiol (E2) and progesterone (P4). RT-PCR was used to evaluate the expression of steroids-related genes (Star, CYP11A1, CYP19A1). Our data suggested that heat stress inhibited GCs proliferation, induced GCs apoptosis, decreased E2 and P4 secretion, reduced the steroids-related genes mRNA expression. Besides, our results indicated that heat treatment-induced apoptosis of GCs through the mitochondrial pathway, which involved caspase-3 and Bax. The reduction in steroids secretion and mRNA expression of Star, CYP11A1, and CYP19A1 might also play a role in heat-induced GCs apoptosis and ovarian injury. PMID:26602771

  7. Role of mycobacteria-induced monocyte/macrophage apoptosis in the pathogenesis of human tuberculosis.

    PubMed

    Bocchino, M; Galati, D; Sanduzzi, A; Colizzi, V; Brunetti, E; Mancino, G

    2005-04-01

    Apoptosis is a physiological programmed cell death process whose dysregulation plays an important role in different human infectious diseases. An increasing number of intracellular pathogens are known to induce target cell apoptosis, while some other parasites inhibit it. Unlike necrosis, apoptosis is a silent immunological event occurring without inflammation. Infection-induced target cell apoptosis may be a successful strategy to eliminate pathogens and assure host survival. Conversely, apoptosis inhibition could represent an adaptive mechanism for pathogen survival, while it may be beneficial for the host to initiate an effective immune response. The worldwide increase in tuberculosis has stimulated more research aimed at defining the interaction between Mycobacterium tuberculosis and the immune system. M. tuberculosis possesses sophisticated strategies to circumvent its fate within target monocytic cells. Apoptosis of alveolar macrophages and monocytes has been described as a consequence of M. tuberculosis infection. Moreover, the observation that mycobacterial lipoproteins activate macrophages through Toll-like receptor (TLR) 2 suggests that innate immune receptors contribute to defence against M. tuberculosis. There is evidence that TLR-induced apoptosis modulates inflammation and immune activation during M. tuberculosis infection. Finally, the role of apoptotic-infected cells as a source of microbial antigens for cross-priming of effector T-cells is also discussed. PMID:15830742

  8. Tumor necrosis factor-alpha-induced apoptosis in hepatocytes in long-term culture.

    PubMed Central

    Bour, E. S.; Ward, L. K.; Cornman, G. A.; Isom, H. C.

    1996-01-01

    Apoptosis occurs naturally in the liver and increases in specific pathogenic processes. We previously described the use of a chemically defined medium supplemented with epidermal growth factor and dimethylsulfoxide to maintain rat hepatocytes in a highly differentiated state for more than 30 days (long-term culture). In this study, we showed that hepatocytes in long-term dimethylsulfoxide culture have definite advantages over using cells in short-term culture (cells in culture for 2 to 4 days) to study apoptosis. We demonstrated that treatment with tumor necrosis factor (TNF)-alpha induced apoptosis (detected morphologically and by formation of an oligonucleosomal DNA ladder) only in hepatocytes that had been subjected to dimethylsulfoxide removal. Neither treatment with TNF-alpha alone or dimethylsulfoxide removal alone induced apoptosis. Apoptosis could be induced by concentrations as low as 500 U of TNF-alpha/ml. Although a DNA ladder was not detected by 12 hours after TNF-alpha treatment, it was easily identified by 24 hours. We conclude that this system can be used 1) to examine the underlying mechanism by which TNF-alpha causes apoptosis in hepatocytes and 2) to study induction of apoptosis in hepatocytes by other agents. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:8579111

  9. Rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways.

    PubMed

    Sarmento, Luciana; Tseggai, Tesfai; Dhingra, Vikas; Fu, Zhen F

    2006-11-01

    Previously, it has been shown that the laboratory attenuated rabies virus CVS-B2C, but not the wild-type virus SHBRV, induces apoptosis in mice and the induction of apoptosis is mediated by viral glycoprotein. Induction of apoptosis by CVS-B2C limits the spread of the virus in the CNS. In the present study, we characterized the pathways by which CVS-B2C induces apoptosis. BSR cells were infected with CVS-B2C or SHBRV and harvested at different time points for detection of apoptosis by immunofluorescence and flow cytometry. Apoptosis was detected only in cells infected with CVS-B2C, but not SHBRV. Caspase activity and expression of several apoptotic proteins were analyzed by fluorometric assay and Western blotting. Activation of caspase-8 and -3, but not of caspase-9, was observed in CVS-B2C-infected cells. In addition, the level of expression of Apaf-1 did not change. Furthermore, PARP was cleaved confirming activation of downstream caspases. All these data suggest that CVS-B2C infection activates the extrinsic, but not the intrinsic, apoptotic pathway. In addition, AIF, a caspase-independent apoptotic protein was up-regulated and translocated from the cytoplasm to the nucleus post-infection, suggesting that apoptosis induced by CVS-B2C also involves the activation of a caspase-independent pathway. PMID:16814422

  10. Anacardic acid induces apoptosis-like cell death in the rice blast fungus Magnaporthe oryzae.

    PubMed

    Muzaffar, Suhail; Bose, Chinchu; Banerji, Ashok; Nair, Bipin G; Chattoo, Bharat B

    2016-01-01

    Anacardic acid (6-pentadecylsalicylic acid), extracted from cashew nut shell liquid, is a natural phenolic lipid well known for its strong antibacterial, antioxidant, and anticancer activities. Its effect has been well studied in bacterial and mammalian systems but remains largely unexplored in fungi. The present study identifies antifungal, cytotoxic, and antioxidant activities of anacardic acid in the rice blast fungus Magnaporthe oryzae. It was found that anacardic acid causes inhibition of conidial germination and mycelial growth in this ascomycetous fungus. Phosphatidylserine externalization, chromatin condensation, DNA degradation, and loss of mitochondrial membrane potential suggest that growth inhibition of fungus is mainly caused by apoptosis-like cell death. Broad-spectrum caspase inhibitor Z-VAD-FMK treatment indicated that anacardic acid induces caspase-independent apoptosis in M. oryzae. Expression of a predicted ortholog of apoptosis-inducing factor (AIF) was upregulated during the process of apoptosis, suggesting the possibility of mitochondria dependent apoptosis via activation of apoptosis-inducing factor. Anacardic acid treatment leads to decrease in reactive oxygen species rather than increase in reactive oxygen species (ROS) accumulation normally observed during apoptosis, confirming the antioxidant properties of anacardic acid as suggested by earlier reports. Our study also shows that anacardic acid renders the fungus highly sensitive to DNA damaging agents like ethyl methanesulfonate (EMS). Treatment of rice leaves with anacardic acid prevents M. oryzae from infecting the plant without affecting the leaf, suggesting that anacardic acid can be an effective antifungal agent. PMID:26381667

  11. Chlorpromazine Protects Against Apoptosis Induced by Exogenous Stimuli in the Developing Rat Brain

    PubMed Central

    Li, Yujun; Zhang, Qingmeng; Chen, Yang; Fu, Yingmei; Fang, Wenjuan; Wang, Jindong; Zhong, Zhaohua; Ling, Hong; Zhang, Liming; Zhang, Fengmin

    2011-01-01

    Background Chlorpromazine (CPZ), a commonly used antipsychotic drug, was found to play a neuroprotective role in various models of toxicity. However, whether CPZ has the potential to affect brain apoptosis in vivo is still unknown. The purpose of this study was to investigate the potential effect of CPZ on the apoptosis induced by exogenous stimuli. Methodology The ethanol treated infant rat was utilized as a valid apoptotic model, which is commonly used and could trigger robust apoptosis in brain tissue. Prior to the induction of apoptosis by subcutaneous injection of ethanol, 7-day-old rats were treated with CPZ at several doses (5 mg/kg, 10 mg/kg and 20 mg/kg) by intraperitoneal injection. Apoptotic cells in the brain were measured using TUNEL analysis, and the levels of cleaved caspase-3, cytochrome c, the pro-apoptotic factor Bax and the anti-apoptotic factor Bcl-2 were assessed by immunostaining or western blot. Findings Compared to the group injected with ethanol only, the brains of the CPZ-pretreated rats had fewer apoptotic cells, lower expression of cleaved caspase-3, cytochrome c and Bax, and higher expression of Bcl-2. These results demonstrate that CPZ could prevent apoptosis in the brain by regulating the mitochondrial pathway. Conclusions CPZ exerts an inhibitory effect on apoptosis induced by ethanol in the rat brain, intimating that it may offer a means of protecting nerve cells from apoptosis induced by exogenous stimuli. PMID:21779358

  12. Autophagy Protects Monocytes from Wolbachia Heat Shock Protein 60–Induced Apoptosis and Senescence

    PubMed Central

    Kamalakannan, Vijayan; Shiny, Abijit; Babu, Subash; Narayanan, Rangarajan Badri

    2015-01-01

    Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60) interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS) induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4–mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence. PMID:25849993

  13. Autophagy protects monocytes from Wolbachia heat shock protein 60-induced apoptosis and senescence.

    PubMed

    Kamalakannan, Vijayan; Shiny, Abijit; Babu, Subash; Narayanan, Rangarajan Badri

    2015-04-01

    Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60) interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS) induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4-mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence. PMID:25849993

  14. The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma.

    PubMed

    van Roosmalen, Ingrid A M; Reis, Carlos R; Setroikromo, Rita; Yuvaraj, Saravanan; Joseph, Justin V; Tepper, Pieter G; Kruyt, Frank A E; Quax, Wim J

    2014-01-01

    Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these

  15. Gambogic acid potentiates clopidogrel-induced apoptosis and attenuates irinotecan-induced apoptosis through down-regulating human carboxylesterase 1 and -2.

    PubMed

    Ning, Rui; Wang, Xiao-Ping; Zhan, Yun-Ran; Qi, Qi; Huang, Xue-Feng; Hu, Gang; Guo, Qing-Long; Liu, Wei; Yang, Jian

    2016-09-01

    1. In this study, we report that gambogic acid (GA), a promising anticancer agent, potentiates clopidogrel-induced apoptosis and attenuates CPT-11-induced apoptosis by down-regulating human carboxylesterase (CES) 1 and -2 via ERK and p38 MAPK pathway activation, which provides a molecular explanation linking the effect of drug combination directly to the decreased capacity of hydrolytic biotransformation. 2. The expression levels of CES1 and CES2 decreased significantly in a concentration- and time-dependent manner in response to GA in Huh7 and HepG2 cells; hydrolytic activity was also reduced. 3. The results showed that pretreatment with GA potentiated clopidogrel-induced apoptosis by down-regulating CES1. Moreover, the GA-mediated repression of CES2 attenuated CPT-11-induced apoptosis. 4. Furthermore, the ERK and p38 MAPK pathways were involved in the GA-mediated down-regulation of CES1 and CES2. 5. Taken together, our data suggest that GA is a potent repressor of CES1 and CES2 and that combination with GA will affect the metabolism of drugs containing ester bonds. PMID:26750665

  16. N-acetyl-L-cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors.

    PubMed

    Kucuksayan, Ertan; Cort, Aysegul; Timur, Mujgan; Ozdemir, Evrim; Yucel, Suleyman Gultekin; Ozben, Tomris

    2013-07-01

    Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N-acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA-2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase-3, -8, -9 activities and Bcl-2, Bax, Cyt-c, Annexin V-FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD50) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD50 ) and H2O2 for 24 h increased Caspase-3, -8, -9 activities, Cyt-c and Bax levels and decreased Bcl-2 levels. The concurrent incubation of NT2 cells with bleomycin/H2O2 and NAC (5 mM) for 24 h abolished bleomycin/H2O2-dependent increases in Caspase-3, -8, -9 activities, Bax and Cyt-c levels and bleomycin/H2O2-dependent decrease in Bcl-2 level. Our results indicate that bleomycin/H2O2 induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H2O2 induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin-induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells. PMID:23386420

  17. Parkia javanica Extract Induces Apoptosis in S-180 Cells via the Intrinsic Pathway of Apoptosis.

    PubMed

    Patra, Kartick; Jana, Samarjit; Sarkar, Arnab; Karmakar, Subrata; Jana, Jagannath; Gupta, Mradu; Mukherjee, Gopeswar; De, Utpal Chandra; Mandal, Deba Prasad; Bhattacharjee, Shamee

    2016-01-01

    Parkia javanica is a leguminous tree, various parts of which are used as food and folklore medicine by the ethnic groups of northeastern India. The present study investigates the in vitro and in vivo anticancer effect of aqueous methanol extract of P. javanica fruit (PJE). HPLC analysis was done to establish the fingerprint chromatogram of PJE and its in vitro radical scavenging activity was measured. PJE caused significant cytotoxicity in sarcoma-180 (S-180), A549, AGS, and MDA-MB435S cancer cells in vitro. Exploration of the mechanistic details in S-180 cells suggested that the reduced cell viability was mediated by induction of apoptosis. Increased expression of proapoptotic proteins such as p53, p21, Bax/Bcl2, cytochrome c (Cyt c), caspase 9, and cleaved poly(ADP-ribose) polymerase, and decrease in proliferative and antiapoptotic markers (Ki-67, Proliferating Cell Nuclear Antigen [PCNA], Bcl-2) validated the anticancer effect of PJE. A decline in the relative fluorescence emission upon staining S-180 cells with Rhodamine 123 (Rh 123), enhanced expression of cytosolic Cyt c and mitochondrial Bax, and inhibition of apoptosis in the presence of caspase-9 inhibitor in PJE-treated cells indicated intrinsic pathway of apoptosis. Liver function test and hepatic antioxidant enzymes demonstrated non-toxicity of PJE. Finally, the detection of PJE in sera by HPLC confirmed its bioavailability. PMID:27144503

  18. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    SciTech Connect

    Huang, Shi-Wei; Wu, Chun-Ying; Wang, Yen-Ting; Kao, Jun-Kai; Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu; Chiu, Husan-Wen; Chang, Chuan-Hsun; Liang, Shu-Mei; Chen, Yi-Ju; Huang, Jau-Ling; Shieh, Jeng-Jer

    2013-02-15

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.

  19. Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis.

    PubMed Central

    Jaffrézou, J P; Levade, T; Bettaïeb, A; Andrieu, N; Bezombes, C; Maestre, N; Vermeersch, S; Rousse, A; Laurent, G

    1996-01-01

    The nature of the signaling pathway(s) which initiate drug-triggered apoptosis remains largely unknown and is of fundamental importance in understanding cell death induced by chemotherapeutic agents. Here we show that in the leukemic cell lines U937 and HL-60, daunorubicin, at concentrations which trigger apoptosis, stimulated two distinct cycles of sphingomyelin hydrolysis (approximately 20% decrease at 1 microM) within 4-10 min and 60-75 min with concomitant ceramide generation. We demonstrate that the increase in ceramide levels, which precedes apoptosis, is mediated by a neutral sphingomyelinase and not by ceramide synthase. Indeed, potent ceramide synthase inhibitors such as fumonisin B1 did not affect daunorubicin-triggered sphingomyelin hydrolysis, ceramide generation or apoptosis. In conclusion, we provide evidence that daunorubicin-triggered apoptosis is mediated by a signaling pathway which is initiated by an early sphingomyelin-derived ceramide production. Images PMID:8665849

  20. Involvement of seven in absentia homolog-1 in ethanol-induced apoptosis in neural crest cells

    PubMed Central

    Sun, Haijing; Chen, Xiaopan; Yuan, Fuqiang; Liu, Jie; Zhao, Yingming; Chen, Shao-yu

    2014-01-01

    Ethanol-induced apoptosis in selected cell populations is a major component of pathogenesis underlying ethanol-induced teratogenesis. However, there is a fundamental gap in understanding how ethanol leads to apoptosis in embryos. In this study, we investigate the role of seven in absentia homolog-1 (Siah1) protein, an E3 ubiquitin ligase, in ethanol-induced apoptosis. Using an in vitro model of neural crest cell (NCC), JoMa1.3 cells, we found that exposure to 100 mM ethanol resulted in a significant increase in Siah1 mRNA expression in NCCs, an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Treatment with 100 mM ethanol for 24 hours also significantly increased the protein expression of Siah1 in JoMa1.3 cells. The nuclear translocation and accumulation of Siah1 was evidenced in the cells exposed to ethanol. In addition, we have found that the inhibition of Siah1 function with siRNA prevents ethanol-induced increase in Siah1 protein expression and nuclear translocation in NCCs. Down-regulation of Siah1 by siRNA also greatly diminished ethanol-induced cell death and caspase-3 activation, indicating that inhibition of Siah1 can attenuate ethanol-induced apoptosis. These results strongly suggest that Siah1 plays an important role in ethanol-induced apoptosis in NCCs. PMID:25193017

  1. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    SciTech Connect

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang Xiao, Shaobo; Chen, Huanchun

    2014-10-03

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.

  2. Bozepinib, a novel small antitumor agent, induces PKR-mediated apoptosis and synergizes with IFNα triggering apoptosis, autophagy and senescence.

    PubMed

    Marchal, Juan Antonio; Carrasco, Esther; Ramirez, Alberto; Jiménez, Gema; Olmedo, Carmen; Peran, Macarena; Agil, Ahmad; Conejo-García, Ana; Cruz-López, Olga; Campos, Joaquin María; García, María Ángel

    2013-01-01

    Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine] is a potent antitumor compound that is able to induce apoptosis in breast cancer cells. In the present study, we show that bozepinib also has antitumor activity in colon cancer cells, showing 50% inhibitory concentration (IC50) values lower than those described for breast cancer cells and suggesting great potential of this synthetic drug in the treatment of cancer. We identified that the double-stranded RNA-dependent protein kinase (PKR) is a target of bozepinib, being upregulated and activated by the drug. However, p53 was not affected by bozepinib, and was not necessary for induction of apoptosis in either breast or colon cancer cells. In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR. Moreover, we report here, for the first time, that in combined therapy, IFNα induces a clear process of autophagosome formation, and prior treatment with chloroquine, an autophagy inhibitor, is able to significantly reduce IFNα/bozepinib-induced cell death. Finally, we observed that a minor population of caspase 3-deficient MCF-7 cells persisted during long-term treatment with lower doses of bozepinib and the bozepinib/IFNα combination. Curiously, this population showed β-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone. Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy. PMID:24194639

  3. Bozepinib, a novel small antitumor agent, induces PKR-mediated apoptosis and synergizes with IFNα triggering apoptosis, autophagy and senescence

    PubMed Central

    Marchal, Juan Antonio; Carrasco, Esther; Ramirez, Alberto; Jiménez, Gema; Olmedo, Carmen; Peran, Macarena; Agil, Ahmad; Conejo-García, Ana; Cruz-López, Olga; Campos, Joaquin María; García, María Ángel

    2013-01-01

    Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine] is a potent antitumor compound that is able to induce apoptosis in breast cancer cells. In the present study, we show that bozepinib also has antitumor activity in colon cancer cells, showing 50% inhibitory concentration (IC50) values lower than those described for breast cancer cells and suggesting great potential of this synthetic drug in the treatment of cancer. We identified that the double-stranded RNA-dependent protein kinase (PKR) is a target of bozepinib, being upregulated and activated by the drug. However, p53 was not affected by bozepinib, and was not necessary for induction of apoptosis in either breast or colon cancer cells. In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR. Moreover, we report here, for the first time, that in combined therapy, IFNα induces a clear process of autophagosome formation, and prior treatment with chloroquine, an autophagy inhibitor, is able to significantly reduce IFNα/bozepinib-induced cell death. Finally, we observed that a minor population of caspase 3-deficient MCF-7 cells persisted during long-term treatment with lower doses of bozepinib and the bozepinib/IFNα combination. Curiously, this population showed β-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone. Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy. PMID:24194639

  4. Hispidulin induces mitochondrial apoptosis in acute myeloid leukemia cells by targeting extracellular matrix metalloproteinase inducer

    PubMed Central

    Gao, Hui; Liu, Yongji; Li, Kan; Wu, Tianhui; Peng, Jianjun; Jing, Fanbo

    2016-01-01

    Acute myeloid leukemia (AML) represents a heterogeneous group of hematological neoplasms with marked heterogeneity in response to both standard therapy and survival. Hispidulin, a flavonoid compound that is anactive ingredient in the traditional Chinese medicinal herb Salvia plebeia R. Br, has recently been reported to have anantitumor effect against solid tumors in vitro and in vivo. The aim of the present study was to investigate the effects of hispidulin on the human leukemia cell line in vitro and the underlying mechanisms of its actions on these cells. Our results showed that hispidulin inhibits AML cell proliferation in a dose- and time-dependent manner, and induces cell apoptosis throughan intrinsic mitochondrial pathway. Our results also revealed that hispidulin treatment significantly inhibits extracellular matrix metalloproteinase inducer (EMMPRIN) expression in both tested AML cell lines in a dose-dependent manner, and that the overexpression of EMMPRIN protein markedly attenuates hispidulin-induced cell apoptosis. Furthermore, our results strongly indicated that the modulating effect of hispidulin on EMMPRIN is correlated with its inhibitory effect on both the Akt and STAT3 signaling pathways. PMID:27158398

  5. Evidence of early involvement of apoptosis inducing factor-induced neuronal death in Alzheimer brain

    PubMed Central

    Lee, Ji-Hye; Cheon, Young-Hee; Woo, Ran-Sook; Song, Dae-Yong; Moon, Cheil

    2012-01-01

    Apoptosis inducing factor (AIF) has been proposed to act as a putative reactive oxygen species scavenger in mitochondria. When apoptotic cell death is triggered, AIF translocates to the nucleus, where it leads to nuclear chromatin condensation and large-scale DNA fragmentation which result in caspase-independent neuronal death. We performed this study to investigate the possibility that, in addition to caspase-dependent neuronal death, AIF induced neuronal death could be a cause of neuronal death in Alzheimer's disease (AD). We have found that AIF immunoreactivity was increased in the hippocampal pyramidal neurons in the Alzheimer brains compared to those of healthy, age-matched control brains. Nuclear AIF immunoreactivity was detected in the apoptotic pyramidal CA1 neurons at the early stage of AD and CA2 at the advanced stage. Nuclear AIF positive neurons were also observed in the amygdala and cholinergic neurons of the basal forebrain (BFCN) from the early stages of AD. The results of this study imply that AIF-induced apoptosis may contribute to neuronal death within the hippocampus, amygdala, and BFCN in early of AD. PMID:22536549

  6. Neuroprotective effects of Rhizoma Dioscoreae polysaccharides against neuronal apoptosis induced by in vitro hypoxia

    PubMed Central

    XIANG, QIN; ZHOU, WEN-YUN; HU, WEI-XU; WEN, ZHU; HE, DAN; WU, XIAO-MU; WEI, HUI-PING; WANG, WEN-DING; HU, GUO-ZHU

    2015-01-01

    Rhizoma Dioscoreae polysaccharides (RDPS) are the primary active ingredient of Rhizoma Dioscoreae, which is a traditional Chinese medicine. RDPS have previously been shown to scavenge reactive oxygen species, and protect against D-galactose-induced mimetic aging. The present study aimed to investigate the neuroprotective effects of RDPS against hypoxia-induced neuronal cell apoptosis. Neuronal cells harvested from pregnant Sprague-Dawley rats were divided into groups, as follows: i) Normal control group; ii) hypoxia-induced apoptosis neuronal cell model; iii) 0.025 g/l RDPS-treated group; iv) 0.05 g/l RDPS-treated group; v) 0.1 g/l RDPS-treated group; and vi) 0.25 g/l RDPS treated group. Neuronal cell viability was investigated using an MTT assay, and neuronal cell apoptosis was analyzed using Annexin V-fluorescein isothiocyanate/propidium iodide double-staining, Hoechst 33342 fluorescent staining, Rhodamine 123 staining, polymerase chain reaction and immunocytochemical staining. The RDPS-treated neuronal cells exhibited improved viability, and decreased hypoxia-induced mitochondrial injury and apoptosis. In addition, the mRNA and protein expression levels of caspase-3 and B-cell lymphoma (Bcl)-2-associated X protein (Bax) were significantly downregulated, whereas the mRNA and protein expression levels of Bcl-2 were significantly upregulated, in the RDPS-treated hypoxic neurons, as compared with the apoptosis model (P<0.05). Furthermore, the ratio of Bcl-2 expression:Bax expression significantly increased following RDPS treatment, as compared with the apoptosis model (P<0.05). The results of the present study suggested that RDPS may attenuate hypoxia-induced neuronal cell apoptosis by altering the expression levels of key apoptosis-regulating proteins in hypoxic neurons. PMID:26668596

  7. Nucleostemin Knockdown Sensitizes Hepatocellular Carcinoma Cells to Ultraviolet and Serum Starvation-Induced Apoptosis.

    PubMed

    Yuan, Fuwen; Cheng, Qian; Li, Guodong; Tong, Tanjun

    2015-01-01

    Nucleostemin (NS) is a GTP-binding protein that is predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS plays an essential role in maintaining the continuous proliferation of stem cells and some types of cancer cells. However, the role of NS in hepatocellular carcinoma (HCC) remains unclear. Therefore, this study aimed to clarify the role of NS in HCC. First, we demonstrated high expression of NS in most HCC cell lines and liver cancer tissues. NS knockdown induced a severe decline in cell viability of MHCC97H cells as detected by MTT and cell proliferation assays. Next, we used ultraviolet (UV) and serum starvation-induced apoptosis models to investigate whether NS suppression or up-regulation affects HCC cell apoptosis. After UV treatment or serum starvation, apoptosis was strongly enhanced in MHCC97H and Bel7402 cells transfected with small interfering RNA against NS, whereas NS overexpression inhibited UV- and serum-induced apoptosis of HCC cells. Furthermore, after UV irradiation, inhibition of NS increased the expression of pro-apoptosis protein caspase 3 and decreased the expression of anti-apoptosis protein Bcl-2. A caspase 3 inhibitor could obviously prevent NS knockdown-induced apoptosis. In conclusion, our study demonstrated overexpression of NS in most HCC tissues compared with their matched surrounding tissues, and silencing NS promoted UV- and serum starvation-induced apoptosis of MHCC97H and Bel7402 cells. Therefore, the NS gene might be a potential therapeutic target of HCC. PMID:26517370

  8. Nucleostemin Knockdown Sensitizes Hepatocellular Carcinoma Cells to Ultraviolet and Serum Starvation-Induced Apoptosis

    PubMed Central

    Li, Guodong; Tong, Tanjun

    2015-01-01

    Nucleostemin (NS) is a GTP-binding protein that is predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS plays an essential role in maintaining the continuous proliferation of stem cells and some types of cancer cells. However, the role of NS in hepatocellular carcinoma (HCC) remains unclear. Therefore, this study aimed to clarify the role of NS in HCC. First, we demonstrated high expression of NS in most HCC cell lines and liver cancer tissues. NS knockdown induced a severe decline in cell viability of MHCC97H cells as detected by MTT and cell proliferation assays. Next, we used ultraviolet (UV) and serum starvation-induced apoptosis models to investigate whether NS suppression or up-regulation affects HCC cell apoptosis. After UV treatment or serum starvation, apoptosis was strongly enhanced in MHCC97H and Bel7402 cells transfected with small interfering RNA against NS, whereas NS overexpression inhibited UV- and serum-induced apoptosis of HCC cells. Furthermore, after UV irradiation, inhibition of NS increased the expression of pro-apoptosis protein caspase 3 and decreased the expression of anti-apoptosis protein Bcl-2. A caspase 3 inhibitor could obviously prevent NS knockdown-induced apoptosis. In conclusion, our study demonstrated overexpression of NS in most HCC tissues compared with their matched surrounding tissues, and silencing NS promoted UV- and serum starvation-induced apoptosis of MHCC97H and Bel7402 cells. Therefore, the NS gene might be a potential therapeutic target of HCC. PMID:26517370

  9. Apoptosis and morphological alterations after UVA irradiation in red blood cells of p53 deficient Japanese medaka (Oryzias latipes).

    PubMed

    Sayed, Alla El-Din Hamid; Watanabe-Asaka, Tomomi; Oda, Shoji; Mitani, Hiroshi

    2016-08-01

    Morphological alterations in red blood cells were described as hematological bioindicators of UVA exposure to investigate the sensitivity to UVA in wild type Japanese medaka (Oryzias latipes) and a p53 deficient mutant. The fewer abnormal red blood cells were observed in the p53 mutant fish under the control conditions. After exposure to different doses of UVA radiation (15min, 30min and 60min/day for 3days), cellular and nuclear alterations in red blood cells were analyzed in the UVA exposed fish compared with non-exposed controls and those alterations included acanthocytes, cell membrane lysis, swollen cells, teardrop-like cell, hemolyzed cells and sickle cells. Those alterations were increased after the UVA exposure both in wild type and the p53 deficient fish. Moreover, apoptosis analyzed by acridine orange assay showed increased number of apoptosis in red blood cells at the higher UVA exposure dose. No micronuclei but nuclear abnormalities as eccentric nucleus, nuclear budding, deformed nucleus, and bilobed nucleus were observed in each group. These results suggested that UVA exposure induced both p53 dependent and independent apoptosis and morphological alterations in red blood cells but less sensitive to UVA than Wild type in medaka fish. PMID:27203565

  10. Hericium erinaceus polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis.

    PubMed

    Zan, Xinyi; Cui, Fengjie; Li, Yunhong; Yang, Yan; Wu, Di; Sun, Wenjing; Ping, Lifeng

    2015-05-01

    HEG-5 is a novel polysaccharide-protein purified from the fermented mycelia of Hericium erinaceus CZ-2. The present study aims to investigate the effects of HEG-5 on proliferation, cell cycle and apoptosis of human gastric cancer cells SGC-7901. Here, we first uncover that HEG-5 significantly inhibited the proliferation and colony formation of SGC-7901 cells by promoting apoptosis and cell cycle arrest at S phase. RT-PCR and Western blot analysis suggested that HEG-5 could decrease the expressions of Bcl2, PI3K and AKT1, while increase the expressions of Caspase-8, Caspase-3, p53, CDK4, Bax and Bad. These findings indicated that the Caspase-8/-3-dependent, p53-dependent mitochondrial-mediated and PI3k/Akt signaling pathways involved in the molecular events of HEG-5 induced apoptosis and cell cycle arrest. Thus, our study provides in vitro evidence that HEG-5 may be taken as a potential candidate for treating gastric cancer. PMID:25703932

  11. Integrated high-throughput analysis identifies Sp1 as a crucial determinant of p53-mediated apoptosis

    PubMed Central

    Li, H; Zhang, Y; Ströse, A; Tedesco, D; Gurova, K; Selivanova, G

    2014-01-01

    The restoration of p53 tumor suppressor function is a promising therapeutic strategy to combat cancer. However, the biological outcomes of p53 activation, ranging from the promotion of growth arrest to the induction of cell death, are hard to predict, which limits the clinical application of p53-based therapies. In the present study, we performed an integrated analysis of genome-wide short hairpin RNA screen and gene expression data and uncovered a previously unrecognized role of Sp1 as a central modulator of the transcriptional response induced by p53 that leads to robust induction of apoptosis. Sp1 is indispensable for the pro-apoptotic transcriptional repression by p53, but not for the induction of pro-apoptotic genes. Furthermore, the p53-dependent pro-apoptotic transcriptional repression required the co-binding of Sp1 to p53 target genes. Our results also highlight that Sp1 shares with p53 a common regulator, MDM2, which targets Sp1 for proteasomal degradation. This uncovers a new mechanism of the tight control of apoptosis in cells. Our study advances the understanding of the molecular basis of p53-mediated apoptosis and implicates Sp1 as one of its key modulators. We found that small molecules reactivating p53 can differentially modulate Sp1, thus providing insights into how to manipulate p53 response in a controlled way. PMID:24971482

  12. Insulin-like growth factor binding protein-3 induces apoptosis in MCF7 breast cancer cells.

    PubMed

    Nickerson, T; Huynh, H; Pollak, M

    1997-08-28

    Insulin-like growth factors (IGFs) are known to have potent antiapoptotic activity. The antiestrogen ICI 182,780 (ICI) is a potent inhibitor of MCF7 human breast cancer cell growth and has recently been reported to act as an antiproliferative agent in part via upregulation of expression of insulin-like growth factor binding proteins (IGFBPs) -3 and -5, which attenuate the bioactivity of IGFs in many experimental systems. We show here that ICI and IGFBP-3 induce apoptosis in MCF7 cells. Treatment of MCF7 cells with 10 nM ICI or 36 nM recombinant human IGFBP. 3 for 72 hours increased apoptosis approximately 3.5-fold relative to control as quantitated by a cell death ELISA which measures DNA fragmentation. Long R3 IGF-I, an IGF-I analogue with greatly reduced affinity for IGFBPs yet similar affinity for IGF-I receptors, was a more potent inhibitor of IGFBP-3-induced and ICI-induced apoptosis than IGF-I. These results suggest that IGFBP-3 enhances apoptosis by reducing bioavailability of ligands for the IGF-I receptor and suggest that modulation of IGFBP-3 expression by ICI contributes to apoptosis induced by this compound. More generally, the data suggest that IGFBPs are regulators of apoptosis. PMID:9299428

  13. NOS1AP O-GlcNAc Modification Involved in Neuron Apoptosis Induced by Excitotoxicity.

    PubMed

    Zhu, Liang; Tao, Tao; Zhang, Dongmei; Liu, Xiaojuan; Ke, Kaifu; Shen, Aiguo

    2015-01-01

    O-Linked N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic and mitochondrial proteins. In addition to cancer and inflammation diseases, O-GlcNAc modification appears to play a critical role during cell apoptosis and stress response, although the precise mechanisms are still not very clear. Here we found that nitric oxide synthase adaptor (NOS1AP), which plays an important part in glutamate-induced neuronal apoptosis, carries the modification of O-GlcNAc. Mass spectrometry analysis identified Ser47, Ser183, Ser204, Ser269, Ser271 as O-GlcNAc sites. Higher O-GlcNAc of NOS1AP was detected during glutamate-induced neuronal apoptosis. Furthermore, with O-GlcNAc sites of NOS1AP mutated, the interaction of NOS1AP and neuronal nitric oxide syntheses (nNOS) decreases. Finally, during glutamate-induced neuronal apoptosis, decreasing the O-GlcNAc modification of NOS1AP results in more severe neuronal apoptosis. All these results suggest that O-GlcNAc modification of NOS1AP exerts protective effects during glutamate-induced neuronal apoptosis. PMID:26197318

  14. Chloride channel protein 2 prevents glutamate-induced apoptosis in retinal ganglion cells

    PubMed Central

    Bi, Miao-Miao; Hong, Sen; Ma, Ling-Jun; Zhou, Hong-Yan; Lu, Jia; Zhao, Jing; Zheng, Ya-Juan

    2016-01-01

    Objective(s): The purpose of this study was to investigate the role of chloride channel protein 2 (ClC-2) in glutamate-induced apoptosis in the retinal ganglion cell line (RGC-5). Materials and Methods: RGC-5 cells were treated with 1 mM glutamate for 24 hr. The expression of ClC-2, Bax, and Bcl-2 was detected by western blot analysis. Cell survival and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Caspase-3 and -9 activities were determined by a colorimetric assay. The roles of ClC-2 in glutamate-induced apoptosis were examined by using ClC-2 complementary deoxyribonucleic acid (cDNA) and small inference ribonucleic acid (RNA) transfection technology. Results: Overexpression of ClC-2 in RGC-5 cells significantly decreased glutamate-induced apoptosis and increased cell viability, whereas silencing of ClC-2 with short hairpin (sh) RNA produced opposite effects. ClC-2 overexpression increased the expression of Bcl-2, decreased the expression of Bax, and decreased caspase-3 and -9 activation in RGC-5 cells treated with glutamate, but silencing of ClC-2 produced opposite effects. Conclusion: Our data suggest that ClC-2 chloride channels might play a protective role in glutamate-induced apoptosis in retinal ganglion cells via the mitochondria-dependent apoptosis pathway.

  15. The mitochondrial and death receptor pathways involved in the thymocytes apoptosis induced by aflatoxin B1.

    PubMed

    Peng, Xi; Yu, Zhengqiang; Liang, Na; Chi, Xiaofeng; Li, Xiaochong; Jiang, Min; Fang, Jing; Cui, Hengmin; Lai, Weimin; Zhou, Yi; Zhou, Shan

    2016-03-15

    Aflatoxin B1 (AFB1) is a potent immunosuppressive agent in endotherms, which can be related to the up-regulated apoptosis of immune organs. In this study, we investigated the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in Aflatoxin B1 induced thymocytes apoptosis. Chickens were fed an aflatoxin B1 containing diet (0.6 mg/kg AFB1) for 3 weeks. Our results showed that (1) AFB1 diet induced the decrease of T-cell subsets, morphological changes, and excessive apoptosis of thymus. (2) The excessive apoptosis involved the mitochondrial pathway (up-regulation of Bax, Bak, cytC and down-regulation of Bcl-2 and Bcl-xL) and death receptor pathway (up-regulation of FasL, Fas and FADD). (3) Oxidative stress, an apoptosis inducer, was confirmed in the thymus. In conclusion, this is the first study to demonstrate that mitochondrial and death receptor pathways involved in AFB1 induced thymocytes apoptosis in broilers. PMID:26933817

  16. PU.1 induces apoptosis in myeloma cells through direct transactivation of TRAIL

    PubMed Central

    Ueno, S; Tatetsu, H; Hata, H; Iino, T; Niiro, H; Akashi, K; Tenen, DG.; Mitsuya, H; Okuno, Y

    2010-01-01

    We previously reported that PU.1 was down-regulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were down-regulated and p21 was up-regulated, while among apoptosis-related genes, TRAIL was found highly up-regulated. When TRAIL was knocked down by siRNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL plays a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Up-regulation of PU.1 induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL. PMID:19749795

  17. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro.

    PubMed

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-05-11

    Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer. PMID:22546556

  18. SARM1, Not MyD88, Mediates TLR7/TLR9-Induced Apoptosis in Neurons.

    PubMed

    Mukherjee, Piyali; Winkler, Clayton W; Taylor, Katherine G; Woods, Tyson A; Nair, Vinod; Khan, Burhan A; Peterson, Karin E

    2015-11-15

    Neuronal apoptosis is a key aspect of many different neurologic diseases, but the mechanisms remain unresolved. Recent studies have suggested a mechanism of innate immune-induced neuronal apoptosis through the stimulation of endosomal TLRs in neurons. TLRs are stimulated both by pathogen-associated molecular patterns as well as by damage-associated molecular patterns, including microRNAs released by damaged neurons. In the present study, we identified the mechanism responsible for TLR7/TLR9-mediated neuronal apoptosis. TLR-induced apoptosis required endosomal localization of TLRs but was independent of MyD88 signaling. Instead, apoptosis required the TLR adaptor molecule SARM1, which localized to the mitochondria following TLR activation and was associated with mitochondrial accumulation in neurites. Deficiency in SARM1 inhibited both mitochondrial accumulation in neurites and TLR-induced apoptosis. These studies identify a non-MyD88 pathway of TLR7/ TLR9 signaling in neurons and provide a mechanism for how innate immune responses in the CNS directly induce neuronal damage. PMID:26423149

  19. The mitochondrial and death receptor pathways involved in the thymocytes apoptosis induced by aflatoxin B1

    PubMed Central

    Chi, Xiaofeng; Li, Xiaochong; Jiang, Min; Fang, Jing; Cui, Hengmin; Lai, Weimin; Zhou, Yi; Zhou, Shan

    2016-01-01

    Aflatoxin B1 (AFB1) is a potent immunosuppressive agent in endotherms, which can be related to the up-regulated apoptosis of immune organs. In this study, we investigated the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in Aflatoxin B1 induced thymocytes apoptosis. Chickens were fed an aflatoxin B1 containing diet (0.6 mg/kg AFB1) for 3 weeks. Our results showed that (1) AFB1 diet induced the decrease of T-cell subsets, morphological changes, and excessive apoptosis of thymus. (2) The excessive apoptosis involved the mitochondrial pathway (up-regulation of Bax, Bak, cytC and down-regulation of Bcl-2 and Bcl-xL) and death receptor pathway (up-regulation of FasL, Fas and FADD). (3) Oxidative stress, an apoptosis inducer, was confirmed in the thymus. In conclusion, this is the first study to demonstrate that mitochondrial and death receptor pathways involved in AFB1 induced thymocytes apoptosis in broilers. PMID:26933817

  20. Anesthetic isoflurane attenuates activated microglial cytokine-induced VSC4.1 motoneuronal apoptosis

    PubMed Central

    Yang, Shuangmei; Liu, Jun; Zhang, Xiaoran; Tian, Jianmin; Zuo, Zhichao; Liu, Jingjing; Yue, Xiuqin

    2016-01-01

    Isoflurane (ISO) exhibits neuroprotective effects against inflammation and apoptosis. However, the role of ISO in motoneuronal apoptosis induced by activated microglia remains poorly studied. We investigated the protective effects of ISO on the apoptosis of ventral spinal cord 4.1 (VSC4.1) motoneurons induced by lipopolysaccharide (LPS)-activated BV-2 microglia. Results indicated that ISO inhibited NF-κB activation and pro-inflammatory cytokine release in LPS-treated BV-2 microglia. Conditioned medium (CM) from activated BV-2 cells treated by ISO directly prevented VSC4.1 motoneurons from LPS-CM-induced neuronal apoptosis, as determined by the following: reductions in caspase-8, caspase-9, and caspase-3 activities; downregulation of pro-apoptotic procaspase-8, cleaved (cl)-caspase-8, procaspase-9, cl-caspase-9, caspase-3, cl-caspase-3, Bid, Bax, and cytochrome c expression; and upregulation of anti-apoptotic Bcl-2 expression in LPS-CM-cultured VSC4.1 motoneurons. Findings demonstrated that ISO inhibits BV-2 microglia activation and alleviates VSC4.1 motoneuronal apoptosis induced by microglial activation. These effects suggest that ISO can be used as an alternative agent for reducing neuronal apoptosis. PMID:27186270

  1. Dominant roles of Fenton reaction in sodium nitroprusside-induced chondrocyte apoptosis.

    PubMed

    Quan, Ying-Yao; Qin, Gui-Qi; Huang, Hao; Liu, Yu-Hong; Wang, Xiao-Ping; Chen, Tong-Sheng

    2016-05-01

    Sodium nitroprusside (SNP) has been widely used as an exogenous nitric oxide (NO) donor to explore the molecular mechanism of NO-mediated chondrocyte apoptosis during the latest two decades. We have recently found that NO-independent ROS play a key role in SNP-induced apoptosis in rabbit chondrocytes. This study aims to investigate what kind of ROS and how the reliable ROS mediators mediate the SNP-induced apoptosis. Data shows that SNP and NO-exhausted SNP (SNPex) induced ROS production or cytotoxicity to identically degree. SNP induced a marked increase in iron ions, superoxide anion (O2(•-)), hydrogen peroxide (H2O2) and hydroxyl radical ((•)OH) level. H2O2 scavenger (CAT) and (•)OH scavenger (DMSO) significantly inhibited SNP-induced chondrocyte apoptosis. Iron ions chelator (DFO) entirely prevented SNP-induced chondrocyte apoptosis. In contrast, O2(•-) scavenger (SOD) and glutathione depletion agent (BSO) promoted SNP-induced cytotoxicity. K3[Fe(CN)6] exhibited no cytotoxicity, and H2O2 alone up to 250µM or iron ions alone up to 90µM is non-cytotoxic to chondrocytes. Combination of 25µM FeSO4 and 100µM H2O2 in the presence of BSO induced chondrocyte death similar to SNP treatment. Fetal bovine serum (FBS) enhanced iron ions release from SNP and the cytotoxicity of SNP. Our data shows that the extracellular Fenton reaction between iron ions released from SNP and H2O2 induced by SNP plays a key role in SNP-induced chondrocyte apoptosis. Overall, our results indicate that the potential of SNP to increase iron ions and ROS should be especially considered for some biological functions and, possibly, also for clinical applications of this drug. PMID:26923801

  2. Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients

    PubMed Central

    2010-01-01

    Background DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Methods Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. Conclusions An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity. PMID:20868468

  3. Somatostatin receptor subtype 2 sensitizes human pancreatic cancer cells to death ligand-induced apoptosis.

    PubMed

    Guillermet, Julie; Saint-Laurent, Nathalie; Rochaix, Philippe; Cuvillier, Olivier; Levade, Thierry; Schally, Andrew V; Pradayrol, Lucien; Buscail, Louis; Susini, Christiane; Bousquet, Corinne

    2003-01-01

    Somatostatin receptor subtype 2 (sst2) gene expression is lost in 90% of human pancreatic adenocarcinomas. We previously demonstrated that stable sst2 transfection of human pancreatic BxPC-3 cells, which do not endogenously express sst2, inhibits cell proliferation, tumorigenicity, and metastasis. These sst2 effects occur as a consequence of an autocrine sst2-dependent loop, whereby sst2 induces expression of its own ligand, somatostatin. Here we investigated whether sst2 induces apoptosis in sst2-transfected BxPC-3 cells. Expression of sst2 induced a 4.4- +/- 0.05-fold stimulation of apoptosis in BxPC-3 through the activation of tyrosine phosphatase SHP-1. sst2 also sensitized these cells to apoptosis induced by tumor necrosis factor alpha (TNFalpha), enhancing it 4.1- +/- 1.5-fold. Apoptosis in BxPC-3 cells mediated by TNF-related apoptosis-inducing ligand (TRAIL) and CD95L was likewise increased 2.3- +/- 0.5-fold and 7.4- +/- 2.5-fold, respectively. sst2-dependent activation and cell sensitization to death ligand-induced apoptosis involved activation of the executioner caspases, key factors in both death ligand- or mitochondria-mediated apoptosis. sst2 affected both pathways: first, by up-regulating expression of TRAIL and TNFalpha receptors, DR4 and TNFRI, respectively, and sensitizing the cells to death ligand-induced initiator capase-8 activation, and, second, by down-regulating expression of the antiapoptotic mitochondrial Bcl-2 protein. These results are of interest for the clinical management of chemoresistant pancreatic adenocarcinoma by using a combined gene therapy based on the cotransfer of genes for both the sst2 and a nontoxic death ligand. PMID:12490654

  4. TSC-22 Promotes Interleukin-2-Deprivation Induced Apoptosis in T-Lymphocytes.

    PubMed

    Pépin, Aurélie; Espinasse, Marie-Alix; Latré de Laté, Perle; Szely, Natacha; Pallardy, Marc; Biola-Vidamment, Armelle

    2016-08-01

    Originally described as a TGF-β-inducible gene, tsc-22 (Transforming growth factor-beta Stimulated Clone 22) encodes a transcriptional regulator affecting biological processes such as cell growth, differentiation, or apoptosis. Along with GILZ (Glucocorticoid-Induced Leucine Zipper), TSC-22 belongs to the evolutionary conserved TSC-22 Domain family. We previously showed that, in T-lymphocytes, GILZ expression was induced upon IL-2 withdrawal, delaying apoptosis through down-regulation of the pro-apoptotic protein BIM expression. The aim of this work was then to elucidate the respective roles of GILZ and TSC-22 upon IL-2 deprivation-induced apoptosis. We report here that these two highly homologous genes are concomitantly expressed in most human tissues and in primary T-lymphocytes and that expression of TSC-22 promotes T-lymphocytes apoptosis by inhibiting GILZ functions. Indeed, we demonstrated that TSC-22 expression in the murine lymphoid CTLL-2 cell line promoted IL-2 deprivation-induced apoptosis. BIM expression and caspases-9 and -3 activities were markedly increased in TSC-22 expressing clones compared to control clones. Analysis of GILZ expression revealed that TSC-22 prevented the induction of the GILZ protein upon IL-2 deprivation, by inhibiting gilz mRNA transcription. These results suggested that TSC-22 could counteract the protective effect of GILZ on IL-2-deprivation-induced apoptosis. Moreover, TSC-22-induced inhibition of GILZ expression was also found in CTLL-2 cells treated with glucocorticoids or TGF-β. In the human NKL cell line deprived of IL-2, TSC-22 showed the same effect and thus may represent a potent repressor of GILZ expression in IL-2-dependent cells, independently of the cell type, or the stimulus, leading to an increase of IL-2-deprived T-cells apoptosis. J. Cell. Biochem. 117: 1855-1868, 2016. © 2016 Wiley Periodicals, Inc. PMID:26752201

  5. Nature and mechanisms of hepatocyte apoptosis induced by d-galactosamine/lipopolysaccharide challenge in mice

    PubMed Central

    WU, YI-HANG; HU, SHAO-QING; LIU, JUN; CAO, HONG-CUI; XU, WEI; LI, YONG-JUN; LI, LAN-JUAN

    2014-01-01

    Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6–10 h after the intraperitoneal injection of d-GalN (700 mg/kg) and LPS (10 μg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering d-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following d-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the d-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by d-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis. PMID:24714963

  6. Resistance to etoposide-induced apoptosis in a Burkitt's lymphoma cell line.

    PubMed

    Zhao, E G; Song, Q; Cross, S; Misko, I; Lees-Miller, S P; Lavin, M F

    1998-08-31

    Burkitt's lymphoma cells that vary in their phenotypic characteristics show significantly different degrees of susceptibility to radiation-induced apoptosis. Propensity to undergo apoptosis is reflected in the degradation of substrates such as DNA-dependent protein kinase but the status of bcl-2, c-myc and p53 has been uninformative. In this study, we have focused on 2 Epstein-Barr virus (EBV)-associated Burkitt's cell lines, one (WW2) susceptible and the other (BL29) resistant to etoposide-induced apoptosis. Differences in expression of BHRF1, an EBV gene that is homologous to the Bcl-2 proto-oncogene and known to inhibit apoptosis, or changes in apoptosis inhibitory proteins (IAPs), did not appear to account for the difference in susceptibility in the 2 cell lines. Cytoplasmic extracts from etoposide-treated WW2 cells caused apoptotic changes in nuclei isolated from either BL29 or WW2 cells, whereas extracts from BL29 cells failed to do so. In addition, extracts from etoposide-treated WW2 cells degraded the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an important indicator of apoptosis, but this protein was resistant to degradation by BL29 extracts. It appears likely that caspase 3 (CPP32) is involved in this degradation since it was activated only in the apoptosis susceptible cells and the pattern of cleavage of DNA-PKcs was similar to that reported previously with recombinant caspase 3. As observed previously, addition of caspase 3 to nuclei failed to induce morphological changes indicative of apoptosis, but addition of caspase 3 to nuclei in the presence of extract from the resistant cells led to apoptotic changes. We conclude that resistance to apoptosis in BL29 cells is due to a failure of etoposide to activate upstream effectors of caspase activity. PMID:9688310

  7. FasL and TRAIL Induce Epidermal Apoptosis and Skin Ulceration Upon Exposure to Leishmania major

    PubMed Central

    Eidsmo, Liv; Fluur, Caroline; Rethi, Bence; Eriksson Ygberg, Sofia; Ruffin, Nicolas; De Milito, Angelo; Akuffo, Hannah; Chiodi, Francesca

    2007-01-01

    Receptor-mediated apoptosis is proposed as an important regulator of keratinocyte homeostasis in human epidermis. We have previously reported that Fas/FasL interactions in epidermis are altered during cutaneous leishmaniasis (CL) and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration. To further investigate the alterations of apoptosis during CL, a keratinocyte cell line (HaCaT) and primary human epidermal keratinocytes were incubated with supernatants from Leishmania major-infected peripheral blood mononuclear cells. An apoptosis-specific microarray was used to assess mRNA expression in HaCaT cells exposed to supernatants derived from L. major-infected cultures. Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression were significantly up-regulated, and apoptosis was detected in both HaCaT and human epidermal keratinocyte cells. The keratinocyte apoptosis was partly inhibited through blocking of Fas or FasL and even more efficiently through TRAIL neutralization. Up-regulation of Fas on keratinocytes in epidermis and the presence of FasL-expressing macrophages and T cells in dermis were previously reported by us. In this study, keratinocytes expressing TRAIL, as well as the proapoptotic receptor TRAIL-R2, were detected in skin biopsies from CL cases. We propose that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL. PMID:17200196

  8. Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis.

    PubMed

    Zhang, Xiaoh