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Development and molecular characterization of HCT116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate  

Microsoft Academic Search

Evidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to

Cara L. Crowley-Weber; Claire M. Payne; Mary Gleason-Guzman; George S. Watts; Bernard Futscher; Caroline N. Waltmire; Cheray Crowley; Katerina Dvorakova; Carol Bernstein; Mary Craven; Harinder Garewal; Harris Bernstein


Galectin-1 and Galectin-3 Mediate Protocadherin-24-Dependent Membrane Localization of ?-catenin in Colon Cancer Cell Line HCT116  

PubMed Central

Protocadherin-24 (PCDH24) is linked to the suppression of tumor growth and the inhibition of cell proliferation in the colon cancer cell line HCT116. We previously observed that ?-catenin is localized to the plasma membrane when PCDH24 is expressed in these cells, but the molecular mechanisms by which PCDH24 induces the membrane localization of ?-catenin remain largely unknown. To clarify these mechanisms, we identified molecules that interact with ectopically expressed PCDH24 in HCT116 cells using a HaloTag® pull-down assay. We found that galectin-1 and galectin-3 physically interact with PCDH24 and are retained at the plasma membrane in association with PCDH24 expression. A luciferase-based pull-down assay using HaloTag-fused galectins revealed that an intracellular region of PCDH24 (amino acids 1186–1280) is essential for this interaction. Furthermore, the over-expression of galectin-1 or -3, or the depletion of endogenous galectins by small interfering RNA modulates ?-catenin translocation. We also revealed that the retention of galectin-1 and -3 at the plasma membrane results in the inactivation of PI3K activity. From these findings, we propose a model in which the galectin-anchoring activity of PCDH24 leads to the suppression of ?-catenin signaling by the localization of ?-catenin at the plasma membrane in PCDH24-expressing HCT116 colon cancer cells.

Ose, Rui; Oharaa, Osamu; Nagase, Takahiro



Human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50 are admixtures of the human colon carcinoma cell line HCT 116  

Microsoft Academic Search

In two recently described human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50, derived from one tumour, we detected identical E-cadherin and ?-catenin gene mutations as in colon carcinoma cell line HCT 116. We demonstrate by HLA-typing, mutation analysis and microsatellite analysis that cell lines JROECL 47 and JROECL 50 are admixtures of the human colon adenocarcinoma cell line

B P L Wijnhoven; M G J Tilanus; A G Morris; S J Darnton; H W Tilanus; W N M Dinjens



Cytotoxicity of aporphines in human colon cancer cell lines HCT-116 and Caco-2: an SAR study  

PubMed Central

A series of synthetic aporphine derivatives structurally related to domesticine and nantenine (ring A, N6 and ring C truncated analogs), was evaluated in MTS cytotoxicity assays against the human colon cancer cell lines, HCT-116 and Caco-2. In general, the C1 position of ring A is tolerant of alkoxy substituents as well as a benzoyl ester functionality. Other modifications evaluated resulted in a decrease in cytotoxic activity. The most potent compounds identified had IC50 values in the range 23?M-38?M, comparable to the known cytotoxic agent, etoposide.

Ponnala, Shashikanth; Chaudhary, Sandeep; Gonzalez-Sarrias, Antonio; Seeram, Navindra P.



Methanolic extract of Indigofera tinctoria induces apoptosis in HCT116 cells  

Microsoft Academic Search

Objective: To determine the anticancer activity of the methanol extract of Indigofera tinctoria, which induces apoptosis in HCT 116 colon cancer cell line. Methodology and results: the effect of the methanolic extract of Indigofera tinctoria on HCT 116 cells was determined by cell viability, DNA fragmentation and comet assay. Treatment of HCT 116 cell lines with various concentrations of the

V. Magesh; G. Yuvaraj; M. Deecaraman; P. T. Kalaichelvan


Rapamycin synergizes with low-dose oxaliplatin in the HCT116 colon cancer cell line by inducing enhanced apoptosis  

PubMed Central

The present study aimed to examine the combined effects of oxaliplatin (L-OHP) and rapamycin (RAPA) in the HCT116 colon cancer cell line. The growth inhibitory effect was evaluated by MTT assay as a monotherapy or combination therapy. IC50 values were determined using CalcuSyn 2.0 software. To determine the interaction of the drugs, the combination index (CI) was calculated using the Chou-Talalay method. Apoptosis was investigated using flow cytometry and Western blotting. Acridine orange staining was employed to observe morphological changes. The results showed the IC50 values of L-OHP and RAPA to be 8.35±0.78 ?M (r=0.99) and 223.44±38.10 nM (r=0.94), respectively. CI was ?1 when L-OHP was used at doses ranging from 1 to 5 ?M plus RAPA at a dose of 10 nM, suggesting synergistic or additive effects. CI was ?1 when 100 nM RAPA was used in combination with low-dose L-OHP, showing additive to antagonistic effects. The combination of L-OHP (1 ?M) and RAPA (10 nM) induced 19.76% Annexin V-positive cells, which was found to be higher than L-OHP (11.45%, p<0.01) or RAPA (6.89%, p<0.01) alone. The cleaved PARP protein expression levels were highest after 48 h of combination treatment. Acridine orange staining showed typical bright red Acidic vesicular organelles in the RAPA group, whereas the green condensed chromatin in the apoptotic bodies was found in both the L-OHP and combination groups. In conclusion, at a cytostatic concentration, RAPA was found to potentiate the anti-tumor effects of low-dose L-OHP in the HCT116 colon cancer cell by inducing enhanced apoptosis.




Live cell catapulting and recultivation does not change the karyotype of HCT116 tumor cells  

Microsoft Academic Search

We used laser microdissection and pressure catapulting (LMPC) to isolate and subsequently recultivate cells from the colorectal cancer cell line HCT116. Cell isolation and recultivation was repeated 5 times from the preceding cell culture. The chromosomal composition of the HCT116 cells was analyzed at the beginning and after each recultivation. We did not observe any additional chromosomal structural or copy

Sabine Langer; Jochen B. Geigl; Susanne Ehnle; Rainer Gangnus; Michael R. Speicher



Transforming Growth Factor Beta Receptor 2 (TGFBR2) Changes Sialylation in the Microsatellite Unstable (MSI) Colorectal Cancer Cell Line HCT116  

PubMed Central

Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc) revealed a significant decline (?30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.

Lee, Jennifer; Ballikaya, Seda; Schonig, Kai; Ball, Claudia R.; Glimm, Hanno; Kopitz, Juergen; Gebert, Johannes



The role of NAD(P)H: quinone oxidoreductase in mitomycin C- and porfiromycin-resistant HCT 116 human colon-cancer cells  

Microsoft Academic Search

A mitomycin C (MMC)- and porfiromycin (PFM)-resistant subline of the HCT 116 human coloncancer cell line was isolated after repeated exposure of HCT 116 cells to increasing concentrations of MMC under aerobic conditions. The MMC-resistant subline (designated HCT 116-R30A) was 5 times more resistant than the parent cells to MMC and PFM under aerobic conditions. Both the MMC-resistant cells and

Su-shu Panl; Steven A. Akman; Gerald L. Forrest; Carlyn Hipsherl; Robin Johnsonl



MicroRNA-18a upregulates autophagy and ataxia telangiectasia mutated gene expression in HCT116 colon cancer cells.  


Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process in which a cell degrades its own long-lived proteins and damaged organelles. Ataxia telangiectasia mutated (ATM) has recently been shown to upregulate the process of autophagy. Previous studies showed that certain microRNAs, including miR-18a, potentially regulate ATM in cancer cells. However, the mechanisms behind the modulation of ATM by miR-18a remain to be elucidated in colon cancer cells. In the present study, we explored the impact of miR-18a on the autophagy process and ATM expression in HCT116 colon cancer cells. To determine whether a preliminary link exists between autophagy and miR-18a, HCT116 cells were irradiated and quantitative (q) PCR was performed to measure miR-18a expression. HCT116 cells were transfected with an miR-18a mimic to study its impact on indicators of autophagy. Western blotting and luciferase assays were implemented to explore the impact of miR-18a on ATM gene expression in HCT116 cells. The results showed that miR-18a expression was strongly stimulated by radiation. Ectopic overexpression of miR-18a in HCT116 cell lines potently enhanced autophagy and ionizing radiation-induced autophagy. Moreover, miR-18a overexpression led to the upregulation of ATM expression and suppression of mTORC1 activity. Results of the present study pertaining to the role of miR-18a in regulating autophagy and ATM gene expression in colon cancer cells revealed a novel function for miR-18a in a critical cellular event and on a crucial gene with significant impacts in cancer development, progression, treatment and in other diseases. PMID:23229340

Qased, Abu Baker; Yi, Heqing; Liang, Nan; Ma, Shumei; Qiao, Shixing; Liu, Xiaodong



NCI60 Cancer Cell Line Panel Data and RNAi Analysis Help Identify EAF2 as a Modulator of Simvastatin and Lovastatin Response in HCT116 Cells  

Microsoft Academic Search

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the

Sevtap Savas; David O. Azorsa; Hamdi Jarjanazi; Irada Ibrahim-Zada; Irma M. Gonzales; Shilpi Arora; Meredith C. Henderson; Yun Hee Choi; Laurent Briollais; Hilmi Ozcelik; Sukru Tuzmen; Eric Bernhard



Induction of a less aggressive phenotype in human colon carcinoma HCT116 cells by chronic exposure to HDAC inhibitor SAHA.  


Histone deacetylase inhibitors (HDACis) are anticancer molecules that epigenetically modulate cell functions. Chronic exposure of HCT116 colon cancer cells to SAHA has been investigated for a better understanding of resistance mechanisms but, surprisingly, a less aggressive tumor phenotype both in vitro and in vivo was obtained after exposure to increasing concentrations of SAHA. Indeed, HCT116/SAHA cells when injected into nude mice showed a reduced engraftment and growth with respect to HCT116 cells. This difference was not observed inoculating the cells into NOD/SCID mice that, differently from nude mice, lack NK activity, thus suggesting the involvement of the native immune response in impairment of HCT116/SAHA cell growth. In agreement with this result, a growing induction of NKG2D ligand expression, MICA and MICB, that are molecular mediators of NK cell killing, was confirmed in HCT116/SAHA chronically exposed to SAHA. A reduced clonogenic efficiency was also observed in HCT116/SAHA with respect to HCT116 cells. Interestingly, even after chronic exposure to SAHA, HCT116/SAHA cells developed only a moderate resistance to SAHA both in vitro and in vivo and they acquired a collateral sensitivity to anthracyclines. These results are of note and probably rely on the fact that, having simultaneously many different targets, HDACis would require many different mutations to display high resistance index. Moreover, to understand the molecular basis of HCT116/SAHA cell phenotype a gene expression profile of cancer genes was evaluated in HCT116 incubated with SAHA for 24 h and in HCT116/SAHA cells to identify selectively regulated genes. PMID:20878117

Bressan, Alessandro; Bigioni, Mario; Bellarosa, Daniela; Nardelli, Federica; Irrissuto, Clelia; Maggi, Carlo Alberto; Binaschi, Monica



Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression  

Microsoft Academic Search

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis.\\u000a To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax+\\/?) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax+\\/? cells following a long delay in the

S.-M. Chiu; L.-Y. Xue; K. Azizuddin; N. L. Oleinick



Antisense activity of 2',4'-BNA targeted to PPAR gamma in THP-1 and HCT116 cells.  


2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) is a nucleic acid analogue that has high affinity binding to its complementary RNA. Peroxisome proliferator-activated receptor (PPAR) y belongs to the nuclear hormone receptor superfamily and is a drug target in the treatment of type 2 diabetes. Here, we show the antisense effects of 2',4'-BNA oligonucleotides against PPARy in the human monocytic leukaemia cell line THP-1 and the human colorectal tumor cell line HCT116. PMID:18029776

Tachibana, Keisuke; Katayama, Tatsuya; Ueda, Chihiro; Sumitomo, Mikako; Tagami, Masayuki; Ishimoto, Kenji; Yamasaki, Daisuke; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Obika, Satoshi; Imanishi, Takeshi; Doi, Takefumi



Tazarotene-induced gene 1 inhibits prostaglandin E2-stimulated HCT116 colon cancer cell growth  

PubMed Central

Background The tazarotene-induced gene 1 (TIG1) is a putative tumor suppressor gene. We have recently demonstrated both TIG1A and TIG1B isoforms inhibited cell growth and induced the expression of G protein-coupled receptor kinase 5 (GRK5) in colon cancer cells. Because elevated prostaglandin E2 (PGE2) signaling plays a significant role in colorectal carcinogenesis, the objective of this study was to explore the effect of TIG1 on PGE2-induced cellular proliferation and signaling in colon cancer cells. Methods HCT116 cells as well as TIG1A and TIG1B stable cells established from HCT116 colon cancer cells using the GeneSwitch system were used. TIG1 isoform expression was induced by mifepristone treatment in stable cells. Cell growth was determined using the WST-1 cell proliferation assay. Activation of ?-catenin/TCF and cyclic adenosine monophosphate (cAMP)/CREB signaling pathways were determined using luciferase reporter assays. Expression and subcellular distribution of ?-catenin were analyzed using Western blot and confocal microscope. Levels of cAMP were measured using an enzyme immunoassay. RNA interference was used to examine the effects of TIG1- and GRK5-mediated changes. Results PGE2-stimulated cell growth was reduced in inducible TIG1A- and TIG1B-stable HCT116 cells. GRK5 expression was upregulated by both TIG1A and TIG1B isoforms, and its expression suppressed PGE2-stimulated HCT116 cell growth. GRK5, TIG1A, and TIG1B expression significantly inhibited PGE2-stimulated ?-catenin/TCF and cAMP signaling pathway reporters and cAMP. Also, PGE2-stimulated nuclear localization of ?-catenin was inhibited by expression of TIG1A and TIG1B, which was ameliorated by both TIG1 and GRK5 siRNAs. Conclusions TIG1 suppressed PGE2-stimulated Wnt and cAMP signaling pathways in colon cancer cells through GRK5.



Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells.  


Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, p<0.05) and maximal (50%, p<0.05) OCR and gene expression of mitochondrial proteins and Bax without affecting cell viability or expression of glycolytic enzymes. Similar changes could be recapitulated by incubating cells with leptin, whereas, leptin-receptor specific antagonist inhibited the reduced OCR induced by conditioned media from obese subjects. We conclude that secreted products from the adipose tissue of obese subjects inhibit mitochondrial respiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues. PMID:24073224

Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty



Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells  

PubMed Central

Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, p<0.05) and maximal (50%, p<0.05) OCR and gene expression of mitochondrial proteins and Bax without affecting cell viability or expression of glycolytic enzymes. Similar changes could be recapitulated by incubating cells with leptin, whereas, leptin-receptor specific antagonist inhibited the reduced OCR induced by conditioned media from obese subjects. We conclude that secreted products from the adipose tissue of obese subjects inhibit mitochondrial respiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues.

Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty



Upregulation of haeme oxygenase-1 by zinc in HCT-116 cells.  


Haeme oxygenase-1 (HO-1) is often viewed as a cytoprotective gene. Toxic heavy metals induce HO-1, but it is unclear whether particular metal micronutrients also induce HO-1. Hence, the ability of exogenously-added copper, iron and zinc to influence HO-1 expression in HCT-116 cells was evaluated. Under the chosen experimental conditions, only zinc noticeably increased the expression of HO-1 mRNA and protein. Concurrently, zinc decreased non-protein thiol levels to a certain extent, but zinc did not increase the production of reactive oxygen species (ROS). Moreover, ascorbate and Trolox did not inhibit zinc-induced HO-1 upregulation. In contrast, deferoxamine blunted the induction of HO-1 mRNA, protein, and enzymatic activity caused by zinc. Additionally, N-acetylcysteine and Tiron inhibited zinc-induced HO-1 upregulation and also nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Collectively, these findings suggest that zinc at above normal levels upregulates HO-1 expression in HCT-116 cells in a ROS-independent manner. PMID:22564156

Smith, Abigail F; Loo, George



?-Tubulin-containing abnormal centrioles are induced by insufficient Plk4 in human HCT116 colorectal cancer cells  

PubMed Central

Summary Cancer cells frequently induce aberrant centrosomes, which have been implicated in cancer initiation and progression. Human colorectal cancer cells, HCT116, contain aberrant centrioles composed of disorganized cylindrical microtubules and displaced appendages. These cells also express unique centrosome-related structures associated with a subset of centrosomal components, including ?-tubulin, centrin and PCM1. During hydroxyurea treatment, these abnormal structures become more abundant and undergo a change in shape from small dots to elongated fibers. Although ?-tubulin seems to exist as a ring complex, the abnormal structures do not support microtubule nucleation. Several lines of evidence suggest that the fibers correspond to a disorganized form of centriolar microtubules. Plk4, a mammalian homolog of ZYG-1 essential for initiation of centriole biogenesis, is not associated with the ?-tubulin-specific abnormal centrosomes. The amount of Plk4 at each centrosome was less in cells with abnormal centrosomes than cells without ?-tubulin-specific abnormal centrosomes. In addition, the formation of abnormal structures was abolished by expression of exogenous Plk4, but not SAS6 and Cep135/Bld10p, which are downstream regulators required for the organization of nine-triplet microtubules. These results suggest that HCT116 cells fail to organize the ninefold symmetry of centrioles due to insufficient Plk4.

Kuriyama, Ryoko; Bettencourt-Dias, Monica; Hoffmann, Ingrid; Arnold, Marc; Sandvig, Lisa



Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells.  


Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone; DMC) is a naturally occurring chalcone, and it is the major compound isolated from the leaves of Syzygium samarangense (Blume) Merr. & L.M. Perry (Myrtaceae). Experiments were conducted to determine the effects of DMC on cell proliferation, cell-cycle distribution, and programmed cell death in cultures of human colorectal carcinoma HCT116 and LOVO cells. Results showed that DMC inhibited HCT116 and LOVO cell proliferation and induced G(2) /M cell cycle arrest, which was associated with the conversion of microtubule associated protein light chain 3 (LC3)-I-LC3-II, an autophagosome marker, and the incorporation of monodansylcadaverine (MDC), a marker for the acidic compartment of autolysosomes or acidic vesicular organelles. The treatment of HCT116 and LOVO cells using a combination of DMC with an autophagy inhibitor, such as 3-methyladenine (3-MA), beclin 1 siRNA, or atg5 siRNA, suppressed the effect of DMC-mediated anti-proliferation. These results imply that DMC can suppress colorectal carcinoma HCT116 and LOVO cell proliferation through a G(2) /M phase cell-cycle delay, and can induce autophagy, the hallmark of Type II programmed cell death (PCD). Taken together, our results suggest that DMC may be an effective chemotherapeutic agent for HCT116 and LOVO colorectal carcinoma cells. PMID:21538483

Ko, Hyeonseok; Kim, Young-Joo; Amor, Evangeline C; Lee, Jong Wha; Kim, Han-Cheon; Kim, Hee Ju; Yang, Hyun Ok



A new in vivo model to analyze hepatic metastasis of the human colon cancer cell line HCT116 in NOD/Shi-scid/IL-2R?(null) (NOG) mice by (18)F-FDG PET/CT.  


Clinically, (18)F-fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG-PET/CT) is useful in the evaluation of various types of human cancers. While PET analysis has been established to evaluate subcutaneous lesions of human cancers in mice, its applications for internal lesions are still being developed. We are currently evaluating new PET approaches for the effective evaluation of in vivo metastatic lesions in the internal organs of small experimental animals. In this study, we analyzed in vivo hepatic metastases of human colonic cancer in immunodeficient mice (NOD/Shi-scid/IL-2R?(null), NOG) using PET imaging. This new PET approach has been proposed for the evaluation of in vivo metastatic lesions in internal organs. The human colon cancer line HCT116 (1.0x10(5) and 1.0x10(6) cells/mouse) was transplanted by intrasplenic injection. (18)F-FDG-PET/CT scans were performed 2 weeks after transplantation. After PET/CT scans, histopathological examinations were performed. PET/CT analysis disclosed multiple metastatic foci and increased standardized uptake values (SUV) of FDG in the livers of NOG mice (control, SUVmean 0.450±0.033, SUVmax 0.635±0.017; 1.0x10(5) cells, 0.853±0.087, 1.254±0.237; 1.0x10(6) cells, 1.211±0.108, 1.701±0.158). There were significant differences in FDG uptakes between the three groups (ANOVA, P=0.017 in SUVmean; P=0.044 in SUVmax, n=2). We clearly and quantitatively detected images of hepatic metastasis in the livers of NOG mice by (18)F-FDG-PET/CT in vivo. PET/CT analysis of internal organ lesions of human cancerous xenografts is a new reliable experimental system to simulate metastases. This model system is useful for analyzing metastatic mechanisms and for developing new novel drugs targeting hepatic metastases of cancer. PMID:23165994

Kawai, Kenji; Tamura, Katsumi; Sakata, Ikuko; Ishida, Jiro; Nagata, Masayoshi; Tsukada, Hideo; Suemizu, Hiroshi; Nakamura, Masato; Abe, Yoshiyuki; Chijiwa, Tsuyoshi



Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells  

Microsoft Academic Search

Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for\\u000a cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate\\u000a intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53+\\/+ and HCT116p53?\\/? colon carcinoma cells were exposed to multiple pulses of 60 kV\\/cm with either 60 ns

Emily H. Hall; Karl H. Schoenbach; Stephen J. Beebe



Both stromal cell and colonocyte epidermal growth factor receptors control HCT116 colon cancer cell growth in tumor xenografts  

PubMed Central

Colon cancer growth requires growth-promoting interactions between malignant colonocytes and stromal cells. Epidermal growth factor receptors (EGFR) are expressed on colonocytes and many stromal cells. Furthermore, EGFR is required for efficient tumorigenesis in experimental colon cancer models. To dissect the cell-specific role of EGFR, we manipulated receptor function on stromal cells and cancer cells. To assess the role of stromal EGFR, HCT116 human colon cancer cells were implanted into immunodeficient mice expressing dominant negative (DN) EgfrVelvet/+ or Egfr+/+. To assess the role of cancer cell EGFR, HCT116 transfectants expressing inducible DN-Egfr were implanted into immunodeficient mice. To dissect EGFR signals in vitro, we examined colon cancer cells in monoculture or in cocultures with fibroblasts for EGFR transactivation and prostaglandin synthase 2 (PTGS2) induction. EGFR signals were determined by blotting, immunostaining and real-time PCR. Tumor xenografts in EgfrVelvet/+ mice were significantly smaller than tumors in Egfr+/+ mice, with decreased proliferation (Ki67) and increased apoptosis (cleaved caspase-3) in cancer cells and decreased stromal blood vessels. Mouse stromal transforming growth factor alpha (TGFA), amphiregulin (AREG), PTGS2 and Il1b and interleukin-1 receptor 1 (Il1r1) transcripts and cancer cell beta catenin (CTNNB1) and cyclin D1 (CCND1) were significantly lower in tumors obtained from EgfrVelvet/+ mice. DN-EGFR HCT116 transfectants also formed significantly smaller tumors with reduced mouse Areg, Ptgs2, Il1b and Il1r1 transcripts. Coculture increased Caco-2 phospho-active ERBB (pERBB2), whereas DN-EGFR in Caco-2 cells suppressed fibroblast PTGS2 and prostaglandin E2 (PGE2). In monoculture, interleukin 1 beta (IL1B) transactivated EGFR in HCT116 cells. Stromal cell and colonocyte EGFRs are required for robust EGFR signals and efficient tumor growth, which involve EGFR–interleukin-1 crosstalk.

Bissonnette, Marc



Curcumin induces DNA damage and caffeine-insensitive cell cycle arrest in colorectal carcinoma HCT116 cells  

Microsoft Academic Search

Curcumin (CUR), a polyphenol derived from the plant Curcuma longa, displays potential anti-cancer activity. One of the mechanisms stems from its ability to elicit cell cycle arrest followed\\u000a by suppression of cell proliferation. Herein, we reported that CUR significantly induced DNA damage and mediated S and G2\\/M\\u000a phase arrest in colorectal carcinoma HCT116 cells. Unlike etoposide, a classical topoisomerase II

Jin-Jian LuYu-Jun; Yu-Jun Cai; Jian Ding



Blastocystis sp. subtype 3 triggers higher proliferation of human colorectal cancer cells, HCT116.  


Blastocystis sp. is a commonly found intestinal microorganism and was reported to cause many nonspecific gastrointestinal symptoms. Various subtypes have been previously reported, and the pathogenicity of different subtypes of Blastocystis is unclear and remains as a controversial issue. A recent study has shown that the Blastocystis antigen isolated from an unknown subtype could facilitate the proliferation of colon cancer cells. Current study was conducted to compare the effect of solubilized antigen isolated from five different subtypes of Blastocystis on colon cancer cells, HCT116. A statistically significant proliferation of these cells was observed when exposed to 1.0 ?g/ml solubilized antigen isolated from subtype 3 Blastocystis (37.22 %, p?cells (p?cells by weakening the cellular immune response. The dysregulation of IFN-? and p53 expression also suggest Blastocystis as a proponent of carcinogenesis. Therefore, it is very likely for subtype 3 Blastocystis to have higher pathogenic potential as it caused an increased propagation of cancer cells and substantial amount of inflammatory reaction compared to other subtypes. PMID:23933809

Kumarasamy, Vinoth; Kuppusamy, Umah R; Samudi, Chandramathi; Kumar, Suresh



A highly polar xanthophyll of 9?- cis -neoxanthin induces apoptosis in HCT116 human colon cancer cells through mitochondrial dysfunction  

Microsoft Academic Search

Highly polar xanthophylls of 9?-cis-neoxanthin (neoxanthin) and fucoxanthin, which have the characteristic structure of an epoxy group and an allenic bond, were\\u000a previously found to induce apoptosis in human prostate cancer cells. In the present study, we found apoptosis induction by\\u000a neoxanthin in HCT116 human colon cancer cells and examined the induction mechanism. The cells exposed to 20 ?M neoxanthin\\u000a clearly

Masaru Terasaki; Akira Asai; Hong Zhang; Akihiko Nagao



Gene Expression Analysis of HCT116 Colon Tumor-derived Cells Treated with the Polyamine Analog PG-11047  

PubMed Central

Background The conformationally restricted polyamine analog PG-11047 has significant growth inhibitory activity against prostate and lung cancer cell lines and is currently under evaluation in several clinical trials, both alone and in combination with other drugs, for the treatment of relapsed or refractory cancer. The objective of this study was to identify the molecular signature of genes responsive to PG-11047 treatment and the biochemical effects of this drug in the HCT116 colon cancer cell line. Materials and Methods Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. Results Treatment of cells with PG-11047 at concentrations ranging from 0.1 to 10 ?M caused inhibition of cell growth. The activity of PG-11047 was found to correlate with its transcriptional effects on cell cycle control, focal adhesion, adherent and gap junction genes, MAPK-, Wnt- and, TGF-? signaling pathways, transport and DNA/RNA transcription factor genes. PG-11047 caused depletion of polyamine pools. Proteomics analysis showed that PG-11047 restricts the modification of eukaryotic translation initiation factor 5A (eIF5A), resulting in suppression of general protein synthesis in PG-11047-treated cells. Conclusion These data show that PG-11047 has a broad spectrum of anticancer activity in colon cancer cells.




Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT116 cells  

Microsoft Academic Search

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.\\u000a The addition of a pan-caspase inhibitor (zVAD-fmk) did not suppress the denbinobin-induced apoptotic effect, and denbinobin-induced\\u000a apoptosis was not accompanied by processing of procaspase-3, -6, -7, -9, and -8. However, denbinobin triggered

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



Mild hyperthermia prior to electroporation increases transfection efficiency in HCT 116, HeLa S3 and SGC 7901 cells  

Microsoft Academic Search

The change in transfection efficiency of electroporation by the combined treatment with mild preheating (40°C for 30 min)\\u000a was investigated. HCT 116, HeLa S3 and SGC 7901 cells were treated with electroporation in medium containing pBKCMV-Luc plasmid\\u000a with or without preheating. After 24 h, luciferase activity was increased by 36, 28 and 77%; luciferase mRNA transcription\\u000a was increased by 45, 50 and

Zheng-Li Wei; Ryohei Ogawa; Ichiro Takasaki; Qing-Li Zhao; Hua-Chuan Zheng; Kanwal Ahmed; Mariame A. Hassan; Takashi Kondo



Cell cycle and apoptotic effects of SAHA are regulated by the cellular microenvironment in HCT116 multicellular tumour spheroids.  


Using multicellular tumour spheroids (MCTS) of HCT116 colon carcinoma cells, we analysed the effects of SAHA (suberoylanilide hydroxamic acid), a histone deacetylase inhibitor (HDACi). We found that, although SAHA-induced histone acetylation and ROS level upregulation occur throughout the spheroid, inhibition of cell cycle progression and induction of apoptosis are dependent on cell microenvironment. SAHA-induced growth inhibition of HCT116 MCTS results from the inhibition of cell cycle progression and induction of apoptosis. At a low concentration SAHA decreases Ki-67 and cyclin A positive cells and increases p21 positive cells in the outer layer while it induces a ROS-dependent apoptosis in the central zone of the spheroid. Coimmunostaining of p21 and apoptotic cells confirms that SAHA effects are different depending on the position of the cells within the spheroid. At a higher dose, SAHA induces mitotic defects and survivin downregulation in the outer layer of cells resulting in an additional cytotoxic effect in this part of the spheroid. Together these findings show that SAHA-induced cytostatic and cytotoxic effects occur in different cell populations, indicating that the cellular microenvironment is an important determinant in the regulation of the effects of SAHA treatment. Consequently, the MCTS model appears to be a valuable advanced tool for evaluating the effects of SAHA treatment in combination with other anticancer agents. PMID:19553104

Lobjois, Valérie; Frongia, Céline; Jozan, Suzanne; Truchet, Isabelle; Valette, Annie



Raman micro-spectroscopic analysis of cultured HCT116 colon cancer cells in the presence of roscovitine  

NASA Astrophysics Data System (ADS)

Raman micro-spectroscopic analysis of cultured HCT116 colon cancer cells in the presence of roscovitine, [seliciclib, 2-(1-ethyl-2-hydroxy-ethylamino)-6-benzylamino-9-isopropylpurine], a promising drug candidate in cancer therapy, has been performed for the first time. The aim of this study was to investigate modulations in colon cancer cells induced by roscovitine. Raman spectra of the cultured HCT116 colon cancer cells treated with roscovitine at different concentrations (0, 5, 10, 25 and 50 ?M) were recorded in the range 400-1850 cm -1. It was shown that the second derivative profile of the experimental spectrum gives valuable information about the wavenumbers and band widths of the vibrational modes of cell components, and it eliminates the appearance of false peaks arising from incorrect baseline corrections. In samples containing roscovitine, significant spectral changes were observed in the intensities of characteristic protein and DNA bands, which indicate roscovitine-induced apoptosis. Roscovitine-induced apoptosis was also assessed by flow cytometry analysis, and analysis of propidium iodide staining. We observed some modifications in amide I and III bands, which arise from alterations in the secondary structure of cell proteins caused by the presence of roscovitine.

Akyuz, S.; Ozel, A. E.; Balci, K.; Akyuz, T.; Coker, A.; Arisan, E. D.; Palavan-Unsal, N.; Ozalpan, A.



Soy soluble polysaccharide induces apoptosis in HCT?116 human colon cancer cells via reactive oxygen species generation.  


Previous studies have suggested that soy sauce contains specific bioactive components and various biological activities of soy sauce have been observed. Soy soluble polysaccharide (SSPS), a predominant bioactive compound in soy sauce, has numerous pharmacological actions, including anti?inflammatory and immunomodulating activities. In the current study, the apoptotic effects of SSPS were investigated in HCT?116 human colon cancer cells. Treatment with SSPS significantly inhibited cell growth in a concentration?dependent manner by inducing apoptosis but not necrosis. This induction was associated with the generation of reactive oxygen species (ROS), mitochondrial dysfunction, activation of caspases and cleavage of the poly (ADP?ribose) polymerase protein. Induction of apoptotic cell death of HCT?116 cells by SSPS showed a correlation with the downregulation of members of the inhibitor of apoptosis protein family, including X?linked inhibitor of apoptosis protein and antiapoptotic Bcl?2, and upregulation of Bax and Bad. Administration of N?acetyl?L?cysteine, a scavenger of ROS, significantly decreased SSPS?induced apoptosis. These results indicate a critical role of signaling cascades involving a ROS?mediated caspase pathway in the anticancer effects of SSPS. PMID:24126443

Ko, Yu-Jin; Jeong, Jin-Woo; Choi, Yung-Hyun; Ryu, Chung-Ho



Curcumin inhibits neurotensin-mediated interleukin-8 production and migration of HCT116 human colon cancer cells  

PubMed Central

Purpose Neurotensin (NT), a gut tridecapeptide, acts as a potent cellular mitogen for various colorectal and pancreatic cancers which possess high-affinity NT receptors (NTR). Cytokine/chemokine proteins are increasingly recognized as important local factors which play a role in the metastasis and invasion of multiple cancers. The purpose of this study was to: (i) determine the effect of NT on cytokine/chemokine gene expression and cell migration in human cancer cells and, (ii) assess the effect of curcumin, a natural dietary product, on NT-mediated processes. Experimental Design The human colorectal cancer, HCT116, was treated with NT, with or without curcumin, and IL-8 expression and protein secretion was measured. Signaling pathways, which contribute to the effects of NT, were assessed. Finally, the effect of curcumin on NT-mediated HCT116 cell migration was analyzed. Results We show that NT, acting through the native high-affinity NTR, induced IL-8 expression in human colorectal cancer cells in a time- and dose-dependent fashion. This stimulation involves Ca2+-dependent PKC, ERK-dependent AP-1 and ERK-independent NF-?B pathways. Curcumin inhibited NT-mediated AP-1 and NF-?B activation and Ca2+ mobilization. Moreover curcumin blocked NT-stimulated IL-8 gene induction and protein secretion and, at a low concentration (ie, 10 ?M), blocked NT-stimulated colon cancer cell migration. Conclusions NT-mediated induction of tumor cell IL-8 expression and secretion may contribute to the procarcinogenic effects of NT on GI cancers. Furthermore a potential mechanism for the chemopreventive and chemotherapeutic effects of curcumin on colon cancers may be through the inhibition of GI hormone (eg, NT)-induced chemokine expression and cell migration.

Wang, Xiaofu; Wang, Qingding; Ives, Kirk L.; Evers, B. Mark



Inhibition of proteasomal degradation of Mcl-1 by cobalt chloride suppresses cobalt chloride-induced apoptosis in HCT116 colorectal cancer cells  

Microsoft Academic Search

Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis\\u000a are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells.\\u000a Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250–750 ?M) which, at higher concentrations (500–750 ?M),\\u000a was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival

Melanie Lee; Abigail Lapham; Matthew Brimmell; Helen Wilkinson; Graham Packham



Diosgenin, a naturally occurring furostanol saponin suppresses 3-hydroxy-3-methylglutaryl CoA reductase expression and induces apoptosis in HCT116 human colon carcinoma cells  

Microsoft Academic Search

A growing body of experimental evidence suggests the therapeutic potential of diosgenin, a furostanol saponin against several cancers. However, precise molecular and cellular mechanisms underlying the modes of action of this compound against colon cancer remain only partially understood. In this study, we investigated if the anticancer mechanism of diosgenin in HCT-116 human colon carcinoma cells involves modulation in the

Jayadev Raju; Ranjana P. Bird



Curcumin induces apoptosis in HCT116 human colon cancer cells in a p21-independent manner  

Microsoft Academic Search

Several micronutrients present in fruits and vegetables exhibit anticancer activity as a result of their actions on molecular targets involved in carcinogenesis and tumor progression. Curcumin, a phenolic phytochemical derived from the rhizome of Curcuma longa, exhibits both cancer-preventative activity and growth inhibitory effects on neoplastic cells. Several studies report that curcumin inhibits cancer cell proliferation and induces apoptosis in

Jane L. Watson; Richard Hill; Patrick W. Lee; Carman A. Giacomantonio; David W. Hoskin



[Effect of free and polymer carrier encapsulated doxorubicin towards HCT116 cells of human colorectal carcinoma].  


Development of novel nanoscale functionalized carriers is nowadays one of the most urgent problems in cancer treatment. The aim of our study was to compare the antineoplastic effect of free doxorubicin and its complex with a nanoscale polymeric carrier towards HTC116 colorectal carcinoma cells. It was established that application of the complex of poly(5-tret-butylperoxy)-5-methyl-1-hexene-3-in-co-glycydyl metacrylat)-graft-polyethyleneglycol (poly(VEP-GMA-PEG)-graft-PEG), where VEP--5-tret-butylperoxy)-5-methyl-1-hexene-3-in; GMA--glycydyl metacrylat; graft-PEG--graft-polyethyleneglycol accordingly, functionalized with phosphatidylcholine for doxorubicin delivery increased 10 times the efficiency of cytotoxic action of this drug, as compared wich such efficiency in case of the action of free doxorubicin. The encapsulated form of doxorubicin caused more intensive cleavage of the reparation enzyme PARP and longer delay in G2/M cell cycle arrest, compared to such effects of free doxorubicin. The developed carrier itself is non-toxic to the used mammalian cells and does not cause impairment in their cell cycle. A deletion in both alleles of p53 gene did not affect the antineoplastic action of doxorubicin that was immobilized on the nanoscale carrier. Thus, p53-dependent signaling pathways are not involved in the cytotoxic action of doxorubicin-carrier complex. It is suggested that novel nanoscale polymeric carrier poly(VEP-GMA-PEG)-graft-PEG functionalized with phosphatidylcholine could be a promising carrier for targeted delivery of anticancer drugs. PMID:23808308

Sen'kiv, Iu V; Heffeter, P; Riabtseva, A O; Bo?ko, N M; Mitina, N Ie; Zaichenko, O S; Berger, W; Sto?ka, R S


The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein  

PubMed Central

Background Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Methods Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. Results HCT116BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Conclusion Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the translocation step of BAX activation may be impaired.



MLH1-deficient HCT116 colon tumor cells exhibit resistance to the cytostatic and cytotoxic effect of the poly(A) polymerase inhibitor cordycepin (3?-deoxyadenosine) in vitro  

PubMed Central

Cordycepin (3?-deoxyadenosine) is an inhibitor of poly(A) polymerase (PAP), an enzyme crucial to mRNA 3?-end processing, which produces the shortening of poly(A) tails, leading to the destabilization of mRNAs. Cordycepin inhibits proliferation and induces apoptosis in tumor cells, indicating its antitumor activity. Defective 3?-end processing is associated with hypersensitivity to UV treatment. We investigated the effects of cordycepin on proliferation and apoptosis in MLH1-deficient and MLH1-proficient HCT116 colon tumor cells. MLH1 is a DNA mismatch repair (MMR) protein involved in the processing of damaged DNA. Cells with defective MMR show resistance to certain anticancer drugs. The results showed that MLH1-deficient HCT116 cells are 2-fold less sensitive to the cytostatic effect of cordycepin, as compared to MLH1-proficient cells. This reduced sensitivity to cordycepin in MLH1-deficient cells was associated with reduced upregulation of the cell cycle inhibitor p21. MLH1-deficient cells also exhibited reduced susceptibility to apoptosis upon treatment with cordycepin, as demonstrated by the reduced PARP-1 cleavage. Our findings showed that MLH1-deficient HCT116 colon tumor cells are resistant to the cytostatic and cytotoxic effect of cordycepin, indicating a possible involvement of MMR in mRNA polyadenylation. The findings also suggest that cordycepin is not suitable to therapeutically encounter tumor cells lacking MLH1 expression.




The anti-cancer activity of dihydroartemisinin is associated with induction of iron-dependent endoplasmic reticulum stress in colorectal carcinoma HCT116 cells  

Microsoft Academic Search

Summary  Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is among the artemisinin derivatives possessing\\u000a potent anti-malarial and anti-cancer activities. In the present study, we found that DHA displayed significant anti-proliferative\\u000a activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis.\\u000a To further elucidate the mechanism of action of DHA,

Jin-Jian Lu; Si-Meng Chen; Xiao-Wei Zhang; Jian Ding; Ling-Hua Meng


Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT116 cells  

Microsoft Academic Search

This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated\\u000a the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays,

Sei-Jung Lee; Kye-Taek Lim



The role of Nrf2 and apoptotic signaling pathways in oroxylin A-mediated responses in HCT-116 colorectal adenocarcinoma cells and xenograft tumors.  


Oroxylin A is a flavonoid found in the roots of Scutellaria baicalensis Georgi, a herbal medicine commonly used as an antipyretic, analgesic, antitumor, and anti-inflammatory agent. It has recently been investigated for its anticancer activities in hepatoma, gastric, and breast tumors. Here, we investigated the antitumor effects of oroxylin A in human colon carcinoma HCT-116 cells in vitro and in vivo. We characterized the proapoptotic effect of oroxylin A using diamidino-phenyl-indole (DAPI) and annexin V/PI staining. We then found that both caspase-3 and caspase-9 were activated, the expression of Bcl-2 protein decreased, and the expression of Bax protein increased after treatment with oroxylin A. In addition, oroxylin A increased nuclear transcription factor erythroid-related factor 2 (Nrf2) expression and induced Nrf2 translocation into the nucleus. Furthermore, we found that oroxylin A treatment elevated intracellular reactive oxygen species levels and increased the protein expression level of two of the Nrf2 target genes heme oxygenase-1 and NADP(H):quinone oxidoreductase-1 in HCT-116 cells. Finally, our study demonstrated that oral administration of oroxylin A significantly decreased tumor volume and weight in immunodeficient mice that were inoculated with HCT-116 cells. The in-vivo chemopreventive efficacy of oroxylin A against HCT-116 human colon cancer was accompanied by its proapoptotic and Nrf2-inducing activities, which correlates with the in-vitro study. This is the first demonstration of oroxylin A-dependent chemoprevention in colon cancer and may offer a potential mechanism for its anticancer action in vivo. PMID:22526619

Hu, Rong; Chen, Nan; Yao, Jing; Zhao, Qing; Zhang, Fengyi; Li, Zhi-Yu; You, Qi-Dong; Guo, Qing-Long



Nocodazole-induced p53-dependent c-Jun N-terminal kinase activation reduces apoptosis in human colon carcinoma HCT116 cells.  


Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53-/- cells in response to short term nocodazole treatment through application of Kinetworks immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53-/- cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53-/- cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53-/- cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function. PMID:12221076

Zhang, Hong; Shi, Xiaoqing; Zhang, Qian-Jin; Hampong, Maggie; Paddon, Harry; Wahyuningsih, Dewi; Pelech, Steven



A 150-kDa glycoprotein isolated from Solanum nigrum L. has cytotoxic and apoptotic effects by inhibiting the effects of protein kinase C alpha, nuclear factor-kappa B and inducible nitric oxide in HCT116 cells  

Microsoft Academic Search

This study was carried out to investigate the anticancer effects of a 150-kDa glycoprotein isolated from Solanum nigrum L. (SNL glycoprotein) on spontaneously and experimentally induced tumor promotion in HCT-116 cells. For spontaneously induced tumor promotion, we evaluated the cytotoxic and apoptotic effects in HCT-116 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT), DNA fragmentation, and H33342 and ethidium bromide staining

Sei-Jung Lee; Phil-Sun Oh; Jeong-Hyeon Ko; Kwang Lim; Kye-Taek Lim



Induction of apoptosis in human colon cancer HCT116 cells treated with an extract of the plant product, Chios mastic gum.  


A hexane extract of the plant product Chios mastic gum (He-CMG) is demonstrated to kill human colon cancer cells in vitro via the process of anoikis. Specifically, the sequence of events includes He-CMG-induced GI-arrest of the cells, detachment of the cells from the substrate and subsequent apoptosis. Anoikis is dependent on the concentration and duration of treatment with He-CMG. Presence of the pan-caspase inhibitor, Z-VAD-fmk, did not prevent cell detachment, but it did prevent apoptosis of the detached cells indicating that the process of cell detachment, but not apoptosis, is independent of caspase activation. He-CMG-induced apoptosis is associated with activation of the initiator caspases-8, and -9 and the effector caspase-3. Caspases are activated in cells at a relatively long time after detachment, and caspase-3 activation may require caspase-8 or caspase-9 activation, as determined by using HCT116 isogenic clones impaired in apoptosis mechanisms that involve these two caspases. Finally, electron microscopy observations indicated a time-dependent appearance of morphological features both typical and non-typical of apoptosis in cells treated with He-CMG for various periods of time. Taken together, the results demonstrated that He-CMG induces apoptosis in HCT116 cells and, therefore, further in vivo and in vitro studies of the anticancer activities of this plant product are warranted. PMID:15796160

Balan, Kannan V; Demetzos, Costas; Prince, Jeffrey; Dimas, Konstantine; Cladaras, Margarita; Han, Zhiyong; Wyche, James H; Pantazis, Panayotis


Double siRNA-targeting of cIAP2 and LIVIN results in synergetic sensitization of HCT-116 cells to oxaliplatin treatment  

PubMed Central

Purpose Most colon cancers show low sensitivity to treatment with oxaliplatin and a specific strategy is needed to overcome this problem. Our approach uses RNA interference to silence the expression of target genes responsible for the development of oxaliplatin resistance. Profile analysis of genes related to the regulation of apoptosis allowed identification of target genes showing the greatest degree of upregulation in response to oxaliplatin exposure. Methods We designed a panel of genes with functions closely related to inactivation of the caspase cascade, endoplasmic reticulum stress reduction, and drug metabolism. The candidate genes were silenced by means of specific small interfering RNA (siRNA) oligonucleotides. Results The caspase 3 and 9 inhibitors of apoptosis 2 (cIAP2) and LIVIN were found to be the most dose-responsive genes during the period of oxaliplatin treatment. Two-fold sensitization of cells to oxaliplatin was observed with independent knockdown of either cIAP2 or LIVIN expression. siRNA-silencing of both targets produced a five-fold increase in oxaliplatin sensitivity of HCT-116 cells. Conclusion A dose-dependent approach revealed reliable targets for siRNA-silencing under low doses of oxaliplatin. Targeting the key proapoptotic chain with several specific siRNAs resulted in synergetic sensitization of HCT-116 cells to oxaliplatin treatment.

Bavykin, Andrey S; Korotaeva, Alexandra A; Poyarkov, Stanislav V; Syrtsev, Alexandr V; Tjulandin, Sergei A; Karpukhin, Alexandr V



Differential growth inhibitory effects of highly oxygenated guaianolides isolated from the Middle Eastern indigenous plant Achillea falcata in HCT-116 colorectal cancer cells.  


Medicinal plants play a crucial role in traditional medicine and in the maintenance of human health worldwide. Sesquiterpene lactones represent an interesting group of plant-derived compounds that are currently being tested as lead drugs in cancer clinical trials. Achillea falcata is a medicinal plant indigenous to the Middle Eastern region and belongs to the Asteraceae family, which is known to be rich in sesquiterpene lactones. We subjected Achillea falcata extracts to bioassay-guided fractionation against the growth of HCT-116 colorectal cancer cells and identified four secotanapartholides, namely 3-?-methoxy-isosecotanapartholide (1), isosecotanapartholide (2), tanaphallin (3), and 8-hydroxy-3-methoxyisosecotanapartholide (4). Three highly oxygenated guaianolides were isolated for the first time from Achillea falcata, namely rupin A (5), chrysartemin B (6), and 1?, 2?-epoxy-3?,4?,10?-trihydroxyguaian-6?,12-olide (7). These sesquiterpene lactones showed no or minor cytotoxicity while exhibiting promising anticancer effects against HCT-116 cells. Further structure-activity relationship studies related the bioactivity of the tested compounds to their skeleton, their lipophilicity, and to the type of functional groups neighboring the main alkylating center of the molecule. PMID:23860275

Tohme, Rita; Al Aaraj, Lamis; Ghaddar, Tarek; Gali-Muhtasib, Hala; Saliba, Najat A; Darwiche, Nadine



Novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivative, MHY-449, induces apoptosis and cell cycle arrest in HCT116 human colon cancer cells.  


Colorectal cancer (CRC) is the second most frequent cancer in men and the third most common cancer in women in Korea. In spite of the significant advances in conventional therapeutic approaches to CRC, most patients ultimately die of their disease. There is a need to develop novel preventive approaches for this malignancy. This study was carried out to investigate the anticancer effect of the diastereoisomeric compounds, MHY-449 and MHY-450, novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivatives, on HCT116 human colon cancer cells. MHY-449 exhibited more potent cytotoxicity than MHY-450, against HCT116 cells. Treatment of cells with MHY-449 resulted in growth inhibition and induction of apoptosis in a concentration-dependent manner, and inhibition of proliferation in a time-dependent manner. The induction of apoptosis was observed by decreased cell viability, DNA fragmentation, activation of protein levels involved in death receptors. Moreover, activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase and alteration in the ratio of Bax/Bcl-2 protein expression was observed. MHY-449 induced G2/M phase arrest in the cell cycle progression which was observed by flow cytometry analysis, and a decrease in the protein expression of cyclin B1 and its activating partners Cdc25c and Cdc2. MHY-449 also caused increase in the expression levels of p53, a tumor suppressor gene, and p21WAF1/CIP and p27KIP, G2/M phase inhibitors. These results suggest that MHY-449 may be a useful candidate for chemo-prevention and/or treatment of colon cancer. PMID:23064444

Hwang, Hye Jung; Kang, Yong Jung; Hossain, Mohammad Akbar; Kim, Dong Hwan; Jang, Jung Yoon; Lee, Sun Hwa; Yoon, Jeong-Hyun; Moon, Hyung Ryong; Kim, Hyung Sik; Chung, Hae Young; Kim, Nam Deuk



B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells  

PubMed Central

Background And Objective The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells. Methods Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells. Results Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition. Conclusions Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.

Guo, Zhang-Yan; Wei, Ming; Meng, Yan-Ling; Yang, An-Gang; Wen, Wei-Hong



Preparation of branched cyclomaltoheptaose with 3-O-?-L-fucopyranosyl-?-D-mannopyranose and changes in fucosylation of HCT116 cells treated with the fucose-modified cyclomaltoheptaose.  


From a mixture of 4-nitrophenyl ?-L-fucopyranoside and D-mannopyranose, 3-O-?-L-fucopyranosyl-D-mannopyranose was synthesised through the transferring action of ?-fucosidase (Sumizyme PHY). 6(I),6(IV)-Di-O-(3-O-?-L-fucopyranosyl-?-D-mannopyranosyl)-cyclomaltoheptaose {8, 6(I),6(IV)-di-O-[?-L-Fuc-(1?3)-?-D-Man]-?CD} was chemically synthesised using the trichloroacetimidate method. The structures were confirmed by MS and NMR spectroscopy. A cell-based assay using the fucosyl ?CD derivatives, including the newly synthesised 8, showed that derivatives with two branches of the ?-L-Fuc or ?-L-Fuc-(1?3)-?-D-Man residues possessed slight growth-promoting effects and lower toxicity in HCT116 cells compared to those with one branch. These compounds may be useful as drug carriers in targeted drug delivery systems. PMID:23623960

Kimura, Madoka; Masui, Yuki; Shirai, Yuko; Honda, Chie; Moriwaki, Kenta; Imai, Taku; Takagi, Uichiro; Kiryu, Takaaki; Kiso, Taro; Murakami, Hiromi; Nakano, Hirofumi; Kitahata, Sumio; Miyoshi, Eiji; Tanimoto, Toshiko



The anti-cancer activity of dihydroartemisinin is associated with induction of iron-dependent endoplasmic reticulum stress in colorectal carcinoma HCT116 cells.  


Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is among the artemisinin derivatives possessing potent anti-malarial and anti-cancer activities. In the present study, we found that DHA displayed significant anti-proliferative activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis. To further elucidate the mechanism of action of DHA, a proteomic study employed two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed. Glucose-regulated protein 78 (GRP78), which is related with endoplasmic reticulum stress (ER stress), was identified to be significantly up-regulated after DHA treatment. Further study demonstrated that DHA enhanced expression of GRP78 as well as growth arrest and DNA-damage-inducible gene 153 (GADD153, another ER stress-associated molecule) at both mRNA and protein levels. DHA treatment also led to accumulation of GADD153 in cell nucleus. Moreover, pretreatment of HCT116 cells with the iron chelator deferoxamine mesylate salt (DFO) abrogated induction of GRP78 and GADD153 upon DHA treatment, indicating iron is required for DHA-induced ER stress. This result is consistent with the fact that the anti-proliferative activity of DHA is also mediated by iron. We thus suggest the unbalance of redox may result in DHA-induced ER stress, which may contribute, at least in part, to its anti-cancer activity. PMID:20607588

Lu, Jin-Jian; Chen, Si-Meng; Zhang, Xiao-Wei; Ding, Jian; Meng, Ling-Hua



N, N-dimethyl-D-erythro-sphingosine increases intracellular Ca2+ concentration via Na+-Ca2+-exchanger in HCT116 human colon cancer cells.  


N,N-dimethyl-D-erythro-sphingosine (DMS), an N-methyl derivative of sphingosine, is an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In previous reports, DMS-induced intracellular Ca2+ increase concentration ([Ca2+]i) was studied in T lymphocytes, monocytes, astrocytes and neuronal cells. In the present study, we studied DMS-induced increase of [Ca2+]i in HCT116 human colon cancer cells. We found that the DMS-induced increase of [Ca2+]i in colon cancer cells is composed of Ca2+ release from intracellular Ca2+ stores and subsequent Ca2+ influx. The Ca2+ release is not related to modulation of inositol 1,4,5-trisphosphate (IP3) receptor or ryanodine receptor. On the other hand, the Ca2+ influx is mediated largely through Ca2+ channels sensitive to verapamil, nifedipine, Ga3+, and La3+. Furthermore, we found that the response is inhibited by bepridil and Ni2+, specific inhibitors of Na+-Ca2+-exchanger, suggesting involvement of Na+-Ca2+ exchanger in the DMS-induced [Ca2+]i increase in colon cancer cells. This inhibition was also observed in U937 monocytes, but not in 1321N1 astrocytes. PMID:18277608

Kim, Hyo-Lim; Im, Dong-Soon



Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells  

Microsoft Academic Search

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which\\u000a are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor\\u000a showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic\\u000a amines involved in many cellular processes, including apoptosis. In

Elif Damla Ar?san; Ajda Çoker; Narçin Palavan-Ünsal


Two Functional S100A4 Monomers Are Necessary for Regulating Nonmuscle Myosin-IIA and HCT116 Cell Invasion  

PubMed Central

S100A4, a member of the Ca2+-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30–60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer–polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.

House, Reniqua P.; Pozzuto, Maria; Patel, Purvi; Dulyaninova, Natalya G.; Li, Zhong-Hua; Zencheck, Wendy D.; Vitolo, Michele I.; Weber, David J.; Bresnick, Anne R.



Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant  

PubMed Central

Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and ?-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer.

Lan, Yu-Hsuan; Chiang, Jo-Hua; Huang, Wen-Wen; Lu, Chi-Cheng; Chung, Jing-Gung; Wu, Tian-Shung; Jhan, Jia-Hua; Lin, Kuei-Li; Pai, Shu-Jen; Chiu, Yu-Jen; Tsuzuki, Minoru; Yang, Jai-Sing



Cytotoxicity and metabolism of 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine in HCT 116 colon cancer cells.  


We examined the cytotoxicity, biochemical effects and metabolism of 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP), a synthetic nucleoside inhibitor of phosphoribosylpyrophosphate synthetase, in HCT 116 human colorectal cancer cells. A 4-hr exposure to 1 and 10 microM MRPP inhibited cell growth over a 72-hr period by 76 and 89%, and inhibited clonogenic capacity by 36 and 65%, respectively. MRPP was avidly metabolized to the 5'-monophosphate derivative (MRPP-MP), and MRPP-MP formation increased with increasing MRPP exposure ( MRPP-MP was stable, and the intracellular half-life was in excess of 48 hr. A 4-hr exposure to 10 microM MRPP resulted in significant decreases in ATP, UTP, GTP, CTP, dATP, dTTP, and PRPP pools. Near maximal ribonucleotide triphosphate depletion was achieved with > or = 24 MRPP, and growth inhibition as a function of MRPP closely reflected the biochemical effects. Ribonucleotide triphosphate pools remained depleted for up to 48 hr after drug removal, apparently as a consequence of the prolonged retention of MRPP-MP. MRPP (10 microM) inhibited the salvage of [3H]guanine, [3H]adenine and [3H]guanosine, and concurrent exposure to MRPP and either 100 microM adenine, hypoxanthine, or guanine did not reverse ATP or GTP depletion. Concurrent exposure to 10 microM MRPP and either 10 microM adenosine, uridine or thymidine was accompanied by repletion of ATP, UTP, and dTTP pools, respectively, but depletion of other nucleotide pools was not corrected. In contrast, 10 microM guanosine did not correct GTP depletion in the presence of MRPP. The combination of 10 microM each of thymidine, uridine, adenosine and guanosine during and following a 24-hr exposure to MRPP provided partial protection against 0.1 or 1 microM MRPP, but did not affect the cytotoxicity associated with 10 microM MRPP. MRPP is a novel antimetabolite that inhibits both de novo and salvage pathways for purine synthesis and de novo pyrimidine synthesis. PMID:7528507

Grem, J L; Daychild, P; Drake, J; Geoffroy, F; Trepel, J B; Pirnia, F; Allegra, C J



Stable suppression of the R2 subunit of ribonucleotide reductase by R2-targeted short interference RNA sensitizes p53(-/-) HCT-116 colon cancer cells to DNA-damaging agents and ribonucleotide reductase inhibitors.  


Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(-/-) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. To evaluate the involvement of R2 in response to DNA damage, p53(-/-) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(-/-) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2. PMID:15096505

Lin, Z Ping; Belcourt, Michael F; Cory, Joseph G; Sartorelli, Alan C



Macrophage inhibitory cytokine-1 (MIC1) and subsequent urokinase-type plasminogen activator mediate cell death responses by ribotoxic anisomycin in HCT116 colon cancer cells  

Microsoft Academic Search

Ribosome-inactivating stresses possess a potent regulatory activity against tumor cell progression. In this study, we demonstrated that macrophage inhibitory cytokine-1 (MIC-1) and its associated signals determined the colon cancer cell response to the chemical ribotoxic stress. The ribotoxic stress agent anisomycin-induced MIC-1 gene expression which was involved in the ribotoxin-induced apoptotic pathway. MIC-1 was also a critical inducer of apoptosis-related

Hyun Yang; Hye Jin Choi; Seong Hwan Park; Jong Sik Kim; Yuseok Moon



Combination of Albendazole and 2-Methoxyestradiol significantly improves the survival of HCT-116 tumor-bearing nude mice  

PubMed Central

Background Albendazole (ABZ) is a microtubule-targeting anthelmintic with a remarkable activity against a variety of human cancer cells. In this study, we examined if the antitumor activity of ABZ could be enhanced by its combination with other microtubule-binding agents. Methods The interactions between ABZ and microtubule-binding agents, paclitaxel, vinblastine, colchicine, and 2-methoxyestradiol were characterized using median effect analysis method in HCT-116 colorectal cancer cells and DU145 prostate cancer cell line. The mechanism underlying the synergistic interaction related to tubulin polymerization and apoptosis was then investigated. Finally, the effect of the combination therapy on the survival of HCT-116 tumor-bearing nude mice was evaluated. Results Among the tested drugs, a synergistic anti-proliferative effect was observed with the combination of low concentrations of ABZ plus colchicine and ABZ plus 2-methoxyestradiol (2ME). Exploring the mechanism of the interaction between ABZ and 2ME revealed that the combination therapy synergistically activated the extrinsic pathway of apoptosis. Consistent with in vitro results, the combination of low concentration of ABZ with 2ME prolonged the survival of mice-bearing HCT-116 tumors. High concentration of ABZ in combination with 2ME, however, proved to be less effective than ABZ alone. Conclusions The combination of low doses of ABZ and 2ME has shown promising results in our pre-clinical model. Additionally, the finding that the combination of two microtubule-binding agents that share the same binding site can act synergistically may lead to the development of new therapeutic strategies in cancer treatment.



p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells  

Microsoft Academic Search

S-ibuprofen which inhibits the cyclooxygenase-1\\/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53wt) or being p(HCT-116 p53?\\/?), we demonstrated that both induction of a cell cycle

Astrid Janssen; Susanne Schiffmann; Kerstin Birod; Thorsten J. Maier; Ivonne Wobst; Gerd Geisslinger; Sabine Groesch



Growth inhibition and antioxidative status induced by selenium-enriched broccoli extract and selenocompounds in DNA mismatch repair-deficient human colon cancer cells.  


The effects of enzymatic-digested Se-enriched broccoli extracts (SeB) and selenocompounds on growth and antioxidative status in human colon cancer cells was investigated in this study. HCT116 and HCT116+Chr.3 cells were treated with selenocompounds (sodium selenite, sodium selenate, Se-Met, MeSeCys) or SeB [high-Se (H-SeB) or low-Se (L-SeB)]. The cytotoxicity induced by selenocompounds in HCT116 cells was not associated with cellular H2O2 level, while the differential cytotoxicity observed by sodium selenite between HCT116 and HCT116+Chr.3 cell lines was related to cellular H2O2 production with the change in antioxidative enzyme activity, and the restoration of chromosome 3. H-SeB was found to reduce the cellular H2O2 content in HCT116+Chr.3 cells. The results in this study indicate that regardless of Se content, the cytotoxicity in HCT116 cells of both SeB forms appeared to be H2O2-independent, whereas the cytotoxicity in HCT116+Chr.3 of either SeB form appeared to be H2O2-dependent with an increase in antioxidative ability for H-SeB. PMID:23561105

Tsai, Cheng-Fang; Ou, Bor-Rung; Liang, Yu-Chuan; Yeh, Jan-Ying



Characterization of the N-methoxyindole-3-carbinol (NI3C)Induced Cell Cycle Arrest in Human Colon Cancer Cell Lines  

Microsoft Academic Search

Recent results have shown that indole-3-carbinol (I3C) inhibits the cellular growth of human cancer cell lines. In some cruciferous vegetables, another indole, N-methoxyindole-3-carbinol (NI3C), is foundbesideI3C.Knowledge aboutthe biologicaleffectsofNI3Cis limited.TheaimofthepresentstudywastoshowtheeffectofNI3C on cell growth of two human colon cancer cell lines, DLD-1 and HCT-116. For the first time it is shown that NI3C inhibits cellular growth of DLD-1 and HCT-116 and that

Antje S. Neave; Sussi M. Sarup; Michel Seidelin; Fritz Duus; Ole Vang



Anomalous dystroglycan in carcinoma cell lines  

Microsoft Academic Search

Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non-tumorigenic mammary human cell lines, the expected ?-dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells

Carmen Losasso; Francesca Di Tommaso; Alessandro Sgambato; Raffaele Ardito; Achille Cittadini; Bruno Giardina; Tamara C. Petrucci; Andrea Brancaccio



Comprehensive sampling of gene expression in human cell lines with massively parallel signature sequencing  

Microsoft Academic Search

Whereas information is rapidly accumulating about the structure and position of genes encoded in the human genome, less is known about the complexity and relative abundance of their expression in individual human cells and tissues. Here, we describe the characteristics of the transcriptomes of two cultured cell lines, HB4a (normal breast epithelium) and HCT-116 (colon adenocarcinoma), using massively parallel signature

C. Victor Jongeneel; Christian Iseli; Brian J. Stevenson; Gregory J. Riggins; Anita Lal; Alan Mackay; Robert A. Harris; Michael J. O'Hare; A. Munro Neville; Andrew J. G. Simpson; Robert L. Strausberg



Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells  

Microsoft Academic Search

Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP\\/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed

G Breuzard; J.-F Angiboust; P Jeannesson; M Manfait; J.-M Millot



4?- O-Alkyaloenin derivatives and their sulfates directed toward overcoming multidrug resistance in tumor cells  

Microsoft Academic Search

The cytotoxic effects on HCT 116, Hep G2 and HCT 116\\/VCR 100-1-1 cell lines of synthetic 4?-O-alkylaloenins (2–17), 4?-O-benzylaloenin (18) and 4?-O-allylaloenin (19) were examined by MTT assay, and compared with that of aloenin (1) isolated from Aloe arborescens Mill. Var. natalensis Berger which showed no marked effect (IC50 value: >100?M). The cytotoxic effects of 4?-O-alkylaloenin sulfates (21–29) were also

Guang-zhu Jin; Hong-Ji Quan; Jyunichi Koyanagi; Kazuhiro Takeuchi; Yoshihiko Miura; Fusao Komada; Setsuo Saito



Apoptotic death in adenocarcinoma cell lines induced by butyrate and other histone deacetylase inhibitors  

Microsoft Academic Search

n-Butyrate inhibits the growth of colon cancer cell lines. In the HCT 116 cell line, butyrateinduced growth inhibition is almost fully reversible, whereas in the VACO 5 cell line, a subpopulation undergoes apoptosis within 30 hr of treatment with butyrate. Concurrent treatment of VACO 5 cells with butyrate and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) accelerates and increases the incidence

John A. McBain; Alan Eastman; C. Stefan Nobel; Gerald C. Mueller



p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells  

SciTech Connect

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53{sup wt}) or being p(HCT-116 p53{sup -/-}), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53{sup -/-} xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53{sup wt} cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53{sup wt} cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75{sup NTR}, p53 and Bax.

Janssen, Astrid; Schiffmann, Susanne; Birod, Kerstin; Maier, Thorsten J.; Wobst, Ivonne; Geisslinger, Gerd [pharmazentrum frankfurt/ZAFES, Institut fuer Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universitaet Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt (Germany); Groesch, Sabine [pharmazentrum frankfurt/ZAFES, Institut fuer Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universitaet Frankfurt, Theodor Stern Kai 7, 60590 Frankfur (Germany)], E-mail:



Redox-sensitive mechanisms of phytochemical-mediated inhibition of cancer cell proliferation 1 1 G. Loo, K. Takahashi, A. Powolny, R.A. Hopkins, Induction of GADD45 gene expression by phenylethyl isothiocyanate in HCT116 human colon adenocarcinoma cells, FASEB J 16 (2002) A264-A265 (abstract #216.15). Also, unpublished data. (review)  

Microsoft Academic Search

Phytochemicals are potential cancer chemopreventive agents, based partly on cellular research establishing that phytochemicals inhibit the proliferation of cancer cells. To elucidate the mechanism of phytochemicals, a basic understanding is needed of what stimulates cancer cell proliferation. Cancer cells, particularly those that are highly invasive or metastatic, may require a certain level of oxidative stress to maintain a balance between

George Loo



The anticancer effect of saffron in two p53 isogenic colorectal cancer cell lines  

PubMed Central

Background Saffron extract, a natural product, has been shown to induce apoptosis in several tumor cell lines. Nevertheless, the p53-dependency of saffron’s mechanism of action in colon cancer remains unexplored. Material and methods In order to examine saffron’s anti-proliferative and pro-apoptotic effects in colorectal cancer cells, we treated two p53 isogenic HCT116 cell lines (HCT wildtype and HCT p53?/?) with different doses of the drug and analyzed cell proliferation and apoptosis in a time-dependent manner. MTT viability and crystal violet assays were performed in order to determine the effective dose of saffron on both cell lines. The cell cycle progress was examined by Flow cytometric analysis. Apoptosis was assessed using Annexin-PI-staining and Western Blotting for caspase 3 and PARP cleavage. Autophagy was determined by Western Blotting of the light chain 3 (LC3)-II and Beclin 1 proteins. The protein content of phospho-H2AX (?H2AX), a sensor of DNA double strand breaks, was also analyzed by Western Blotting. Results Saffron extract induced a p53-dependent pattern of cell cycle distribution with a full G2/M stop in HCT116 p53 wildtype cells. However, it induced a remarkable delay in S/G2 phase transit with entry into mitosis in HCT116 p53 ?/? cells. The apoptotic Pre-G1 cell fraction as well as Annexin V staining and caspase 3 cleavage showed a more pronounced apoptosis induction in HCT116 p53 wildtype cells. Obviously, the significantly higher DNA-damage, reflected by ?H2AX protein levels in cells lacking p53, was coped by up-regulation of autophagy. The saffron-induced LC3-II protein level was a remarkable indication of the accumulation of autophagosomes, a response to the cellular stress condition of drug treatment. Conclusions This is the first study showing the effect of saffron in HCT116 colorectal cancer cells with different p53 status. Saffron induced DNA-damage and apoptosis in both cell lines. However, autophagy has delayed the induction of apoptosis in HCT116 p53 ?/? cells. Considering the fact that most tumors show a functional p53 inactivation, further research is needed to elucidate the long-term effects of saffron in p53 ?/? tumors.



Cellular response to 5-fluorouracil (5FU) in 5FU-resistant colon cancer cell lines during treatment and recovery  

Microsoft Academic Search

BACKGROUND: Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number

Paula M De Angelis; Debbie H Svendsrud; Katherine L Kravik; Trond Stokke



Glutathione S-Transferase P1 Promotes Tumorigenicity in HCT116 Human Colon Cancer Cells  

Microsoft Academic Search

GSTP1 is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles with glutathione in the process of detoxification. GSTP1 is widely overexpressed in colorectal cancer, from aberrant crypt foci to advanced carcinomas. Increased expression of GSTP1 is associated with multidrug resistance and a worse clinical prognosis. However, GSTP1-null mice have an increased risk of tumor

Duyen T. Dang; Manu Kohli; Carlo Rago



The in vitro effects of three lysosomotropic detergents against three human tumor cell lines  

Microsoft Academic Search

Three lysosomotropic detergents, N-dodecylimidazole (NDI) 1, and two new analogues, serine dodecylamide (SDA) 2 and O-methyl serine dodecylamide (MSD) 3, were tested against three human tumor cell lines in vitro: HCT116, RCA and MCF-7. All three demonstrated dose-related cytotoxicity which, except for a few cases, was also dependent on incubation time, and visually resembled cell death by apoptosis.

Gene M. Dubowchik; Susan L. Gawlak; Raymond A. Firestone



Spectral karyotype analysis of colon cancer cell lines of the tumor suppressor and mutator pathway  

Microsoft Academic Search

Background and aims: Microsatellite instability (MSI) is characterized by the size variation of microsatellites in tumor DNA as compared to matching normal DNA due to defects in the mismatch repair system. To examine the chromosomal differences in microsatellite-stable (MSS) and -unstable (MSI) tumors in detail, we analyzed MSS (Caco-2, Colo-205, SW948) and MSI (HCT-15, HCT-116, LoVo) cell lines by spectral

R. Melcher; S. Koehler; C. Steinlein; M. Schmid; C. R. Mueller; H. Luehrs; T. Menzel; W. Scheppach; H. Moerk; M. Scheurlen; J. Koehrle; O. Al-Taie



Trisubstituted and tetrasubstituted pyrazolines as a novel class of cell-growth inhibitors in tumor cells with wild type p53.  


Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted pyrazoline were synthesized and tested for their inhibitory effects versus the p53(+/+) HCT116 and p53(-/-) H1299 human tumor cell lines. Several compounds were active against the two cell lines displaying IC50 values in the low micromolar range with a clearly more pronounced effect on the p53(+/+) HCT116 cells. The compound class shows excellent developability due to the modular synthesis, allowing independent optimization of all three to four key substituents to improve the properties of the molecules. PMID:24139845

Abdel-Halim, Mohammad; Keeton, Adam B; Gurpinar, Evrim; Gary, Bernard D; Vogel, Simon M; Engel, Matthias; Piazza, Gary A; Boeckler, Frank M; Hartmann, Rolf W; Abadi, Ashraf H



Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells.  


The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells. PMID:12466962

Jaiswal, Aruna S; Marlow, Benjamin P; Gupta, Nirupama; Narayan, Satya



Suppression of manganese superoxide dismutase augments sensitivity to radiation, hyperthermia and doxorubicin in colon cancer cell lines by inducing apoptosis  

PubMed Central

Increased expression of manganese superoxide dismutase (Mn-SOD), one of the mitochondrial enzymes involved in the redox system, has been shown to diminish the cytotoxic effects of several anti-cancer modalities, including tumour necrosis factor-?, ionizing radiation, certain chemotherapeutic agents and hyperthermia. We asked if Mn-SOD is a potential target to augment the sensitivity of cancer cells to various anti-cancer treatments and for this we established stable Mn-SOD antisense RNA expressing cell clones from two human colon cancer cell lines, HCT116 (p53 wild-type) and DLD1 (p53 mutant-type). Suppression of Mn-SOD in HCT116 was accompanied by an increased sensitivity to radiation, hyperthermia and doxorubicin, as compared with findings in controls. The mitochondrial permeability transition, as measured by a decrease of the mitochondrial transmembrane potential was more intensely induced by radiation in HCT116 antisense clones than in the control, an event followed by a greater extent of DNA fragmentation. Apoptosis was also induced by hyperthermia more intensely in HCT116 antisense clones than in the control. On the other hand, DLD1 antisense clones did not exhibit any enhancement of sensitivity to any of these treatments. These data support the possibility that inhibition of Mn-SOD activity renders colon cancer cells with wild-type p53 susceptible to apoptosis induced by radiation, hyperthermia and selected anti-cancer drugs. Therefore, we suggest that Mn-SOD could be a target molecule to overcome the resistance to anti-cancer treatments in some colon cancer cells carrying wild-type p53. © 2000 Cancer Research Campaign

Kuninaka, S; Ichinose, Y; Koja, K; Toh, Y



In vivo and in vitro antitumor activity of oxaliplatin in combination with cetuximab in human colorectal tumor cell lines expressing different level of EGFR  

Microsoft Academic Search

This study aimed to assess the effect of cetuximab (C225, Erbitux®, a chimeric anti-epidermal growth factor receptor (EGFR)\\u000a monoclonal antibody) in combination with oxaliplatin in vitro and in vivo on four colon cancer cell lines (HCT-8; HT-29, SW620,\\u000a HCT-116) expressing different levels of EGFR. In vitro, cetuximab combined with oxaliplatin significantly decreased the IC50 values of oxaliplatin in HCT-8 (EGF-R

Diane Balin-Gauthier; Jean-Pierre Delord; Philippe Rochaix; Valérie Mallard; Fabienne Thomas; Isabelle Hennebelle; Roland Bugat; Pierre Canal; Cuider Allal



Sphingosine kinase 1 plays a role in the upregulation of CD44 expression through extracellular signal-regulated kinase signaling in human colon cancer cells.  


Our previous study has shown that the activity and expression of sphingosine kinase (SPHK) regulated the sensitivity of human colon cancer cells to the chemotherapeutic oxaliplatin (L-OHP). In addition, the cancer stem cell marker CD44 increases cell resistance to anticancer drugs. Here, we use colon cancer cell lines to examine the relationship between SPHK1 activity and CD44 expression.CD44 expression was measured by western blotting and quantitative PCR in two human colon cancer cell lines: L-OHP-resistant RKO and L-OHP-sensitive HCT116. The regulation of CD44 by SPHK1 was examined by either blocking or overexpressing SPHK1 and by using an L-OHP-resistant HCT116 clone (HCT116-R).The levels of SPHK1, CD44, phosphorylated-Akt, and phosphorylated-extracellular signal-regulated kinase (ERK) were much higher in the RKO cells than in the HCT116 cells. The treatment of RKO cells with the SPHK inhibitor or SPHK1 silencing by RNA interference suppressed CD44 protein expression. SPHK1 and CD44 levels were much higher in HCT116-R cells compared with the parental HCT116 cells. Transfection of HCT116 cells with SPHK1 cDNA enhanced the expression of both CD44 and phosphorylated-ERK. The increase in the CD44 protein level was abolished by the inhibition of ERK phosphorylation. Treatment of RKO cells with the sphingosine-1-phosphate (S1P)2 receptor antagonist suppressed ERK phosphorylation and the expression of CD44 mRNA and protein. Exogenous stimulation with S1P increased ERK phosphorylation and CD44 protein expression in HCT116 cells, but treatment with an MEK inhibitor and S1P2 receptor antagonist blocked this effect.These findings indicate that SPHK1 and its product, S1P, contribute toward the regulation of CD44 protein expression through the ERK signaling pathway through S1P2 in human colon cancer cells. PMID:23426175

Kawahara, Satomi; Otsuji, Yoko; Nakamura, Mitsuhiro; Murakami, Masashi; Murate, Takashi; Matsunaga, Toshiyuki; Kanoh, Hiroyuki; Seishima, Mariko; Banno, Yoshiko; Hara, Akira



The farnesyl transferase inhibitor RPR-130401 does not alter radiation susceptibility in human tumor cells with a K-Ras mutation in spite of large changes in ploidy and lamin B distribution  

Microsoft Academic Search

BACKGROUND: Growth inhibition by RPR-130401, a non-peptidomimetic farnesyltransferase inhibitor, was investigated without or with combined exposure to ionizing radiation in three human tumor cell lines (HCT-116, MiAPaCa-2 and A-549) bearing a point mutation in the K-Ras gene. RESULTS: RPR-130401 inhibited cell growth with an IC50 of 50 nM (HCT-116), 120 nM (MiAPaCa-2) and 710 nM (A-549), with a poor incidence

Frédérique Mégnin-Chanet; François Lavelle; Vincent Favaudon



Allicin Purified From Fresh Garlic Cloves Induces Apoptosis in Colon Cancer Cells Via Nrf2  

Microsoft Academic Search

Allicin (diallyl thiosulfinate) is the best-known biologically active component in freshly crushed garlic extract. We developed a novel, simple method to isolate active allicin, which yielded a stable compound in aqueous solution amenable for use in in vitro and in vivo studies. We focused on the in vitro effects of allicin on cell proliferation of colon cancer cell lines HCT-116,

Wolf Bat-Chen; Tal Golan; Irena Peri; Zvi Ludmer; Betty Schwartz



The bisphosphonate zoledronic acid inhibits the growth of HCT116 colon carcinoma cells and induces tumor cell apoptosis  

Microsoft Academic Search

Besides its preventive action on bone resorption the third generation bisphosphonate zoledronic acid (ZOL) has been shown\\u000a to display potent inhibitory action on the formation of bone metastases of various human cancers. Recent research also indicates\\u000a an antitumoral effect on primary tumors and visceral metastases. Here we investigate for the first time the effect of ZOL\\u000a on the human colon

Lilian Sewing; Florian Steinberg; Harald Schmidt; Rüdiger Göke



Meloxicam inhibits the growth of colorectal cancer cells  

Microsoft Academic Search

Cyclooxygenase-2 has been reported to play an important role in colorectal carcinogenesis. The effects of meloxicam (a COX-2 inhibitor) on the growth of two colon cancer cell lines that express COX-2 (HCA-7 and Moser-S) and a COX-2 negative cell line (HCT-116) were evaluated. The growth rate of these cells was measured following treatment with meloxicam. HCA-7 and Moser-S colony size

Angela P. Goldman; Christopher S. Williams; Hongmiao Sheng; Laura W. Lamps; Vanessa P. Williams; Michel Pairet; Jason D. Morrow; Raymond N. DuBois


Targeted disruption of the K-Ras oncogene in an invasive colon cancer cell line down-regulates urokinase receptor expression and plasminogen-dependent proteolysis  

Microsoft Academic Search

The urokinase receptor, overexpressed in invasive colon cancer, promotes tumour cell invasion. Since K-Ras is activated in many colon cancers, we determined if urokinase receptor overexpression is a consequence of this activated oncogene. Accordingly, urokinase receptor expression was compared in HCT 116 colon cancer cells containing either a mutation-activated K-Ras or disrupted for this oncogene (by homologous recombination). HCT 116

H Allgayer; H Wang; S Shirasawa; T Sasazuki; D Boyd



Differential Expression of Sialic Acid and N-acetylgalactosamine Residues on the Cell Surface of Intestinal Epithelial Cells According to Normal or Metastatic Potential  

Microsoft Academic Search

In this study we investigated the levels of expression of sialic acid and N-acetylgalactosamine residues on the cell surface of a normal intestinal epithelium cell line, IEC-6, and in two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116. Glycoprotein expression was estimated initially by cytochemistry with WGA and HPA lectins and biochemistry with isolated plasma membrane fractions

Patricia de Albuquerque Garcia Redondo; Celso Vataru Nakamura; Wanderley de Souza; José Andrés Morgado-Díaz



Ganoderic acid Me induces G 1 arrest in wild-type p53 human tumor cells while G 1\\/S transition arrest in p53-null cells  

Microsoft Academic Search

The mechanism of cell cycle arrest of tumor cells induced by ganoderic acid Me (GA-Me) is not understood. In this work, GA-Me was found to possess remarkable cytotoxicity on highly metastatic lung carcinoma 95-D cell line in both dose- and time-dependent manners. The effect of GA-Me on cell cycle arrest was found in 95-D, p53-null lung cancer cells H1299, HCT-116

Nian-Hong Chen; Jian-Jiang Zhong



DNA damage signaling in response to 5-fluorouracil in three colorectal cancer cell lines with different mismatch repair and TP53 status.  


We studied patterns of DNA damage signaling and cell cycle response to clinically-relevant (bolus) and high doses of 5-fluorouracil (5-FU) in three colorectal cancer cell lines with differing MMR and TP53 status in an attempt to better understand how 5-FU exerts its cytotoxicity. The ATM/CHEK2/ CHEK1 signaling pathway was not activated in response to bolus 5-FU in the MMR-deficient cell lines HCT116 (TP53-proficient or TP53-depleted) and HCT15 (TP53-deficient), consistent with negligible/reparable DNA damage and no cell death. The pattern of DNA damage checkpoint activation in bolus 5-FU-treated HT29 (TP53-deficient/MMR-proficient) cultures suggested SSB formation (CHEK1 activation) followed by DSB formation (CHEK2 activation and increased phospho-H2AX levels), but no cell death suggested that DNA repair capacity was not overwhelmed. High-dose 5-FU treatment led to activation of ATM/CHEK2/TP53 (not CHEK1) in TP53-proficient and TP53-depleted HCT116 (later CHEK2 activation relative to TP53-proficient) cultures; HCT15 cultures had ATM activation only. These data and increased phospho-H2AX levels indicated DSB formation; apoptosis was induced in both cell lines indicating irreparable DNA damage. TP53-depleted HCT116 cultures also had DSBs after high-dose 5-FU treatment but experienced a (transient) G1/S cell cycle arrest that protected them from apoptosis. TP53 phosphorylation at Ser20/33/37 was seen in TP53-proficient HCT116 cultures regardless of 5-FU concentration at ?4 h following treatment, indicating TP53 stabilization/transcriptional activation. Overall, activation of ATM, CHEK1 and/or CHEK2 and phospho-H2AX levels reflected the nature of 5-FU-induced DNA damage and indi-cated when DNA damage was significant (5-FU-dose-dependent). DNA repair and cell cycle responses to 5-FU-induced DNA damage were distinctly affected by MMR and TP53 (role in BER/NER) functionalities, but MMR deficiency especially seemed to confer less overall sensitivity to 5-FU. PMID:21674128

Adamsen, Birgitte L; Kravik, Katherine L; De Angelis, Paula M



Mutant K-ras Regulates Cathepsin B Localization on the Surface of Human Colorectal Carcinoma Cells1  

Microsoft Academic Search

Cathepsin B protein and activity are known to localize to the basal plasma membrane of colon carcinoma cells following the appearance of K-ras mutations. Using immunofluorescence and subcellular fractiona- tion techniques and two human colon carcinoma cell lines—one with a mutated K-ras allele (HCT 116) and a daughter line in which the mutated allele has been disrupted (HKh-2)—we demonstrate that

Dora Cavallo-Medved; Julie Dosescu; Bruce E. Linebaugh; Mansoureh Sameni; Debbie Rudy; Bonnie F. Sloane


LINE-1 and Alu retrotransposition exhibit clonal variation  

PubMed Central

Background The non-long terminal repeat (non-LTR) retrotransposons, long interspersed element-1 (LINE-1) and Alu are currently active retroelements in humans. We, and others, have observed that different populations of HeLa cells from different laboratories support retrotransposition of LINE-1 and Alu to varying degrees. We therefore tested whether individual cell clones of HeLa and HCT116 cell lines supported different levels of LINE-1 and Alu retrotransposition, and whether these variations were stable upon re-cloning. Findings Standard retrotransposition tissue culture assays were used to measure a cell’s ability to support LINE-1 and Alu retrotransposition in clonal HeLa and HCT116 cell lines. We observed that both LINE-1 and Alu retrotransposition exhibited clonal variation in HeLa cells, with certain HeLa cell clones supporting high levels of LINE-1 and Alu retrotransposition and other cell clones being essentially retrotransposition-dead. This clonal variation was similarly observed in HCT116 cells, although possibly not to the same extent. These patterns of clonal variation are relatively consistent upon re-cloning. Conclusions Observations of the variability of LINE-1 and Alu retrotransposition in different populations of the same cell line are supported by our results that indicate in some cell types, individual cell clones can have dramatically differing capacity for retrotransposition. The mixed populations of cells commonly used in laboratories have often been passaged for many generations and accumulated significant genetic and epigenetic diversity. Our results suggest that the clonal variability observed by our cloning experiments may lead to a homogenization of retrotransposition capacity, with the resulting mixed population of cells being composed of individual variants having either increased or decreased retrotransposition potential compared to the starting population.



E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation  

Microsoft Academic Search

BACKGROUND: PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. METHODS: PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines

Hongying Hao; Yanbin Dong; Maria T Bowling; Jorge G Gomez-Gutierrez; H Sam Zhou; Kelly M McMasters



Tart cherry anthocyanins inhibit tumor development in Apc(Min) mice and reduce proliferation of human colon cancer cells.  


Anthocyanins, which are bioactive phytochemicals, are widely distributed in plants and especially enriched in tart cherries. Based on previous observations that tart cherry anthocyanins and their respective aglycone, cyanidin, can inhibit cyclooxygenase enzymes, we conducted experiments to test the potential of anthocyanins to inhibit intestinal tumor development in Apc(Min) mice and growth of human colon cancer cell lines. Mice consuming the cherry diet, anthocyanins, or cyanidin had significantly fewer and smaller cecal adenomas than mice consuming the control diet or sulindac. Colonic tumor numbers and volume were not significantly influenced by treatment. Anthocyanins and cyanidin also reduced cell growth of human colon cancer cell lines HT 29 and HCT 116. The IC(50) of anthocyanins and cyanidin was 780 and 63 microM for HT 29 cells, respectively and 285 and 85 microM for HCT 116 cells, respectively. These results suggest that tart cherry anthocyanins and cyanidin may reduce the risk of colon cancer. PMID:12706854

Kang, Soo-Young; Seeram, Navindra P; Nair, Muraleedharan G; Bourquin, Leslie D



Impact of Oncogenes in Tumor Angiogenesis: Mutant K-ras up-Regulation of Vascular Endothelial Growth Factor\\/Vascular Permeability Factor is Necessary, but not Sufficient for Tumorigenicity of Human Colorectal Carcinoma Cells  

Microsoft Academic Search

Targeted disruption of the single mutant K-ras allele in two human colorectal carcinoma cell lines (DLD-1 and HCT-116) leads to loss of tumorigenic competence in nude mice with retention of ability to grow indefinitely in monolayer culture. Because expression of the mutant K-ras oncogene in these cell lines is associated with marked up-regulation of vascular endothelial growth factor\\/vascular permeability factor

Futoshi Okada; Janusz W. Rak; Brad St. Croix; Blandine Lieubeau; Mitsunori Kaya; Luba Roncari; Senji Shirasawa; Takehiko Sasazuki; Robert S. Kerbel



N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA  

Microsoft Academic Search

BACKGROUND: Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is able to induce senescence in the HCT-116 colon cancer cell line. RESULTS: Cells treated with

Aruna S Jaiswal; Asha S Multani; Sen Pathak; Satya Narayan



Therapeutic Targeting of Neuropilin-2 on Colorectal Carcinoma Cells Implanted in the Murine Liver  

Microsoft Academic Search

Methods Immunohistochemistry and immunoblotting were used to assess NRP2 expression levels in colorectal tumors and colorectal cancer cell lines, respectively. HCT-116 colorectal cancer cells stably transfected with short hairpin RNA (shRNAs) against NRP2 or control shRNAs were assayed for proliferation by the tetrazo- lium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for activation of the VEGFR1 pathway by immuno blotting. Soft

Michael J. Gray; George Van Buren; Nikolaos A. Dallas; Ling Xia; Xuemei Wang; Anthony D. Yang; Ray J. Somcio; Yvonne G. Lin; Sherry Lim; Fan Fan; Lingegowda S. Mangala; Thiruvengadam Arumugam; Craig D. Logsdon; Gabriel Lopez-Berestein; Anil K. Sood; Lee M. Ellis



Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases  

Microsoft Academic Search

Hypermethylation associated silencing of the CpG islands of tumor suppressor genes is a common hallmark of human cancer. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using two molecular screenings for differentially methylated loci (differential methylation

Maria F. Paz; Susan Wei; Juan C. Cigudosa; Sandra Rodriguez-Perales; Miguel A. Peinado; Tim Hui-Ming Huang; Manel Esteller



Tumor cell proliferation and cyclooxygenase enzyme inhibitory compounds in Amaranthus tricolor.  


Amaranthus tricolor is consumed as a vegetable in Asia. Bioassay-directed isolation of leaves and stems of A. tricolor yielded three galactosyl diacylglycerols (1-3) with potent cyclooxygenase and human tumor cell growth inhibitory activities. The purified compounds were characterized by spectroscopic methods. In addition, the fatty acid moieties in diacyl galactosyl glyerols were characterized by GC-MS analyses. The galactosyl diacylglycerols 1-3 inhibited the cyclooxygenase-1 (COX-1) enzyme by 78, 63, and 93% and the cyclooxygenase-2 (COX-2) enzyme by 87, 74, and 95%, respectively. These compounds were tested for antiproliferative activity using human AGS (gastric), CNS (central nervous system; SF-268), HCT-116 (colon), NCI-H460 (lung), and MCF-7 (breast) cancer cell lines. Compound 1 inhibited the growth of AGS, SF-268, HCT-116, NCI-H460, and MCF-7 tumor cell lines with IC50 values of 49.1, 71.8, 42.8, 62.5, and 39.2 mug/mL, respectively. For AGS, HCT-116, and MCF-7 tumor cell lines, the IC50 values of compounds 2 and 3 were 74.3, 71.3, and 58.7 microg/mL and 83.4, 73.1, and 85.4, respectively. This is the first report of the COX enzyme inhibitory activity for galactosyl glycerols and antiproliferative activities against human colon, breast, lung, stomach, and CNS tumor cell lines. PMID:15537300

Jayaprakasam, Bolleddula; Zhang, Yanjun; Nair, Muraleedharan G



Celecoxib inhibits the expression of survivin via the suppression of promoter activity in human colon cancer cells  

Microsoft Academic Search

We investigated the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on human colon cancer cell lines to clarify the mechanisms underlying the chemopreventive effect of NSAIDs. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, induced apoptosis and strongly reduced the expression of an anti-apoptotic protein, survivin, in both protein and mRNA levels in HCT-116 cells. Subsequently, we conducted luciferase reporter assay using a

Naoko Sakoguchi-Okada; Fumi Takahashi-Yanaga; Kazuhiro Fukada; Fumie Shiraishi; Yoji Taba; Yoshikazu Miwa; Sachio Morimoto; Mitsuo Iida; Toshiyuki Sasaguri



Cox2 is needed but not sufficient for apoptosis induced by Cox2 selective inhibitors in colon cancer cells  

Microsoft Academic Search

The role of Cox-2 in NSAID-induced apoptosis is debated. We studied the role of Cox-2 inhibition in apoptosis induced by a selective Cox-2 inhibitor, SC236 (a structural analogue of celecoxib) in two colon cancer cell lines, HT29 (expressing Cox-2 protein) and HCT116 (not expressing Cox-2 protein). Apoptosis was quantified by flow cytometry. SC236 0–75 µM decreased cell numbers and induced

B. Agarwal; P. Swaroop; P. Protiva; S. V. Raj; H. Shirin; P. R. Holt



Synthesis of novel ciprofloxacin analogues and evaluation of their anti-proliferative effect on human cancer cell lines.  


A series of twenty two novel 1-cyclopropyl-6-fluoro-4-oxo-7-(4-substituted piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid analogues have been synthesized, characterized ((1)H NMR, (13)C NMR and LCMS) and evaluated for their inhibitory activity on the proliferation of human caucasian acute lymphoblastic leukemia cells (CCRF-CEM), breast adenocarcinoma cells (MDA-MB-468) and human colon carcinoma cells (HCT-116). Among all the synthesized ciprofloxacin analogues 3t at 50?M showed comparable potency to doxorubicin (10?M) in all three cell lines and 3j inhibited proliferation of MDA-MB-468 up to 35% selectively over other two cell lines. PMID:24138941

Suresh, Narva; Nagesh, Hunsur Nagendra; Chandra Sekhar, Kondapalli Venkata Gowri; Kumar, Anil; Shirazi, Amir N; Parang, Keykavous



Lipid alterations in human colon epithelial cells induced to differentiation and\\/or apoptosis by butyrate and polyunsaturated fatty acids  

Microsoft Academic Search

The present study highlights the important association between lipid alterations and differentiation\\/apoptotic responses in human colon differentiating (FHC) and nondifferentiating (HCT-116) cell lines after their treatment with short-chain fatty acid sodium butyrate (NaBt), polyunsaturated fatty acids (PUFAs), and\\/or their combination. Our data from GC\\/MS and LC\\/MS\\/MS showed an effective incorporation and metabolization of the supplemented arachidonic acid (AA) or docosahexaenoic

Ji?ina Hofmanová; Miroslav Ciganek; Josef Slavík; Alois Kozubík; Lenka Stixová; Alena Vaculová; Ladislav Dušek; Miroslav Machala


Resveratrol inhibits proliferation, angiogenesis and induces apoptosis in colon cancer cells: Calorie restriction is the force to the cytotoxicity.  


The aim of this study was to examine the antitumour activity of resveratrol in human colorectal cancer cell lines (HCT116 and Caco2) and to explore its mechanism of action assuming that it is by calorie-restriction effect. Resveratrol inhibited the proliferation of colon cancer cells with half maximal inhibitory concentration (IC50) equal to 50 and 130 ?M for HCT116 and Caco2, respectively. Caco2 cells appeared with significant time-dependent increase in the glycolytic pathway, a behaviour that was absent in HCT116 cells. Resveratrol (100 ?M) significantly decreased the glycolytic enzymes (pyruvate kinase and lactate dehydrogenase) in Caco2 cells, while an increase in citrate synthase activity and a decrease in glucose consumption were observed in both cell lines. Moreover, resveratrol downregulated the expressions of leptin and c-Myc, and decreased the content of vascular endothelial growth factor. The apoptotic markers, caspases 3 and 8, were activated and the Bax/BCl2 ratio was increased. The study suggested a promising anticancer activity of resveratrol, calorie-restriction pathway may be one of the driving forces for this activity. PMID:23536519

Fouad, Ma; Agha, Am; Merzabani, Mm Al; Shouman, Sa



Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells  

PubMed Central

Accumulating evidence suggests a role for microRNAs in human carcinogenesis as novel types of tumor suppressors or oncogenes. However, their precise biological role remains largely elusive. In the present study, we aimed to identify microRNA species involved in the regulation of cell proliferation. Using quantitative RT-PCR analysis, we demonstrated that miR-34a was highly up-regulated in a human colon cancer cell line, HCT 116, treated with a DNA-damaging agent, adriamycin. Transient introduction of miR-34a into two human colon cancer cell lines, HCT 116 and RKO, caused complete suppression of cell proliferation and induced senescence-like phenotypes. Moreover, miR-34a also suppressed in vivo growth of HCT 116 and RKO cells in tumors in mice when complexed and administered with atelocollagen for drug delivery. Gene-expression microarray and immunoblot analyses revealed down-regulation of the E2F pathway by miR-34a introduction. Up-regulation of the p53 pathway was also observed. Furthermore, 9 of 25 human colon cancers (36%) showed decreased expression of miR-34a compared with counterpart normal tissues. Our results provide evidence that miR-34a functions as a potent suppressor of cell proliferation through modulation of the E2F signaling pathway. Abrogation of miR-34a function could contribute to aberrant cell proliferation, leading to colon cancer development.

Tazawa, Hiroshi; Tsuchiya, Naoto; Izumiya, Masashi; Nakagama, Hitoshi



The -1195G allele increases the transcriptional activity of cyclooxygenase-2 gene (COX-2) in colon cancer cell lines.  


Up-regulation of cyclooxygenase-2 (COX-2) is an early and key event in human colorectal carcinogenesis (CRC). Nevertheless, the molecular mechanisms leading to this over-expression are largely unknown. We previously reported an association between the -1195G allele and higher predisposition for CRC in a Caucasian population. The biological explanation for the involvement of this polymorphism in CRC remains elusive. We aimed to functionally characterize the influence of the -1195A>G promoter region polymorphism on COX-2 transcription activity in colon cancer cell lines. Luciferase reporter assays were performed to assess whether the -1195A/G alleles influenced COX-2 transcription. The COX-2 promoter's region containing either the -1195A or -1195G alleles was cloned into pGL3-basic reporter vector. The reporter vectors were transiently co-transfected with the pGL4.73 control plasmid to HCT-116 and HCA-7 colon cancer cell lines. The levels of reporter gene expression driven by the -1195G allele-containing COX-2 promoter were significantly higher in both colon cancer cell lines. A 2.2-fold increase in promoter activity was observed in the HCT-116 cell line (P?cell line with a threefold higher transcriptional activity (P?=?0.001). The -1195G allele appeared to enhance COX-2 transcription, providing a molecular basis underlying the increased susceptibility for CRC and potentially a new mechanism for COX-2 overexpression. © 2013 Wiley Periodicals, Inc. PMID:23776069

Pereira, Carina; Sousa, Hugo; Silva, Joana; Brandão, Carla; Elgueta-Karstegl, Claudio; Farrell, Paul J; Medeiros, Rui; Dinis-Ribeiro, Mário



DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines  

PubMed Central

Background Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes. Methods HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity® Pathway Analysis. Results Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed. Conclusion This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.



150 kDa glycoprotein isolated from Solanum nigrum Linne stimulates caspase-3 activation and reduces inducible nitric oxide production in HCT116 cells  

Microsoft Academic Search

This study was carried out to investigate the apoptotic effects of glycoprotein (SNL glycoprotein, 150-kDa) isolated from Solanum nigrum Linne, which has been used as an antipyretic and anticancer agent in folk medicine. We found that SNL glycoprotein consists of carbohydrate content (69.74%) and protein content (30.26%), which contains more than 50% hydrophobic amino acids such as glycine and proline.

Sei-Jung Lee; Kye-Taek Lim



Cancer stem cell sorting from colorectal cancer cell lines by sedimentation field flow fractionation.  


Recently, cancer stem cells (CSCs) have been identified in many types of cancers, such as colorectal cancer (CRC). CSCs seem to be involved in initiation, growth, and tumor metastasis, as well as in radio- and chemotherapy failures. CSCs appears as new biological targets for cancer therapy, requiring the development of noninvasive cell sorting methods. In this study, we used sedimentation field flow fractionation (SdFFF) to prepare enriched populations of CSCs from eight cell lines corresponding to different CRC grades. On the basis of phenotypic and functional characterizations, "hyperlayer" elution resulted in a fraction overexpressing CSC markers (CD44, CD166, EpCAM) for all cell lines. CSCs were eluted in the last fraction for seven out of eight cell lines, but in the first for HCT116. These results suggest, according to the literature, that two different pools of CSCs exist, quiescent and activated, which can both be sorted by SdFFF. Moreover, according to CSC properties, enriched fractions are able to form colonies. PMID:22236375

Mélin, Carole; Perraud, Aurélie; Akil, Hussein; Jauberteau, Marie-Odile; Cardot, Philippe; Mathonnet, Muriel; Battu, Serge



Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines.  


In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer. PMID:19927377

Knutsen, Turid; Padilla-Nash, Hesed M; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J; Ried, Thomas



Multiple antitumor effects of picropodophyllin in colon carcinoma cell lines: Clinical implications  

PubMed Central

Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1R-mediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 ?M PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease.




Multiple antitumor effects of picropodophyllin in colon carcinoma cell lines: clinical implications.  


Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1R-mediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 µM PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease. PMID:22159423

Feng, Xiaoying; Aleem, Eiman; Lin, Yingbo; Axelson, Magnus; Larsson, Olle; Strömberg, Thomas



Desipramine Induces Apoptotic Cell Death through Nonmitochondrial and Mitochondrial Pathways in Different Types of Human Colon Carcinoma Cells  

Microsoft Academic Search

Cytotoxic effects of desipramine on human colon carcinoma HT29 and HCT116 cells were examined. Desipramine reduced the viability of HT29 cells in a concentration-dependent manner, but failed to cause any significant change in the viability of HCT116 cells by the concentration up to 50 ?mol\\/l, at which an approximately 60% reduction of the viability of HT29 cells was observed. Despite

Hideki Arimochi; Kyoji Morita



5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint  

PubMed Central

Background & Aims Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53?/?, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis.




Stable Karyotypes in Epithelial Cancer Cell Lines Despite High Rates of Ongoing Structural and Numerical Chromosomal Instability  

PubMed Central

Abstract Most human tumors and tumor cell lines exhibit numerical and structural chromosomal abnormalities. The goal of this study was to determine the ongoing rates of structural and numerical instability in selected cancer cell lines and to investigate the consequences of these rates to karyotypic progression. We studied two colorectal (HCT-116 and HT-29) and two ovarian (SKOV-3 and OVCAR-8) cancer cell lines and their single cell subclones. We found that the signature karyotypes of all four cell lines were distinct and each aberrant. Whereas high rates of ongoing structural and/or numerical chromosomal instability could be demonstrated in all cell lines, there was a relative stability of the consensus karyotype over many generations. No new clonal structural chromosomal reconfigurations emerged and the few numerical changes of karyotypes were restricted to abnormal chromosomes. This implies a kind of genomic optimization under the conditions of cell culture and suggests a link between genomic stabilization and cell propagation. We have been able to support this possibility by computer modeling. We did not observe a profound difference in the rates of numerical or structural instability in the cell lines with a replication error phenotype (RER+) versus the other cell lines.

Roschke, Anna V; Stover, Kristen; Tonon, Giovanni; Schaffer, Alejandro A; Kirsch, Ilan R



Mechanism of Transport and Intracellular Binding of Porfiromycin in MCI 116 Human Colon Carcinoma Cells1  

Microsoft Academic Search

The mechanism of uptake and efflux of porfiromycin (PFM) by IK T 116 human colon carcinoma cells or freshly obtained human RBC was investigated. The time course of uptake of radioactivity upon exposure of HCT 116 cells to |\\

Su-shu Pan; Robin Johnson; Hector Gonzalez; Vinay Thohan


Dissimilar cytokine patterns in different human liver and colon cancer cell lines.  


An accurate and simultaneous estimate of cellular levels of a large cytokine number is very useful to obtain information about an organ dysfunction leading to cancer because through the understanding of the evolution of cytokine patterns we can recognize and predict the disease progression. Cancer cell lines are commonly used to study the cancer microenvironment, to analyze their chemosensitivity and carcinogenesis as well as to test in vitro the effect of molecules, such as drugs or anti-oxidants, on the inflammation status and its progression. We noted that various cell lines commonly used as a model for studies on liver and colon cancer possess different patterns of cytokines. This aspect may generate data not comparable in laboratories using different cell lines; thus, to investigate the origin of these abnormalities we compared the cell lines HepG2 and Huh7, and HT-29 and HCT-116, for liver and colon cancer, respectively. In this context we have evaluated and compared the levels of cytokines, chemokines and growth factors in the supernatants of these cellular lines. Our aim was to identify what cytokines were significantly different correlating similarities and differences to the specific inflammation status of each cellular model of cancer. PMID:24064000

Guerriero, Eliana; Capone, Francesca; Rusolo, Fabiola; Colonna, Giovanni; Castello, Giuseppe; Costantini, Susan



Characterization of a human colorectal carcinoma cell line with acquired resistance to flavopiridol.  


Flavopiridol is a broad-spectrum inhibitor of cyclin-dependent kinases (cdks) and represents the first in this anticancer class to enter clinical trials. In anticipation of the likelihood that, as with other cancer drugs, acquired resistance may limit the drug's efficacy, an acquired resistance model has been established by in vitro drug exposure of the human colon carcinoma cell line HCT116. This stably resistant line, possessing 8-fold resistance to flavopiridol, showed a lack of cross-resistance to the anticancer agents etoposide, doxorubicin, paclitaxel, topotecan, and cisplatin, and notably to other chemical classes of cdk inhibitors: the aminopurines roscovitine and purvalanol A, 9-nitropaullone, and hymenialdisine. Resistance did not seem to be related to differences in the levels of multidrug resistance drug efflux proteins, P-glycoprotein, and MRP1. Moreover, there were no changes in overall drug accumulation between the resistant and sensitive cell lines. Flavopiridol induced cell cycle arrest, apoptosis, and inhibition of retinoblastoma gene product phosphorylation on serine 780 in both parental and resistant lines, but the latter required 8-fold higher concentrations to achieve these effects. Cyclin E protein levels and cyclin E-associated kinase activity were increased in the resistant line, suggesting that overexpression of cyclin E may be the mechanism of resistance to flavopiridol. However, transfection of cyclin E to increase expression of this protein did not result in an increase in resistance to flavopiridol. Thus, up-regulation of cyclin E alone does not seem to cause resistance to this cdk inhibitor. PMID:11641415

Smith, V; Raynaud, F; Workman, P; Kelland, L R



Curcumin causes superoxide anion production and p53-independent apoptosis in human colon cancer cells  

Microsoft Academic Search

Curcumin from the rhizome of theCurcuma longa plant has chemopreventative activity and inhibits the growth of neoplastic cells. Since p53 has been suggested to be important for anticancer activity by curcumin, we investigated curcumin-induced cytotoxicity in cultures of p53+\\/+ and p53?\\/? HCT-116 colon cancer cells, as well as mutant p53 HT-29 colon cancer cells. Curcumin killed wild-type p53 HCT-116 cells

Jane L. Watson; Richard Hill; Paul B. Yaffe; Anna Greenshields; Mark Walsh; Patrick W. Lee; Carman A. Giacomantonio; David W. Hoskin



Crocin from Crocus Sativus Possesses Significant Anti-Proliferation Effects on Human Colorectal Cancer Cells  

PubMed Central

Aim To investigate the anti-proliferative effects of Crocus sativus extract and its major constituent, crocin, on three colorectal cancer cell lines (HCT-116, SW-480, and HT-29). The cell growth inhibition effect was compared to that of non-small cell lung cancer (NSCLC) cells. In addition, Crocus sativus' effect on non-cancer cells was evaluated. Methods Using high performance liquid chromatography (HPLC), the purity of crocin and the content of crocin extract were determined. Anti-proliferative effects of Crocus sativus extract and crocin on test cells was evaluated by MTS assay. Results The purity of crocin was found to be 95.9% and the content of crocin in the extract was 22.9%. Significant concentration-related inhibition effects of the extract on all three colorectal cancer cell lines were observed (P < 0.01). The proliferation was reduced most significantly in HCT-116 cells, to 45.5% at 1.0 mg/ml and to 6.8 % at 3.0 mg/ml. Crocin at 1.0 mM, significantly reduced HCT-116, SW-480, and HT-29 cell proliferation to 2.8%, 52%, and 16.8%, respectively (P < 0.01). Since 3.0 mg/ml Crocus sativus extract contained approximately 0.6 mM crocin, the observed effects suggest that crocin is a major responsible constituent in the extract. Significant anti-proliferative effects were also observed in non-small cell lung cancer cells. However, Crocus sativus extract did not significantly affect the growth of non-cancer young adult mouse colon cells. Conclusion Data from this study demonstrated that Crocus sativus extract and its major constituent, crocin, significantly inhibited the growth of colorectal cancer cells while not affecting normal cells. Crocus sativus extract should be investigated further as a viable option in the treatment of colorectal cancer.

Aung, H.H.; Wang, C.Z.; Ni, M.; Fishbein, A.; Mehendale, S.R.; Xie, J.T.; Shoyama, A.Y.; Yuan, C.S.



Cells lacking CIP1\\/WAF1 genes exhibit preferential sensitivity to cisplatin and nitrogen mustard  

Microsoft Academic Search

We have previously shown that p53 disruption sensitizes certain cancer cell types to cisplatin (CDDP) (Fan et al., 1995). In the present study we investigated the role of the p53 downstream effector, p21CIP1\\/WAF1 (p21), in this sensitization. Studies were performed in human colon cancer HCT-116 cells and murine embryonic fibroblasts (MEF) with intact versus disrupted p21 genes. For comparison, HCT-116

Saijun Fan; Johnny K Chang; Martin L Smith; Diane Duba; Albert J Fornace Jr; Patrick M O'Connor



Fucoidan present in brown algae induces apoptosis of human colon cancer cells  

Microsoft Academic Search

BACKGROUND: Fucoidan is a sulfated polysaccharide found in brown algae; it has been shown to exhibit a number of biological effects, including anti-tumor effects. In this study, we evaluated the effects of fucoidan on apoptosis in HT-29 and HCT116 human colon cancer cells. METHODS: HT-29 and HCT116 cells were cultured with various concentrations of fucoidan (0 - 20 ?g\\/mL). Apoptosis

Eun Ji Kim; So Young Park; Jae-Yong Lee; Jung Han Yoon Park



In vitro anti-proliferative effects of Zuojinwan on eight kinds of human cancer cell lines.  


Zuojinwan (ZJW), a famous Chinese medicinal formula, contains two medicinal herbs Coptis chinese Frach and Evodia rutaecarpa (Juss.) Benth in the ratio of 6: 1. The inhibitory effects of ZJW on eight kinds of human cancer cell lines including SMMC-7721, BEL-7402, BEL-7404, HepG2, A549, NCI-H446, NCI-H460 and HCT- 116 cells were evaluated, and the possible mechanism was investigated. The growths of the eight kinds of cancer cells were inhibited by ZJW assessed through MTT assay. Flow cytometry assay revealed a sub-G1 peak with reduced DNA content was formed. The cell cycle was arrested in the G0/G1 phase in ZJW-treated SMMC-7721 and HepG2 cells, and in the S phase for NCI-H460 cells. Significant DNA damage was produced by ZJW assessed with single-cell gel electrophoresis assay. Morphological changes were also observed. Caspase-3 and -9 activities were increased following ZJW treatment. Western blot analysis showed that Bax and Bak protein levels were increased after ZJW treatment, while Bcl-2 and Bcl-xl protein levels were decreased. Our results suggest that ZJW has significant anti-cancer activities due to induction of mitochondria- dependent apoptosis pathway. Therefore, ZJW has the potential to be a novel chemotherapy drug to treat hepatoma, lung cancer and colon cancer by suppressing tumor growth. PMID:23397442

Xu, Lina; Qi, Yan; Lv, Linlin; Xu, Youwei; Zheng, Lingli; Yin, Lianhong; Liu, Kexin; Han, Xu; Zhao, Yanyan; Peng, Jinyong



Changes in adsorption and permeability of mitoxantrone on plasma membrane of BCRP\\/MXR resistant cells  

Microsoft Academic Search

A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP\\/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption

G. Breuzard; O. Piot; J.-F. Angiboust; M. Manfait; L. Candeil; M. Del Rio; J.-M. Millot



BAI, a novel cyclin-dependent kinase inhibitor induces apoptosis in A549 cells through activation of caspases and inactivation of Akt.  


Previously, we have synthesized a novel cyclin-dependent kinase (CDK) inhibitor, 2-[1,1'biphenyl]-4-yl-N-[5-(1,1-dioxo-1?(6) -isothiazolidin-2-yl)-1H-indazol-3-yl]acetamide (BAI) and reported its anti-cancer activity in head and neck cancer cells. In this study, we further evaluated the effect of BAI on growth of various human cancer cell lines, including A549 (nonsmall cell lung cancer), HCT116 (colon), and Caki (kidney). Profoundly, results of XTT and clonogenic assays demonstrated that BAI at nanomolar concentrations (20-60 nM) inhibited growth of A549, HCT116, and Caki cells, suggesting the anti-cancer potency. We show that BAI induced a dose-dependent apoptotic cell death in these human cancer cells, as measured by fluorescence-activated cell sorting (FACS). Interestingly, further biochemical analysis showed that treatment with BAI at 20?nM induced apoptosis in A549 cells in association with activation of caspases, cleavage of phospholipase C-?1 (PLC-?1), and inhibition of Akt in A549 cells. Importantly, pharmacological inhibition study revealed that pretreatment with z-VAD-fmk, a pan caspase inhibitor strongly blocked the BAI-induced apoptosis in A549 cells. Transfection analysis with Akt cDNA encoding constitutively active Akt further addressed the significance of Akt inhibition in the BAI-induced apoptosis in A549 cells. Notably, disruption of the PI3K/Akt pathway by LY294002, a PI3K/Akt inhibitor potentiated apoptosis in A549 cells by BAI at a subcytotoxic concentration. These findings collectively suggest that BAI potently inhibits growth of A549, HCT116, and Caki cells, and that the BAI-induced apoptosis in A549 cells is associated with activation of caspases, and inhibition of Akt. PMID:22887215

Kim, Shin; Lee, Jinho; Jang, Byeong-Churl; Kwon, Taeg Kyu; Park, Jong-Wook



Induction of apoptosis against cancer cell lines by four ascomycetes (endophytes) from Malaysian rainforest.  


Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with IC(50) values less than 17 ?g/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis. PMID:22397996

Hazalin, Nurul Aqmar Mohamad Nor; Ramasamy, Kalavathy; Lim, Siong Meng; Cole, Anthony L J; Majeed, Abu Bakar Abdul



Critical Role of p53 Upregulated Modulator of Apoptosis in Benzyl Isothiocyanate-Induced Apoptotic Cell Death  

PubMed Central

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.

Antony, Marie Lue; Kim, Su-Hyeong; Singh, Shivendra V.



MicroRNA-218 Inhibits Cell Cycle Progression and Promotes Apoptosis in Colon Cancer by Downregulating BMI1 Polycomb Ring Finger Oncogene  

PubMed Central

Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3? untranslated region (3?UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3?UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.

He, Xinqi; Dong, Yujuan; Wu, Chung Wah; Zhao, Zengren; Ng, Simon S M; Chan, Francis K L; Sung, Joseph J Y; Yu, Jun



Enhanced clearance of topoisomerase I inhibitors from human colon cancer cells by glucuronidation.  


As part of a program to identify novel mechanisms of resistance to topoisomerase I (topo I) inhibitors, the cellular pharmacology of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of clinically used irinotecan (CPT-11) and NU/ICRF 505, an anthraquinone-tyrosine conjugate, has been investigated in two human colorectal cancer (CRC) cell lines. Two novel metabolites of NU/ICRF 505 (M1 and M2) and a single metabolite of SN-38 (M1) were detected by high performance liquid chromatography in the culture medium of HT29 cells but were absent in HCT116 cells. Identities of all three metabolites were established by a combination of biochemical and physicochemical techniques. M1 of SN-38 was the C10-(beta)-glucuronide of the parent lactone while M1 of NU/ICRF 505 was the C4-O-glucuronide and M2 the tyrosine-O-glucuronide, both of the parent compound. Drug transport studies revealed that by 24hr HT29 cells had effectively cleared 82.5% of NU/ICRF 505 (10 microM) into the culture medium as the two glucuronides. In contrast, intracellular concentrations of NU/ICRF 505 were maintained in HCT116 cells in the absence of glucuronidation at a level 550 times greater than in HT29 cells. HT29 cells cleared 40.9% of SN-38 (1 microM) as the glucuronide to the culture medium, while the parent drug was maintained at a level 2-fold greater in HCT116 cells. Enhanced drug clearance due to glucuronidation may contribute to intrinsic drug resistance of human CRC. PMID:11992628

Cummings, Jeffrey; Boyd, Gary; Ethell, Brian T; Macpherson, Janet S; Burchell, Brian; Smyth, John F; Jodrell, Duncan I



The cytotoxicity of methyl protoneodioscin (NSC-698791) against human cancer cell lines in vitro.  


Methyl protoneodioscin (NSC-698791) is a furostanol saponin isolated from the rhizome of Dioscorea collettii var. hypoglauca (Dioscoreaceae), a Chinese herbal remedy for the treatment of cervical carcinoma, carcinoma of the urinary bladder and renal tumor for centuries. In order to systematically evaluate its potential anticancer activity, methyl protoneodioscin cytotoxicity was tested in vitro against 60 human cancer cell lines in the NCI's (National Cancer Institute, USA) anticancer drug screen. As a result, methyl protoneodioscin was cytotoxic against all the test cell lines from leukemia and solid tumors in the NCI's human cancer panel, especially selectively against one non-small cell lung cancer (NSCLC) line (A549/ATCC), one colon cancer line (HCT-116), two central nenous system (CNS) cancer lines (SF-539 and SNB-75), one melanoma line (M14), one renal cancer line (CAKI-1), one prostate cancer (DU-145) and two breast cancer lines (HS 578T and MDA-MB-435) with GI50 < or = 2.0 microM. The selectivity between these nine most sensitive lines and the least sensitive line (TK-10) was from 22- to 30- fold. In the same cancer subpanel, a selectivity at GI50 level of more than 15-fold was observed between A549/ATCC and EKVX (NSCLC), between CAKI-1 and TK-10, A498 (renal cancer), respectively. In general the CNS cancer was the most sensitive subpanel, while renal cancer was the least sensitive subpanel. Based on an analysis of COMPARE computer program with methyl protoneodioscin as a seed compound, no compounds in the NCIs anticancer drug screen database have similar cytotoxicity patterns (mean graphs) to that of methyl protoneodioscin, indicating a potential novel mechanism of anticancer action involved. PMID:12014616

Hu, K; Yao, X S


Chromenylchalcones showing cytotoxicity on human colon cancer cell lines and in silico docking with aurora kinases.  


Due to toxicity problems, various plant-derived compounds have been screened to find the chemotherapeutic agents. As anticancer therapeutic agents, chalcones have advantages such as poor interaction with DNA and low risk of mutagenesity. Chromenones show anticancer activities too. Therefore, hybrids of chalcone and chromenone may be potent chemotherapeutic agents. We prepared 16 synthetic chromenylchalcones and applied a clonogenic long-term survival assay method for them on HCT116 human colorectal cancer cell lines. One of chromenylchalcones tested here, chromenylchalcone 11, showed IC50 of 93.1nM which can be competed with the IC50 values of well-known flavonoids such as catechin gallate and epicatechin gallate. Further biological experiments including cell cycle analysis, apoptosis assay, Western blot analysis, and immunofluorescent microscopy were carried out for this compound. In addition, in vitro kinases binding assay performed to explain its molecular mechanism demonstrated the compound inhibited aurora kinases. The binding modes between chromenylchalcone 11 and aurora kinases were elucidated using in silico docking experiments. These findings could be used for designing cancer therapeutic or preventive plant-derived chromenylchalcone agents. PMID:23719279

Shin, Soon Young; Yoon, Hyuk; Ahn, Seunghyun; Kim, Dong-Wook; Kim, Sang Ho; Koh, Dongsoo; Lee, Young Han; Lim, Yoongho



Cell lines  

US Patent & Trademark Office Database

Genomic instability in T-antigen expressing cells can be overcome by modifying the gene expressing T-antigen so that it lacks Bub1 binding. Stable cell lines can be produced by incorporation of the modified T-antigen gene, preferably together with the catalytic sub-unit of the telomerase construct.



Whole-body imaging of tumor cells by azaelectrocyclization: visualization of metastasis dependence on glycan structure.  


Noninvasive imaging of cancer metastasis through the efficient cell labeling constitutes a major technological breakthrough for cancer research and patient monitoring post-surgery. In the current work, we expanded our cell surface labeling technique on the whole-body fluorescence imaging of tumor metastasis in BALB/c nude mice. Four kinds of human cancer cells (two cancer cell lines, MKN45 and HCT116, and their transfected versions expressing surface glycan-related genes, MKN45-GnT-V and HCT116-GMDS) were labeled by azaelectrocyclization with Hilyte Fluor 750 for 10 min and without affecting cell viability. Fluorescence-labeled cancer cells were injected into the abdominal cavities of BALB/c mice and whole-body scans were performed with an eXplore Optix device. In accordance with previous findings, the fluorescence imaging clearly showed that tumor metastasis was dependent upon the cell surface glycans: A larger polylactosamine structure or the loss of fucosylation on the cancer cell surfaces, respectively, enhanced the metastatic potential of the tumor cells. Our noninvasive technique provides the landmark opportunity for sensitively monitoring the dynamics of the cancer cells depending on their surface structures and/or the host environments, thus impacts on the cancer prognosis and the therapeutic applications. PMID:23375093

Tanaka, Katsunori; Moriwaki, Kenta; Yokoi, Satomi; Koyama, Koichi; Miyoshi, Eiji; Fukase, Koichi



?-Glutamyl hydrolase modulation and folate influence chemosensitivity of cancer cells to 5-fluorouracil and methotrexate.  


Background:?-Glutamyl hydrolase (GGH) regulates intracellular folate and antifolates for optimal nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. The modulation of GGH may therefore affect chemosensitivity of cancer cells, and exogenous folate levels may further modify this effect.Methods:We generated a novel model of GGH modulation in human HCT116 and MDA-MB-435 cancer cells and investigated the effect of GGH modulation on chemosensitivity to 5-fluorouracil (5FU) and methotrexate (MTX) at different folate concentrations in vitro and in vivo.Results:Overexpression of GGH significantly decreased chemosensitivity of MDA-MB-435 cells to 5FU and MTX at all folate concentrations as expected. In contrast, in HCT116 cells this predicted effect was observed only at very high folate concentration, and as the folate concentration decreased this effect became null or paradoxically increased. This in vitro observation was confirmed in vivo. Inhibition of GGH significantly increased chemosensitivity of both cancer cells to 5FU at all folate concentrations. Unexpectedly, GGH inhibition significantly decreased chemosensitivity of both cancer cells to MTX at all folate concentrations. In both GGH modulation systems and cell lines, the magnitude of chemosensitivity effect incrementally increased as folate concentration increased.Conclusion:Modulation of GGH affects chemosensitivity of cancer cells to 5FU and MTX, and exogenous folate levels can further modify the effects. PMID:24045662

Kim, S-E; Cole, P D; Cho, R C; Ly, A; Ishiguro, L; Sohn, K-J; Croxford, R; Kamen, B A; Kim, Y-I



p53 is important for the anti-invasion of ganoderic acid T in human carcinoma cells.  


The function of p53 induced by ganoderic acids (GAs) in anti-invasion was unknown, although our previous work reported the inhibition of tumor invasion and metastasis by Ganoderic acid T (GA-T). This work indicated that GA-T promoted cell aggregation, inhibited cell adhesion and surpressed cell migration with a dose-dependent manner in human colon tumor cell lines of HCT-116 p53(+/+) and p53(-/-). Furthermore, comparing the ratios of HCT-116 p53(+/+) and p53(-/-) cells, p53 modified GA-T inhibition of migration and adhesion and GA-T promotion of cell aggregation, and p53 also modified GA-T inhibition of NF-?B nuclear translocation, I?B? degradation, and down-regulation of urokinase-type plaminogen activator (uPA), matrix metalloproteinase-2/9 (MMP-2/9), inducible nitric oxide synthase (iNOS/NOS2) protein expression and inducible nitric oxide (NO) production. The results indicated that p53 played an important role in anti-invasion of GA-T in human carcinoma cells. p53 may be an important target for GA-T inhibiting human carcinoma cells anti-invasion. PMID:21353507

Chen, Nian-Hong; Zhong, Jian-Jiang



Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines  

PubMed Central

Background CD26 (dipeptidyl peptidase IV, DPPIV) is a 110 kDa surface glycoprotein expressed in most normal tissues, and is a potential novel therapeutic target for selected cancers. Our work evaluates the mechanism involved in confluence-dependent CD26 expression in colon cancer. Methods Colon adenocarcinoma cells were grown to confluence, and expression of CD26 and transcription factors implicated in its regulation was confirmed by immunofluorescence and Western blotting. Real-time PCR was also performed to evaluate CD26 upregulation at the transcriptional level. The influence of c-Myc on CD26 expression during different growth conditions was further evaluated following transient transfection of a c-Myc-expressing plasmid and a c-Myc specific siRNA. Results We found that the colon cancer cell lines HCT-116 and HCT-15 exhibited a confluence-dependent increase in CD26 mRNA and protein, associated with decreased expression of c-Myc, increased USF-1 and Cdx 2 levels, and unchanged HNF-1? expression. Meanwhile, ectopic expression of c-Myc in both cell lines led to decreased CD26 expression. In contrast, transfection of a siRNA targeted to Cdx2 resulted in decreased CD26 level. Importantly, culturing of cells in serum-depleted media, but not acidic conditions, upregulated CD26. While HIF-1? level also increased when cells were cultured in serum-depleted media, its expression was required but not sufficient for CD26 upregulation. Conclusions CD26 mRNA and protein levels increase in a confluence-dependent manner in colon carcinoma cell lines, with c-Myc acting as a repressor and Cdx2 acting as an enhancer of CD26 expression. The enhanced expression of CD26 in serum-depleted media and a requirement for HIF-1? suggest a role for nutrients or growth factors in the regulation of CD26 protein expression.



Expression analysis of secreted and cell surface genes of five transformed human cell lines and derivative xenograft tumors  

PubMed Central

Background Since the early stages of tumorigenesis involve adhesion, escape from immune surveillance, vascularization and angiogenesis, we devised a strategy to study the expression profiles of all publicly known and putative secreted and cell surface genes. We designed a custom oligonucleotide microarray containing probes for 3531 secreted and cell surface genes to study 5 diverse human transformed cell lines and their derivative xenograft tumors. The origins of these human cell lines were lung (A549), breast (MDA MB-231), colon (HCT-116), ovarian (SK-OV-3) and prostate (PC3) carcinomas. Results Three different analyses were performed: (1) A PCA-based linear discriminant analysis identified a 54 gene profile characteristic of all tumors, (2) Application of MANOVA (Pcorr < .05) to tumor data revealed a larger set of 149 differentially expressed genes. (3) After MANOVA was performed on data from individual tumors, a comparison of differential genes amongst all tumor types revealed 12 common differential genes. Seven of the 12 genes were identified by all three analytical methods. These included late angiogenic, morphogenic and extracellular matrix genes such as ANGPTL4, COL1A1, GP2, GPR57, LAMB3, PCDHB9 and PTGER3. The differential expression of ANGPTL4 and COL1A1 and other genes was confirmed by quantitative PCR. Conclusion Overall, a comparison of the three analyses revealed an expression pattern indicative of late angiogenic processes. These results show that a xenograft model using multiple cell lines of diverse tissue origin can identify common tumorigenic cell surface or secreted molecules that may be important biomarker and therapeutic discoveries.

Stull, Robert A; Tavassoli, Roya; Kennedy, Scot; Osborn, Steve; Harte, Rachel; Lu, Yan; Napier, Cheryl; Abo, Arie; Chin, Daniel J



Synthesis and antitumor evaluation of a novel series of triaminotriazine derivatives  

Microsoft Academic Search

A series of triaminotriazine derivatives (compounds 5a–f, 6a–x, and 7a–g) was designed, synthesized, and evaluated for their inhibition activities to colorectal cancer (CRC) cell lines (HCT-116 and HT-29). Most of the synthesized compounds demonstrated moderate anti-proliferatory effects on both HCT-116 and HT-29 cell lines at the concentration of 10?M. The inhibitory activities against HCT-116 and HT-29 cell lines were discussed

Mingfang Zheng; Chenghui Xu; Jianwei Ma; Yan Sun; Feifei Du; Hong Liu; Liping Lin; Chuan Li; Jian Ding; Kaixian Chen; Hualiang Jiang



Hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells.  


In this paper, a targeted drug delivery system has been developed based on hyaluronic acid (HA) modified mesoporous silica nanoparticles (MSNs). HA-MSNs possess a specific affinity to CD44 over-expressed on the surface of a specific cancer cell line, HCT-116 (human colon cancer cells). The cellular uptake performance of fluorescently labelled MSNs with and without HA modification has been evaluated by confocal microscopy and fluorescence-activated cell sorter (FACS) analysis. Compared to bare MSNs, HA-MSNs exhibit a higher cellular uptake via HA receptor mediated endocytosis. An anticancer drug, doxorubicin hydrochloride (Dox), has been loaded into MSNs and HA-MSNs as drug delivery vehicles. Dox loaded HA-MSNs show greater cytotoxicity to HCT-116 cells than free Dox and Dox-MSNs due to the enhanced cell internalization behavior of HA-MSNs. It is expected that HA-MSNs have a great potential in targeted delivery of anticancer drugs to CD44 over-expressing tumors. PMID:23076766

Yu, Meihua; Jambhrunkar, Siddharth; Thorn, Peter; Chen, Jiezhong; Gu, Wenyi; Yu, Chengzhong



Targeting colorectal cancer cells with single-walled carbon nanotubes conjugated to anticancer agent SN-38 and EGFR antibody.  


In this study, single-walled carbon nanotubes (SWNTs) conjugated with antibody C225 were used to achieve targeted therapy against EGFR over-expressed colorectal cancer cells. In addition, the control release of the chemotherapeutic drug, 7-Ethyl-10-hydroxy-camptothecin (SN38), was studied. We used three different colorectal cancer cell lines, HCT116, HT29, and SW620, listed in the order of decreasing expression levels of EGFR. Our results showed that SWNT could use C225 to specifically bind to EGFR-expressed cells. The cellular uptakes of SWNT of EGFR over-expressed cells (HCT116 and HT29) were much higher than that of the negative control (SW620). We, next, demonstrated that receptor-mediated endocytosis was the primary cell entry route for SWNT. As a consequence, abundant amount of SN38 was released and EGFR over-expressed cells were killed. The drug control release process was studied by utilizing human carboxylesterase enzyme (hCE) that would break the bond linking SN38 and SWNT-carrier in cytoplasm. The intracellular SN38 release observed by confocal microscopy showed that SN38 actually dissociated from the SWNT-carrier first. SN38's entry to nucleus was then followed while the SWNT-carrier still remained in the cytoplasm. Overall, all these data suggested that SWNT could be a good carrier for targeting controlled release therapy. PMID:23937913

Lee, Pei-Chi; Chiou, Yu-Chi; Wong, Jau-Min; Peng, Cheng-Liang; Shieh, Ming-Jium



Evaluation of copper-64-labeled somatostatin agonists and antagonist in sstr2-transfected cell lines that are positive and negative for p53: implications for cancer therapy  

PubMed Central

Objectives Radiolabeled somatostatin analogs have become important agents for molecular imaging and targeted radiotherapy of somatostatin receptor-positive tumors. Here we determine the effect of the tumor suppressor protein, p53, on trafficking 64Cu to tumor cell nuclei from DOTA vs.CB-TE2A-conjugated agonist Y3-TATE and the antagonist 64Cu-CB-TE2A-sst2-ANT in cell lines that are positive or negative for p53. Methods Receptor binding, internalization, cAMP and nuclear localization studies were performed with the SSTr2 agonists, 64Cu-CB-TE2A-Y3-TATE and 64Cu-DOTA-Y3-TATE vs. antagonist, 64Cu-CB-TE2A-sst2-ANT, in SSTr2-transfected p53 +/+ and ?/? HCT116 colorectal carcinoma cells. Results The antagonist, 64Cu-CB-TE2A-sst2-ANT, bound 8-9-fold more SSTr2 binding sites than did the 64Cu-labeled agonists. 64Cu-CB-TE2A-Y3-TATE was more efficiently internalized than 64Cu-DOTA-Y3-TATE, while 64Cu-CB-TE2A-sst2-ANT showed lower, yet significant levels of internalization. CB-TE2A-Y3-TATE acted as a full agonist, inhibiting cAMP production, whereas CB-TE2A-sst2-ANT showed no inhibition of cAMP production.The 64Cu from agonists 64Cu-DOTA-Y3-TATE and 64Cu-CB-TE2A-Y3-TATE showed greater nuclear localization at 24 h in p53 +/+ vs. ?/? cells; however, there was no difference in the levels of 64Cu from the antagonist based on p53 status. Surprisingly, the DOTA and CB-TE2A-conjugated agonists showed similar nuclear localization in the p53 +/+ and ?/? cells, suggesting no difference in 64Cu release from these chelators in the HCT116 cell lines. Conclusion Based on thesein vitro data, the agonist 64Cu-CB-TE2A-Y3-TATE demonstrated the most promise as an agent for targeted radiotherapy in p53 positive, SSTr2-positive tumors.

Nguyen, Kim; Parry, Jesse J.; Rogers, Buck E.; Anderson, Carolyn J.



Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.  


In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy. PMID:23345168

Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela



p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line  

SciTech Connect

We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.

Orre, Lukas M. [Cancer Centrum Karolinska Institutet, Department of Oncology and Pathology, Division of Medical Radiation Biology, Stockholm (Sweden)]. E-mail:; Stenerloew, Bo [Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Stockholm (Sweden); Dhar, Sumeer [Division of Clinical Pharmacology, Department of Medical Sciences, University Hospital, Uppsala (Sweden); Larsson, Rolf [Division of Clinical Pharmacology, Department of Medical Sciences, University Hospital, Uppsala (Sweden); Lewensohn, Rolf [Cancer Centrum Karolinska Institutet, Department of Oncology and Pathology, Division of Medical Radiation Biology, Stockholm (Sweden); Lehtioe, Janne [Cancer Centrum Karolinska Institutet, Department of Oncology and Pathology, Division of Medical Radiation Biology, Stockholm (Sweden)



Cacospongionolide and Scalaradial, Two Marine Sesterterpenoids as Potent Apoptosis-Inducing Factors in Human Carcinoma Cell Lines  

PubMed Central

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-?B subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.

Malik, Shoaib Ahmad; Iodice, Carmine; De Rosa, Salvatore; Maiuri, Maria Chiara; Carnuccio, Rosa



UDN glycoprotein regulates activities of manganese-superoxide dismutase, activator protein-1, and nuclear factor-?B stimulated by reactive oxygen radicals in lipopolysaccharide-stimulated HCT116 cells  

Microsoft Academic Search

This study was carried out to investigate the anti-inflammatory effects of glycoprotein (UDN glycoprotein, 116-kDa) isolated from Ulmus davidiana Nakai, which has been used to heal inflammatory diseases in Korean herbal medicine. We found that UDN glycoprotein has strong scavenging effect on the production of intracellular superoxide anion (O2-), hydrogen peroxides (H2O2), and nitric oxide (NO) without any cytotoxicity, and

Sei-Jung Lee; Kye-Taek Lim



Cancer Cell Cytotoxicities of 1-(4-Substitutedbenzoyl)-4-(4-chlorobenzhydryl)piperazine Derivatives  

PubMed Central

A series of novel 1-(4-substitutedbenzoyl)-4-(4-chlorobenzhydryl)piperazine derivatives 5a–g was designed by a nucleophilic substitution reaction of 1-(4-chlorobenzhydryl)piperazine with various benzoyl chlorides and characterized by elemental analyses, IR and 1H nuclear magnetic resonance spectra. Cytotoxicity of the compounds was demonstrated on cancer cell lines from liver (HUH7, FOCUS, MAHLAVU, HEPG2, HEP3B), breast (MCF7, BT20, T47D, CAMA-1), colon (HCT-116), gastric (KATO-3) and endometrial (MFE-296) cancer cell lines. Time-dependent cytotoxicity analysis of compound 5a indicated the long-term in situ stability of this compound. All compounds showed significant cell growth inhibitory activity on the selected cancer cell lines.

Yarim, Mine; Koksal, Meric; Durmaz, Irem; Atalay, Rengul



Cancer Cell Cytotoxicities of 1-(4-Substitutedbenzoyl)-4-(4-chlorobenzhydryl)piperazine Derivatives.  


A series of novel 1-(4-substitutedbenzoyl)-4-(4-chlorobenzhydryl)piperazine derivatives 5a-g was designed by a nucleophilic substitution reaction of 1-(4-chlorobenzhydryl)piperazine with various benzoyl chlorides and characterized by elemental analyses, IR and (1)H nuclear magnetic resonance spectra. Cytotoxicity of the compounds was demonstrated on cancer cell lines from liver (HUH7, FOCUS, MAHLAVU, HEPG2, HEP3B), breast (MCF7, BT20, T47D, CAMA-1), colon (HCT-116), gastric (KATO-3) and endometrial (MFE-296) cancer cell lines. Time-dependent cytotoxicity analysis of compound 5a indicated the long-term in situ stability of this compound. All compounds showed significant cell growth inhibitory activity on the selected cancer cell lines. PMID:22942690

Yarim, Mine; Koksal, Meric; Durmaz, Irem; Atalay, Rengul



Curcumin cytotoxicity is enhanced by PTEN disruption in colorectal cancer cells  

PubMed Central

AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency on the cytotoxicity of chemotherapeutic agents toward colorectal cancer cells. METHODS: PTEN-deficient colorectal cancer (CRC) cells were generated by human somatic cell gene targeting using the adeno-associated virus system. The cytotoxic effects of compounds including curcumin, 5-fluorouracil (5-FU), dihydroartemisinin (DHA), irinotecan (CPT-11) and oxaliplatin (OXA) on cancer cells were determined using the MTT assay. Enhanced cytotoxicity of curcumin in PTEN-deficient CRC cells was observed, and this was confirmed using clonogenic assays. Apoptosis and cell cycle progression were analyzed by flow cytometry. Levels of apoptosis and cell cycle-related proteins were examined by Western blotting. RESULTS: We developed an isogenic set of CRC cell lines that differed only in their PTEN status. Using this set of cell lines, we found that disruption of the PTEN gene had no effect on the sensitivity of CRC cells to 5-FU, CPT-11, DHA, or OXA, whereas PTEN disruption increased the sensitivity of CRC cells to curcumin. Loss of PTEN did not alter the curcumin-induced apoptosis in CRC cells. However, PTEN deficiency led to an altered pattern of curcumin-mediated cell cycle arrest. In HCT116 PTEN+/+ cells, curcumin caused a G2/M phase arrest, whereas it caused a G0/G1 phase arrest in HCT116 PTEN-/- cells. Levels of cell cycle-related proteins were consistent with these respective patterns of cell cycle arrest. CONCLUSION: Curcumin shows enhanced cytotoxicity toward PTEN-deficient cancer cells, suggesting that it might be a potential chemotherapeutic agent for cancers harboring PTEN mutations.

Chen, Lin; Li, Wen-Feng; Wang, Hong-Xiao; Zhao, Hai-Na; Tang, Jia-Jia; Wu, Chang-Jie; Lu, Li-Ting; Liao, Wan-Qin; Lu, Xin-Cheng



Systematic Analyses of the Cytotoxic Effects of Compound 11a, a Putative Synthetic Agonist of Photoreceptor-Specific Nuclear Receptor (PNR), in Cancer Cell Lines.  


Photoreceptor cell-specific receptor (PNR/NR2E3) is an orphan nuclear receptor that plays a critical role in retinal development and photoreceptor maintenance. The disease-causing mutations in PNR have a pleiotropic effect resulting in varying retinal diseases. Recently, PNR has been implicated in control of cellular functions in cancer cells. PNR was reported to be a novel regulator of ER? expression in breast cancer cells, and high PNR expression correlates with favorable response to tamoxifen treatment. Moreover, PNR was shown to increase p53 stability in HeLa cells, implying that PNR may be a therapeutic target in this and other cancers that retain a wild type p53 gene. To facilitate further understanding of PNR functions in cancer, we characterized compound 11a, a synthetic, putative PNR agonist in several cell-based assays. Interestingly, we showed that 11a failed to activate PNR and its cytotoxicity was independent of PNR expression, excluding PNR as a mediator for 11a cytotoxicity. Systematic analyses of the cytotoxic effects of 11a in NCI-60 cell lines revealed a strong positive correlation of cytotoxicity with p53 status, i.e., p53 wild type cell lines were significantly more sensitive to 11a than p53 mutated or null cell lines. Furthermore, using HCT116 p53+/+ and p53-/- isogenic cell lines we revealed that the mechanism of 11a-induced cytotoxicity occurred through G1/S phase cell cycle arrest rather than apoptosis. In conclusion, we observed a correlation of 11a sensitivity with p53 status but not with PNR expression, suggesting that tumors expressing wild type p53 might be responsive to this compound. PMID:24066170

Zhao, Zibo; Wang, Lu; Wen, Zhi; Ayaz-Guner, Serife; Wang, Yidan; Ahlquist, Paul; Xu, Wei



Systematic Analyses of the Cytotoxic Effects of Compound 11a, a Putative Synthetic Agonist of Photoreceptor-Specific Nuclear Receptor (PNR), in Cancer Cell Lines  

PubMed Central

Photoreceptor cell-specific receptor (PNR/NR2E3) is an orphan nuclear receptor that plays a critical role in retinal development and photoreceptor maintenance. The disease-causing mutations in PNR have a pleiotropic effect resulting in varying retinal diseases. Recently, PNR has been implicated in control of cellular functions in cancer cells. PNR was reported to be a novel regulator of ER? expression in breast cancer cells, and high PNR expression correlates with favorable response to tamoxifen treatment. Moreover, PNR was shown to increase p53 stability in HeLa cells, implying that PNR may be a therapeutic target in this and other cancers that retain a wild type p53 gene. To facilitate further understanding of PNR functions in cancer, we characterized compound 11a, a synthetic, putative PNR agonist in several cell-based assays. Interestingly, we showed that 11a failed to activate PNR and its cytotoxicity was independent of PNR expression, excluding PNR as a mediator for 11a cytotoxicity. Systematic analyses of the cytotoxic effects of 11a in NCI-60 cell lines revealed a strong positive correlation of cytotoxicity with p53 status, i.e., p53 wild type cell lines were significantly more sensitive to 11a than p53 mutated or null cell lines. Furthermore, using HCT116 p53+/+ and p53-/- isogenic cell lines we revealed that the mechanism of 11a-induced cytotoxicity occurred through G1/S phase cell cycle arrest rather than apoptosis. In conclusion, we observed a correlation of 11a sensitivity with p53 status but not with PNR expression, suggesting that tumors expressing wild type p53 might be responsive to this compound.

Zhao, Zibo; Wang, Lu; Wen, Zhi; Ayaz-guner, Serife; Wang, Yidan; Ahlquist, Paul; Xu, Wei



Peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) ligands do not potentiate growth of human cancer cell lines.  


Ligands for peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) increase skeletal muscle fatty acid catabolism, improve insulin sensitivity, increase serum high-density lipoprotein cholesterol, elicit anti-inflammatory activity and induce terminal differentiation. Contradictory findings are also reported suggesting that PPARbeta/delta ligands potentiate tumorigenesis by increasing cell proliferation, by inhibiting apoptosis through phosphorylation of Akt and by increasing cyclooxygenase-2 (COX2) and vascular endothelial growth factor (VEGF) expression. The contradictory findings could be due to differences in the model system (cancer cell line versus in vivo), differences in cell culture conditions (with and without serum) or differences in ligands. The present study examined the effect of two different PPARbeta/delta ligands (GW0742 and GW501516) in human cancer cell lines (HT29, HCT116, LS-174T, HepG2 and HuH7) cultured in the presence or absence of serum and compared in vitro analysis with in vivo analysis. Neither PPARbeta/delta ligand increased cell growth or phosphorylation of Akt and no increase in the expression of VEGF or COX2 were detected in any cancer cell line in the presence or absence of serum. Similarly, liver, colon and colon polyps from mice administered these PPARbeta/delta ligands in vivo did not exhibit changes in these markers. Results from these studies demonstrate that serum withdrawal and/or differences in ligands do not underlie the disparity in responses reported in the literature. The quantitative nature of the present findings are inconsistent with the hypothesis that cancer cell lines respond differentially as compared with normal cells, and provide further evidence that PPARbeta/delta ligands do not potentiate tumorigenesis. PMID:17693664

Hollingshead, Holly E; Killins, Renee L; Borland, Michael G; Girroir, Elizabeth E; Billin, Andrew N; Willson, Timothy M; Sharma, Arun K; Amin, Shantu; Gonzalez, Frank J; Peters, Jeffrey M



Quantitative phase imaging of living cells: application of the phase volume and area functions to the analysis of "nucleolar stress".  


We applied coherent phase microscopy to develop a method of quantitative evaluation of functional state of eukaryotic cells using the coordinates of characteristic points (CP) in the functions of the phase volume W and area S. In a fragment of a single cell image (HCT116 human colon carcinoma cell line) with detectable nucleolus, the values of the phase thickness, area, and volume were calculated. These values dramatically changed within the initial minutes of cell exposure to the transcriptional inhibitor actinomycin D. The positions of CP in the graphs of S and W functions allowed for monitoring the time-dependent decrease of nucleolar contrast, a major optical hallmark of "nucleolar stress." Given that the area and volume functions reflect optical heterogeneity of the cell and are independent of its optical model, these functions can be applicable as general mathematical tools for the analysis of cell morphology and physiology. PMID:23974227

Tychinsky, Vladimir; Kretushev, Alexander V; Klemyashov, Ivan V; Zverzhkhovskiy, Vladislav D; Vyshenskaya, Tatyana V; Shtil, Alexander A



Induction of Apoptosis in Human Cancer Cells by Candidaspongiolide, a Novel Sponge Polyketide  

PubMed Central

Background Candidaspongiolide (CAN), a novel polyketide from a marine sponge, is the active component of a mixture that was found to be potently cytotoxic in the National Cancer Institute’s 60-cell-line screen. Methods Effects of CAN on U251 glioma and HCT116 colorectal cancer cells and on normal fibroblasts were assessed using radiolabeling studies to measure protein synthesis, clonogenic assays to measure cell survival, flow cytometry of annexin V– and propidium iodide–stained cells to measure apoptosis, and western blots in the presence or absence of specific inhibitors to assess accumulation and phosphorylation of potential downstream target proteins. Results CAN inhibited protein synthesis and potently induced apoptosis in both U251 and HCT116 cells, the latter in part by a caspase 12–dependent pathway. For example, 25%–30% of U251 or HCT116 cells became apoptotic after 24 hours of treatment with 100 nM CAN. CAN also rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)-? at Ser51 and of the translation elongation factor eEF2 at Thr56, which could contribute to its dose-dependent inhibition of protein synthesis. Stable expression of dominant-negative eIF2? was sufficient to prevent CAN-induced eIF2? phosphorylation and induction of apoptosis but insufficient to prevent inhibition of protein synthesis. CAN induction of eIF2? phosphorylation did not occur by a classic endoplasmic reticulum stress pathway. However, an inhibitor of and small-interfering RNAs to the double-stranded RNA–dependent protein kinase PKR prevented CAN-mediated eIF2? phosphorylation and apoptosis, respectively. Although CAN inhibited protein synthesis in both cancer cells and normal human fibroblasts, it induced eIF2? phosphorylation and apoptosis only in cancer cells. Conclusions CAN triggers PKR/eIF2?/caspase 12–dependent apoptosis and inhibits protein synthesis in cancer cells but only inhibits protein synthesis in normal cells.

Trisciuoglio, Daniela; Uranchimeg, Badarch; Cardellina, John H.; Meragelman, Tamara L.; Matsunaga, Shigeki; Fusetani, Nobuhiru; Del Bufalo, Donatella; Shoemaker, Robert H.



NO-donating aspirin inhibits the activation of NF-kappaB in human cancer cell lines and Min mice.  


Nitric oxide-donating aspirin (NO-ASA) is a promising agent for the control of cancer, whose mechanism of action remains unclear. NF-kappaB is an important signaling molecule in the pathogenesis of cancer. We studied in several human colon (HT-29, HCT-15, LoVo, HCT116 and SW-480), pancreatic (BxPC-3, MIA PaCa-2) and breast (MDA-MB-231 and MCF-7) cancer cell lines, the effect of NO-ASA on NF-kappaB activation, determined by electrophoretic mobility shift assays, immunofluorescence and western blot analyses of nuclear proteins. NO-ASA inhibited NF-kappaB activation, as early as 30 min and with IC(50)s ranging between 0.83 and 64 microM. Such inhibition was also observed at NO-ASA concentrations that had an insignificant or marginal effect on cell growth. The effect of NO-ASA on NF-kappaB binding to DNA was significantly correlated with its effect on cell growth (P < 0.05) indicating that the growth inhibitory effect of NO-ASA may be mediated by its effect on NF-kappaB. Compared with control, NO-ASA decreased NF-kappaB activation in intestinal epithelial cells of APC(min+/-) mice by 38.4% (P < 0.01). Western blot and immunofluorescence analyses revealed that the nuclear levels of the p50 and p65 NF-kappaB subunits were virtually unaffected, suggesting an inhibitory mechanism different from suppressed subunit translocation into the nucleus. Inhibition of NF-kappaB activation by NO-ASA may account, at least in part, for its chemopreventive efficacy. PMID:18174252

Williams, Jennie L; Ji, Ping; Ouyang, Nengtai; Liu, Xiaoping; Rigas, Basil



Antiproliferative and cytotoxic effects of sodium selenite in human colon cancer cells  

Microsoft Academic Search

Sodium selenite has been reported to interfere with cell growth and proliferation and to induce cell death. Despite of our current knowledge, details about its effects on growth and behavior of colonocytes with differing p53 status remain unknown. In our study, we evaluated the antiproliferative, cell cycle specific and proapoptotic potential of sodium selenite in HCT-116 colorectal cells with wild

V?ra Králová; Kate?ina Brigulová; Miroslav ?ervinka; Emil Rudolf



In Vitro Characterization of a Liposomal Formulation of Celecoxib Containing 1,2-Distearoyl-sn-Glycero-3-Phosphocholine, Cholesterol, and Polyethylene Glycol and its Functional Effects Against Colorectal Cancer Cell Lines.  


Nanosized liposomal drug delivery systems are well suited for selective drug delivery at tumor sites. Celecoxib (CLX) is a highly hydrophobic cyclooxygenase-2 inhibitor that can reduce the incidence of colorectal polyps; however, the adverse cardiovascular effects limit its applicability. Here, we report a liposomal formulation of CLX using 1,2-Distearoyl-sn-glycero-3-phosphocholine, cholesterol, and polyethylene glycol. Encapsulation efficiency of the drug was greater than 70%; the release was slow and sustained with only 12%-20% of CLX released in the first 12 h. Flow cytometry and confocal microscopy studies using the colon cancer cell lines HCT-116 and SW620 showed significantly higher cellular association and internalization of the liposomes after incubation for 6 h when compared with 30 min. The liposomes did not colocalize with transferrin, but had a punctuate appearance, indicating vesicular localization. Cell proliferation was inhibited by 95% and 78%, respectively, in SW620 and HT29 cells after incubation with 600 ?M liposomal CLX for 72 h. Moreover, cellular motility, as shown by a scratch wound healing assay, was also significantly (p = 0.006) inhibited when SW620 cells were incubated with 400 ?M liposomal CLX. This is the first report of the successful encapsulation of CLX in a long-circulating liposomal formulation that could be effective against colorectal cancer. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3666-3677, 2013. PMID:23897281

Erdo?, Asli; Putra Limasale, Yanuar Dwi; Keskin, Dilek; Tezcaner, Ay?en; Banerjee, Sreeparna



Differential ganciclovir-mediated cell killing by glutamine 125 mutants of herpes simplex virus type 1 thymidine kinase.  


The therapeutic combination of the herpesvirus simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and the prodrug, ganciclovir (GCV), has found great utility for the treatment of many types of cancer. After initial phosphorylation of GCV by HSV-1 TK, cellular kinases generate the toxic GCV-triphosphate metabolite that is incorporated into DNA and eventually leads to tumor cell death. The cellular and pharmacological mechanisms by which metabolites of GCV lead to cell death are still poorly defined. To begin to address these mechanisms, different mutated forms of HSV-1 TK at residue Gln-125 that have distinct substrate properties were expressed in mammalian cell lines. It was found that expression of the Asn-125 HSV-1 TK mutant in two cell lines, NIH3T3 and HCT-116, was equally effective as wild-type HSV-1 TK for metabolism and sensitivity to GCV, bystander effect killing and induction of apoptosis. The major difference between the two enzymes was the lack of deoxypyrimidine metabolism in the Asn-125 TK-expressing cells. In HCT-116 cells expressing the Glu-125 TK mutant, GCV metabolism was greatly attenuated, yet at higher GCV concentrations, cell sensitivity to the drug and bystander effect killing were diminished but still effective. Cell cycle analysis, 4', 6'-diamidine-2'-phenylindoledihydrochloride staining, and caspase 3 activation assays indicated different cell death responses in the Glu-125 TK-expressing cells as compared with the wild-type HSV-1 TK or Asn-125 TK-expressing cells. A mechanistic hypothesis to explain these results based on the differences in GCV-triphosphate metabolite levels is presented. PMID:10601281

Drake, R R; Wilbert, T N; Hinds, T A; Gilbert, K M



HY-1 induces G(2)/M cell cycle arrest in human colon cancer cells through the ATR-Chk1-Cdc25C and Weel pathways.  


The novel aroylthiourea analogue of podophyllotoxin HY-1 (4?-[benzoyl-thioureido]-4-deoxypodophyllotoxin) was synthesized in our laboratory with the aim of developing multitargeted DNA topoisomerase II inhibitors. The compound showed significant antiproliferative effects on seven cancer cell lines and induced G2 /M phase arrest in HCT116 cells. Moreover, HY-1 showed a potent inhibitory effect on topoisomerase II-mediated kinetoplast DNA decatenation in a dose-dependent manner. Our results showed that cdc2 phosphorylation and decreased cdc2 kinase acitivity through the ATR-Chk1-Cdc25C and Weel pathways were the central mechanisms for G2 /M phase arrest in human colon cancer cells. PMID:23600770

Zhao, Yu; Wu, Zhonghua; Zhang, Yuting; Zhu, Li



Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes  

PubMed Central

Putative cancer stem cell (CSC) populations efflux dyes such as Hoechst 33342 giving rise to side populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoechst 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non-toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (CloP). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress and proliferation in several cancer cell lines when stained whereas Hoechst 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53?/? cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.

Allen, Joshua E.; Hart, Lori S.; Dicker, David T.; Wang, Wenge; El-Deiry, Wafik S.



Simultaneous Aurora-A/STK15 overexpression and centrosome amplification induce chromosomal instability in tumour cells with a MIN phenotype  

PubMed Central

Background Genetic instability is a hallmark of tumours and preneoplastic lesions. The predominant form of genome instability in human cancer is chromosome instability (CIN). CIN is characterized by chromosomal aberrations, gains or losses of whole chromosomes (aneuploidy), and it is often associated with centrosome amplification. Centrosomes control cell division by forming a bipolar mitotic spindle and play an essential role in the maintenance of chromosomal stability. However, whether centrosome amplification could directly cause aneuploidy is not fully established. Also, alterations in genes required for mitotic progression could be involved in CIN. A major candidate is represented by Aurora-A/STK15 that associates with centrosomes and is overexpressed in several types of human tumour. Methods Centrosome amplification were induced by hydroxyurea treatment and visualized by immunofluorescence microscopy. Aurora-A/STK15 ectopic expression was achieved by retroviral infection and puromycin selection in HCT116 tumour cells. Effects of Aurora-A/STK15 depletion on centrosome status and ploidy were determined by Aurora-A/STK15 transcriptional silencing by RNA interference. Changes in the expression levels of some mitotic genes were determined by Real time RT-PCR. Results We investigated whether amplification of centrosomes and overexpression of Aurora-A/STK15 induce CIN using as a model system a colon carcinoma cell line (HCT116). We found that in HCT116 cells, chromosomally stable and near diploid cells harbouring a MIN phenotype, centrosome amplification induced by hydroxyurea treatment is neither maintained nor induces aneuploidy. On the contrary, ectopic overexpression of Aurora-A/STK15 induced supernumerary centrosomes and aneuploidy. Aurora-A/STK15 transcriptional silencing by RNA interference in cells ectopically overexpressing this kinase promptly decreased cell numbers with supernumerary centrosomes and aneuploidy. Conclusion Our results show that centrosome amplification alone is not sufficient to induce chromosomal instability in colon cancer cells with a MIN phenotype. Alternatively, centrosome amplification has to be associated with alterations in genes regulating mitosis progression such as Aurora-A/STK15 to trigger CIN.

Lentini, Laura; Amato, Angela; Schillaci, Tiziana; Di Leonardo, Aldo



DNMT1 as a molecular target in a multimodality-resistant phenotype in tumor cells.  


We have previously shown that hydrogen peroxide-resistant permanent (OC-14) cells are resistant to the cytotoxicity of several exogenous oxidative and anticancer agents including H(2)O(2), etoposide, and cisplatin; and we refer to this process as an oxidative multimodality-resistant phenotype (MMRP). Furthermore, OC-14 cells contain increased activator protein 1 activity, and inhibition of activator protein 1 reversed the MMRP. In this study, we show that permanent Rat-1 cell lines genetically altered to overexpress c-Fos also displayed a similar MMRP to H(2)O(2), etoposide, and cisplatin as OC-14 cells. Gene expression analysis of the OC-14 cells and c-Fos-overexpressing cells showed increased DNMT1 expression. Where OC-14 and c-Fos-overexpressing cells were exposed to 5-aza-2'-deoxycytidine, which inhibits DNMT activity, a significant but incomplete reversal of the MMRP was observed. Thus, it seems logical to suggest that DNMT1 might be at least one target in the MMRP. Rat-1 cells genetically altered to overexpress DNMT1 were also shown to be resistant to the cytotoxicity of H(2)O(2), etoposide, and cisplatin. Finally, somatic HCT116 knockout cells that do not express either DNMT1 (DNMT1(-/-)) or DNMT3B (DNMT3B(-/-)) were shown to be more sensitive to the cytotoxicity of H(2)O(2), etoposide, and cisplatin compared with control HCT116 cells. This work is the first example of a role for the epigenome in tumor cell resistance to the cytotoxicity of exogenous oxidative (H(2)O(2)) or systemic (etoposide and cisplatin) agents and highlights a potential role for DNMT1 as a potential molecular target in cancer therapy. PMID:18314485

Mishra, Mark V; Bisht, Kheem S; Sun, Lunching; Muldoon-Jacobs, Kristi; Awwad, Rania; Kaushal, Aradhana; Nguyen, Phuongmai; Huang, Lei; Pennington, J Daniel; Markovina, Stephanie; Bradbury, C Matthew; Gius, David



Colon cancer mesenchymal stem cells modulate the tumorigenicity of colon cancer through interleukin 6.  


Multipotent mesenchymal stem cells (MSCs) have been isolated from several tumors and are implicated to play critical roles to increase malignant cell growth, invasion and metastasis. Here, we show that the MSC-like cells were isolated from human colon cancer tissues. These isolated hCC-MSCs (human colon cancer-derived mesenchymal stem cells) shared similar characteristic features with bone marrow-derived MSCs, which include cell morphology, surface antigens and specific gene expression. Additionally, the hCC-MSCs could differentiate into osteocytes or adipocytes under appropriate culture conditions. The conditioned medium collected from the cultured hCC-MSCs was shown to enhance the migration and invasive activity of HCT-116 colon cancer cells in vitro. Besides, transplantation of HCT-116 cells along with hCC-MSCs in nude mice increased the tumor growth and metastasis. Further study revealed that IL-6 present in the hCC-MSC-conditioned medium sufficiently induced the levels of Notch-1 and CD44 in HCT-116 and HT-29 cells, which contribute to enhance tumorigenic activity of HCT-116 and HT-29 cells. By using immunohistochemical staining, the intense co-expression of IL-6, Notch-1 and CD44 was predominantly detected in human colon cancer tissues. Taken together, our findings suggest the importance of the IL-6/Notch-1/CD44 signaling axis in the interaction between hCC-MSCs and colon cancer cells. PMID:23751564

Lin, Jen-Tsun; Wang, Jeng-Yi; Chen, Mu-Kuan; Chen, Hong-Chang; Chang, Tung-Hao; Su, Bi-Wen; Chang, Pey-Jium



Curcumin induces permanent growth arrest of human colon cancer cells: link between senescence and autophagy.  


Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-?-galactosidase activation was observed both in p53+/+ and p53-/- cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53-/- HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells. PMID:22613224

Mosieniak, Grazyna; Adamowicz, Marek; Alster, Olga; Jaskowiak, Hubert; Szczepankiewicz, Andrzej A; Wilczynski, Grzegorz M; Ciechomska, Iwona A; Sikora, Ewa



A novel synthetic C-1 analogue of 7-deoxypancratistatin induces apoptosis in p53 positive and negative human colorectal cancer cells by targeting the mitochondria: enhancement of activity by tamoxifen.  


The natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis plant, specifically induces apoptosis in many cancer cell lines. Unlike many other chemotherapeutics, PST is not genotoxic and has minimal adverse effects on non-cancerous cells. However, its availability for preclinical and clinical work is limited due to its low availability in its natural source and difficulties in its chemical synthesis. Several synthetic analogues of 7-deoxypancratistatin with different modifications at C-1 were synthesized and screened for apoptosis inducing activity in human colorectal cancer (CRC) cells. We found that a C-1 acetoxymethyl derivative of 7-deoxypancratistatin, JC-TH-acetate-4 (JCTH-4), was effective in inducing apoptosis in both p53 positive (HCT 116) and p53 negative (HT-29) human CRC cell lines, demonstrating similar efficacy to that of natural PST. JCTH-4 was able to decrease mitochondrial membrane potential (MMP), increase levels of reactive oxygen species in isolated mitochondria, cause release of the apoptogenic factor cytochrome c (Cyto c) from isolated mitochondria, and induce autophagy in HCT 116 and HT-29 cells. Interestingly, when JCTH-4 was administered with tamoxifen (TAM), there was an enhanced effect in apoptosis induction, reactive oxygen species (ROS) production and Cyto c release by isolated mitochondria, and autophagic induction by CRC cells. Minimal toxicity was exhibited by a normal human fetal fibroblast (NFF) and a normal colon fibroblast (CCD-18Co) cell line. Hence, JCTH-4 is a novel compound capable of selectively inducing apoptosis and autophagy in CRC cells alone and in combination with TAM and may serve as a safer and more effective alternative to current cancer therapies. PMID:21494837

Ma, Dennis; Tremblay, Phillip; Mahngar, Kevinjeet; Akbari-Asl, Pardis; Collins, Jonathan; Hudlicky, Tomas; McNulty, James; Pandey, Siyaram



CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells.  


It has been shown that excess stress to the endoplasmic reticulum (ER) triggers apoptosis, but the mechanisms underlying these processes remain unclear. We and others have reported previously that DR5 expression is up-regulated in thapsigargin (THG)-treated human cancer cells. Here, we provide evidence that CHOP is involved in THG up-regulation of DR5, which is a critical step for ER stress-induced apoptosis in human cancer cells. In human colon cancer HCT116 cells, knockdown of DR5 by siRNA blocked THG-induced Bax conformational change along with caspase-3 activation and cell death. Moreover, inhibition of CHOP expression attenuated DR5 up-regulation and apoptosis induced by THG, whereas ectopic expression of DR5 restored the sensitivity of CHOP siRNA-transfected cells to THG-induced apoptosis. In addition to HCT116 cells, inhibition of CHOP or DR5 induction also attenuated THG-induced cell death in other cancer cell lines including LNCaP, A2780S, and DU145, indicating that CHOP and DR5 are critical for ER stress-mediated apoptosis in human carcinoma cells. Furthermore, we identified a potential CHOP-binding site in the 5'-flanking region of the DR5 gene. Mutation of this site abrogated the enhanced reporter activity in response to THG treatment. Together, our findings suggest that CHOP regulates ER stress-induced apoptosis, at least in part, through enhancing DR5 expression in some types of human cancer cells. PMID:15322075

Yamaguchi, Hirohito; Wang, Hong-Gang



Depletion of Securin Induces Senescence After Irradiation and Enhances Radiosensitivity in Human Cancer Cells Regardless of Functional p53 Expression  

SciTech Connect

Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, gamma-H2AX, and phospho-Chk2. Senescent cells were analyzed using a beta-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced gamma-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation.

Chen Wenshu; Yu Yichu [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Lee Yijang [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan (China); Chen, J.-H. [Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien, Taiwan (China); Hsu, H.-Y. [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Chiu, S.-J., E-mail: [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Institute of Radiation Sciences, Tzu Chi Technology College, Hualien, Taiwan (China)



Anti-carcinogenic properties of omeprazole against human colon cancer cells and azoxymethane-induced colonic aberrant crypt foci formation in rats.  


Omeprazole is a proton pump inhibitor, a widely used drug to treat ulcers and gastroesophageal refluxdisease. We have evaluated colon cancer chemopreventive properties of omeprazole using azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats and analyzed cell growth inhibition and apoptosis induction in human colon cancer cells. Five-week-old male F344 rats were fed a control or experimental diet containing two doses of omeprazole (200 and 400 ppm). After one week, all animals were s.c. injected with AOM (15 mg/kg body weight, once weekly for two weeks). Rats continued on experimental diets for seven more weeks before being sacrificed. Colons were histopathologically evaluated for ACF. Human colon cancer HCT-116 and HCA-7 cells treated with omeprazole were evaluated for different markers associated with proliferation and apoptotic markers using Western blot technique. Rats fed with 200 and 400 ppm of omeprazole significantly suppressed total colonic ACF formation (~30%, P<0.001) and showed significant suppression of multi-crypt foci (~30-50%, P<0.05-0.001). Omeprazole produced significant dose-response effects on inhibition of multi-crypt foci (?4). Omeprazole treatment in human colon cancer cell lines HCT-116 and HCA-7 cells resulted in induction of p21waf1/cip1 and decreased the expression of anti-apoptotic proteins Bcl-2, Bcl-XL and survivin in a dose-dependent manner. Anticancer properties observed in colon cancer cell lines suggest that omeprazole may induce key signaling molecules of antiproliferation and inhibition of anti-apoptotic proteins. PMID:21956158

Patlolla, Jagan M R; Zhang, Yuting; Li, Qian; Steele, Vernon E; Rao, Chinthalapally V



Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL  

Microsoft Academic Search

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells

Ramachandran Rashmi; Santhosh Kumar; Devarajan Karunagaran



Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines  

PubMed Central

Introduction Activator protein-2 (AP-2) ? and AP-2? transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity. Methods Ku proteins were identified as AP-2? interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression. Results Nuclear proteins from BT-474 cells overexpressing AP-2? and AP-2? were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2? activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2? on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2? and AP-2? siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2? and AP-2? or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2? and AP-2?. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter. Conclusions Ku proteins in interaction with AP-2 (? and ?) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.



Celecoxib Induced Tumor Cell Radiosensitization by Inhibiting Radiation Induced Nuclear EGFR Transport and DNA-Repair: A COX-2 Independent Mechanism  

SciTech Connect

Purpose: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. Methods and Materials: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by {gamma}H{sub 2}AX foci assay. Results: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observed radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual {gamma}H2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. Conclusions: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.

Dittmann, Klaus H. [Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen (Germany)], E-mail:; Mayer, Claus; Ohneseit, Petra A. [Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen (Germany); Raju, Uma [Department of Experimental Radiation Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX (United States); Andratschke, Nickolaus H. [Klinik und Poliklinik fuer Strahlentherapie und Radiologische Onkologie, Klinikum Rechts Der Isar, Technische Universitaet Muenchen, Munich (Germany); Milas, Luka [Department of Experimental Radiation Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX (United States); Rodemann, H. Peter [Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen (Germany)



Selenium compounds activate ATM-dependent DNA damage responses via the mismatch repair protein hMLH1 in colorectal cancer cells  

Technology Transfer Automated Retrieval System (TEKTRAN)

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR) process. Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells ...


Overexpression of Arginine Transporter CAT-1 Is Associated with Accumulation of L-Arginine and Cell Growth in Human Colorectal Cancer Tissue.  


We previously showed that L-arginine (Arg) accumulates in colorectal cancer tissues. The aim of this study was to investigate the mechanism by which Arg accumulates and determine its biological significance. The concentration of Arg and Citrulline (Cit) in sera and tumor tissues from colorectal cancer (CRC) patients was analyzed by high-performance liquid chromatography (HPLC). The expression of Arg transporters was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis of tissue microarray. We also transfected the colon cancer cell line HCT-116 with siRNA specific for the Arg transporter CAT-1 and measured the induction of apoptosis by flow cytometry and cell proliferation by MTT assay. Consistent with our previous results, serum Arg and Cit concentrations in colorectal cancer patients were significantly lower than those in normal volunteers, while Arg and Cit concentrations in colorectal cancer tissues were significantly higher than in matched adjacent normal colon tissues. Quantitative RT-PCR showed that the CAT-1 gene was highly overexpressed in 70.5% of colorectal cancer tissue samples relative to adjacent normal colon tissues in all 122 patients with colorectal cancer. Immunohistochemical analysis of tissue microarray confirmed that the expression of CAT-1 was higher in all 25 colorectal cancer tissues tested. CAT-1 siRNA significantly induced apoptosis of HCT-116 cells and subsequently inhibited cell growth by 20-50%. Our findings indicate that accumulation of L-Arg and Cit and cell growth in colorectal cancer tissues is associated with over-expression of the Arg transporter gene CAT-1. Our results may be useful for the development of molecular diagnostic tools and targeted therapy for colorectal cancer. PMID:24040099

Lu, Ying; Wang, Weimin; Wang, Junchen; Yang, Chunzhang; Mao, Huiming; Fu, Xuelian; Wu, Yanling; Cai, Jingping; Han, Junyi; Xu, Zengguang; Zhuang, Zhengping; Liu, Zhongmin; Hu, Hai; Chen, Bingguan



Overexpression of Arginine Transporter CAT-1 Is Associated with Accumulation of L-Arginine and Cell Growth in Human Colorectal Cancer Tissue  

PubMed Central

We previously showed that L-arginine (Arg) accumulates in colorectal cancer tissues. The aim of this study was to investigate the mechanism by which Arg accumulates and determine its biological significance. The concentration of Arg and Citrulline (Cit) in sera and tumor tissues from colorectal cancer (CRC) patients was analyzed by high-performance liquid chromatography (HPLC). The expression of Arg transporters was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis of tissue microarray. We also transfected the colon cancer cell line HCT-116 with siRNA specific for the Arg transporter CAT-1 and measured the induction of apoptosis by flow cytometry and cell proliferation by MTT assay. Consistent with our previous results, serum Arg and Cit concentrations in colorectal cancer patients were significantly lower than those in normal volunteers, while Arg and Cit concentrations in colorectal cancer tissues were significantly higher than in matched adjacent normal colon tissues. Quantitative RT-PCR showed that the CAT-1 gene was highly overexpressed in 70.5% of colorectal cancer tissue samples relative to adjacent normal colon tissues in all 122 patients with colorectal cancer. Immunohistochemical analysis of tissue microarray confirmed that the expression of CAT-1 was higher in all 25 colorectal cancer tissues tested. CAT-1 siRNA significantly induced apoptosis of HCT-116 cells and subsequently inhibited cell growth by 20–50%. Our findings indicate that accumulation of L-Arg and Cit and cell growth in colorectal cancer tissues is associated with over-expression of the Arg transporter gene CAT-1. Our results may be useful for the development of molecular diagnostic tools and targeted therapy for colorectal cancer.

Wang, Junchen; Yang, Chunzhang; Mao, Huiming; Fu, Xuelian; Wu, Yanling; Cai, Jingping; Han, Junyi; Xu, Zengguang; Zhuang, Zhengping; Liu, Zhongmin; Hu, Hai; Chen, Bingguan



Binding of the Phage Display Derived Peptide CaIX-P1 on Human Colorectal Carcinoma Cells Correlates with the Expression of Carbonic Anhydrase IX.  


Phage display represents an attractive screening strategy for the identification of novel, specific binding ligands that could be used for tumor targeting. Recently, a new peptide (CaIX-P1) with affinity for human carbonic anhydrase IX (CAIX) was identified and evaluated. The aim of the present study is to characterize the properties of CaIX-P1 for targeting human colorectal carcinoma and investigate the correlation of peptide binding with the expression of carbonic anhydrase IX. Human colorectal carcinoma HCT116 and HT29 cells were investigated for CAIX expression using Western Blot analysis. Binding and competition studies of 125I-radiolabeled CaIX-P1 were performed on HCT116 cells in vitro. FACS analysis and fluorescence microscopy studies were carried out after cell incubation with fluorescein-labeled CaIX-P1 and rhodamine-labeled anti-human CAIX-mAb. Our studies revealed an enhanced in vitro expression of carbonic anhydrase IX in HCT116 and HT29 cells with increasing cell density. Binding of 125I-labeled-CaIX-P1 on HCT116 cells increased with increasing cell density and correlated to the CAIX expression. FACS analysis demonstrated a correlation of cell labeling between FITC-CaIX-P1 and rhodamine-labeled anti-CAIX-mAb in both HCT116 and HT29 cells. The results of our study indicate that the phage display identified peptide CaIX-P1 might be an attractive candidate for the development of a ligand targeting CAIX in colorectal cancer. PMID:23202936

Askoxylakis, Vasileios; Ehemann, Volker; Rana, Shoaib; Krämer, Susanne; Rahbari, Nuh N; Debus, Jürgen; Haberkorn, Uwe



Binding of the Phage Display Derived Peptide CaIX-P1 on Human Colorectal Carcinoma Cells Correlates with the Expression of Carbonic Anhydrase IX  

PubMed Central

Phage display represents an attractive screening strategy for the identification of novel, specific binding ligands that could be used for tumor targeting. Recently, a new peptide (CaIX-P1) with affinity for human carbonic anhydrase IX (CAIX) was identified and evaluated. The aim of the present study is to characterize the properties of CaIX-P1 for targeting human colorectal carcinoma and investigate the correlation of peptide binding with the expression of carbonic anhydrase IX. Human colorectal carcinoma HCT116 and HT29 cells were investigated for CAIX expression using Western Blot analysis. Binding and competition studies of 125I-radiolabeled CaIX-P1 were performed on HCT116 cells in vitro. FACS analysis and fluorescence microscopy studies were carried out after cell incubation with fluorescein-labeled CaIX-P1 and rhodamine-labeled anti-human CAIX-mAb. Our studies revealed an enhanced in vitro expression of carbonic anhydrase IX in HCT116 and HT29 cells with increasing cell density. Binding of 125I-labeled-CaIX-P1 on HCT116 cells increased with increasing cell density and correlated to the CAIX expression. FACS analysis demonstrated a correlation of cell labeling between FITC-CaIX-P1 and rhodamine-labeled anti-CAIX-mAb in both HCT116 and HT29 cells. The results of our study indicate that the phage display identified peptide CaIX-P1 might be an attractive candidate for the development of a ligand targeting CAIX in colorectal cancer.

Askoxylakis, Vasileios; Ehemann, Volker; Rana, Shoaib; Kramer, Susanne; Rahbari, Nuh N.; Debus, Jurgen; Haberkorn, Uwe



Colon Cancer Cell Separation by Dielectrophoresis  

NASA Astrophysics Data System (ADS)

Separation of cancer cells from the other biological cells can be useful for clinical cancer diagnosis and cancer treatment. In this presentation, conventional dielectrophoresis (c-DEP) is used in a microfluidic chip to manipulate and collect colorectal cancer HCT116 cell, which is doped with Human Embryonic Kidney 293 cells (HEK 293). It is noticed that, the HCT116 cell are deflected to a side channel from a main channel clearly by apply electric field at particular AC frequency band. This motion caused by negative DEP can be used to separate the cancer cell from others. In this manuscript, chip design, flow condition, the DEP spectrum of the cancer cell are reported respectively, and the separation and collection efficiency are investigated as well. The sorter is microfabricated using plastic laminate technology. -/abstract- This work has been financially supported by the NSF RII funding (EP

Yang, Fang; Yang, Xiaoming; Jiang, H.; Wood, P.; Hrushesky, W.; Wang, Guiren



Pharmacogenomics of the polyamine analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride, CGC-11093, in the colon adenocarcinoma cell line HCT1161.  


Polyamine analogs are known to inhibit tumorigenesis at least in part by mimicking some of the regulatory roles of natural polyamines. To begin the identification of those signaling pathways that are involved in differential cellular responses to the synthetic conformationally restricted polyamine analog CGC-11093, we conducted gene expression profiling, proteomic, and genome-wide DNA methylation and histone acetylation analyses of the HCT116 colon adenocarcinoma cell line after treatment with this analog. Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. DNA methylation was measured using 6,800 element CpG island microarrays. Treatment of cells with CGC-11093 at concentrations ranging from 0.1 to 10 microM caused inhibition of cell growth and metabolic activity, but only minimally affected cell viability. Gene expression analysis showed concentration-dependent effects of CGC-11093 on the DNA/RNA binding transcription factor, cell cycle, signaling, transport, cytoskeletal/structural, and serine protease genes. Functional gene analysis revealed distinct expression patterns related to inhibition of cell cycle control, TGF beta signaling, proteasome and RNA polymerase pathways, upregulation of the aminoacyl-tRNA synthesis pathway, and perturbations in the MAPK and Wnt signaling pathways. Microarray results were validated for selected genes with real time RT PCR. Proteomics analysis showed correlative changes in the expression of proteins involved in the regulation of proteasome function (proteasome subunit Y) and tRNA synthesis. CGC-11093 treatment did not produce any detectable changes in DNA methylation or histone acetylation in cells. This study validates specific target pathways for a specific conformationally restricted polyamine analog and suggests the utility of combined gene and DNA methylation microarrays along with proteomic analyses as a useful approach to the evaluation of the mechanisms of action of anticancer drugs. PMID:17121431

Ignatenko, Natalia A; Yerushalmi, Hagit F; Watts, George S; Futscher, Bernard W; Stringer, David E; Marton, Laurence J; Gerner, Eugene W



Mouse cell line authentication.  


The scientific community has responded to the misidentification of human cell lines with validated methods to authenticate these cells; however, few assays are available for nonhuman cell line identification. We have developed a multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines. PMID:23430347

Almeida, Jamie L; Hill, Carolyn R; Cole, Kenneth D



Novel herbal flavonoids promote apoptosis but differentially induce cell cycle arrest in human colon cancer cell  

Microsoft Academic Search

Summary  Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus, a medicinal plant that possesses antitumorigenic property. We attempted to compare the anticarcinogenic mechanism of formononetin\\u000a with that of the known proapoptotic flavonoid isoliquiritigenin (ISL) in human cancer cells. We first evaluated the effects\\u000a of formononetin and ISL on HCT 116 colon cancer cell viability. Immunofluorescence staining was then performed

Kathy Ka-Wai Auyeung; Joshua Ka-Shun Ko



Protection of human colon epithelial cells against deoxycholate by rottlerin  

Microsoft Academic Search

The bile salt, deoxycholate (DOC), can harm cells and cause disease. Hence, there is interest in identifying compounds capable\\u000a of protecting cells against DOC. In HCT-116 colon epithelial cells, DOC increased generation of reactive oxygen species and\\u000a caused DNA damage and apoptosis. These effects of DOC were inhibited by rottlerin, which is a phenolic compound of plant origin.\\u000a In elucidating

Jennifer M. Longpre; George Loo



Optimized pregelatinized starch technique for cell block preparation in cell cultures.  


The aim of the present study was to optimize the pregelatinized starch technique for cell block preparation and apply this approach in cultured cells of all types of growing forms, suspension and adherent. In order to evenly mix the starch powder and the cell suspension, we crafted a special plastic dropper. To prove the effectiveness of this optimized technique we used different cell lines, NCI-H69, NCI-H345, HCT-116, SKBR3 and MDA-MB-231. The morphology features, immunocytochemistry (ICC) and fluorescent/chromogenic in-situ hybridization (FISH/CISH) on the cell block sections were evaluated. The morphology features, the ICC and ISH results of cell block sections prepared by the new method were satisfactory comparing with the results obtained in biopsies, the gold standard test for this kind of analysis. The most attractive advantage of our optimized pregelatinized starch technique is that this new method is based on cell suspensions instead of cell sediment, so with our technique every section will contain cells due to the even distribution of the starch powder and the cells forming a homogeneous cell block. To the authors' knowledge, this is the first description on cell block preparation based on cell suspension. PMID:23797005

Zhu, Ya-Zhen; Cui, Feng-Yun; Yang, Yu; Peng, Hui; Li, Wei-Ping; Huang, Zhen-Dong; Zhu, Hong-Guang; He, Qing-Lian; Zheng, Guang-Juan



PRIMA-1 induces autophagy in cancer cells carrying mutant or wild type p53.  


PRIMA-1 is a chemical compound identified as a growth suppressor of tumor cells expressing mutant p53. We previously found that in the MDA-MB-231 cell line expressing high level of the mutant p53-R280K protein, PRIMA-1 induced p53 ubiquitination and degradation associated to cell death. In this study, we investigated the ability of PRIMA-1 to induce autophagy in cancer cells. In MDA-MB-231 and HCT116 cells, expressing mutant or wild type p53, respectively, autophagy occurred following exposure to PRIMA-1, as shown by acridine orange staining, anti-LC3 immunofluorescence and immunoblots, as well as by electron microscopy. Autophagy was triggered also in the derivative cell lines knocked-down for p53, although to a different extent than in the parental cells expressing mutant or wild type p53. In particular, while wild type p53 limited PRIMA-1 induced autophagy, mutant p53 conversely promoted autophagy, thus sustaining cell viability following PRIMA-1 treatment. Therefore, the autophagic potential of PRIMA-1, besides being cell context dependent, could be modulated in a different way by the presence of wild type or mutant p53. Furthermore, since both cell lines lacking p53 were more sensitive to the cytotoxic effect of PRIMA-1 than the parental ones, our findings suggest that a deregulated autophagy may favor cell death induced by this drug. PMID:23545415

Russo, Debora; Ottaggio, Laura; Foggetti, Giorgia; Masini, Matilde; Masiello, Pellegrino; Fronza, Gilberto; Menichini, Paola



Decitabine, a DNA methyltransferases inhibitor, induces cell cycle arrest at G2/M phase through p53-independent pathway in human cancer cells.  


Decitabine (5-aza-2'-deoxycytidine), an inhibitor of DNA methyltransferases, has a wide range of anti-metabolic and anti-cancer activities. Decitabine also induces cell cycle arrest at G2/M phase and apoptosis in human cancer cells. However, the cellular and molecular mechanisms of this cell cycle arrest are poorly understood. In the present study, we investigated the roles of the tumor suppressor p53 and the cyclin-dependent kinase (Cdk) inhibitor p21 following decitabine-induced G2/M arrest in human cancer cells. DNA flow cytometric analyses indicated that decitabine induced a G2/M arrest in AGS gastric and A549 lung carcinoma cell lines, which have wild type p53. Western blot analyses using whole cell lysates from AGS cells demonstrated that decitabine treatment did not change the steady-state level of Cdks and Cdk inhibitor p27, but it partially inhibited expression of cyclin A, cyclin B1, and Cdc25C proteins. However, similar results were found using the A549 cell line, where decitabine induced a dramatic up-regulation of both p53 and p21 expression, and the increased levels of p21 were associated with increased binding of p21 with Cdks, cyclin A, and cyclin B1. Knockdown of p53 by small interfering RNA (siRNA) markedly abolished p53 induction by decitabine in AGS cells, yet p53 siRNA had no attenuating effect on p21 induction. In addition, depletion of p21 expression with siRNA, but not p53, significantly attenuated decitabine-induced G2/M arrest. We also observed that decitabine strongly induced G2/M arrest associated with p21 induction in both p53 allele-null (-/-) HCT116 and wild type p53 (+/+) HCT116 cell lines. Therefore, our data indicated that p21 plays a crucial role in decitabine-induced G2/M arrest and operates in a p53-independent manner. PMID:23582784

Shin, Dong Yeok; Sung Kang, Ho; Kim, Gi-Young; Kim, Wun-Jae; Yoo, Young Hyun; Choi, Yung Hyun



Sorafenib and Vorinostat Kill Colon Cancer Cells by CD95-Dependent and -Independent Mechanisms  

PubMed Central

We examined the interaction between the multikinase inhibitor sorafenib and histone deacetylase inhibitors. Sorafenib and vorinostat synergized (sorafenib + vorinostat) to kill HCT116 and SW480 cells. In SW480 cells, sorafenib + vorinostat increased CD95 plasma membrane levels and promoted death-inducing signal complex (DISC) formation, and drug toxicity was blocked by knockdown of CD95 or overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s). In SW620 cells that are patient-matched to SW480 cells, sorafenib + vorinostat toxicity was significantly lower, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing, whereas knockdown of LASS6 in SW480 cells suppressed CD95 activation. Knocking down LASS6 expression also suppressed CD95 activation in hepatoma, pancreatic, and ovarian cancer cells. In HCT116 cells, sorafenib + vorinostat treatment caused DISC formation without reducing c-FLIP-s expression and did not increase CD95 plasma membrane levels; sorafenib + vorinostat exposure killed HCT116 cells via an intrinsic pathway/caspase 9-dependent mechanism. In HCT116 cells, knockdown of CD95 enhanced sorafenib + vorinostat lethality, which correlated with less drug-induced CD95-dependent autophagy. Sorafenib + vorinostat treatment activated the c-Jun NH2-terminal kinase pathway, which was causal in promoting dissociation of Beclin1 from BCL-2, and in promoting autophagy. Knockdown of Beclin1 expression blocked autophagy and enhanced drug toxicity. Our data demonstrate that treatment of colon cancer cells with sorafenib + vorinostat activates CD95 via de novo ceramide synthesis that promotes viability via autophagy or degrades survival via either the extrinsic or intrinsic pathways.

Walker, Teneille; Mitchell, Clint; Park, Margaret A.; Yacoub, Adly; Graf, Martin; Rahmani, Mohamed; Houghton, Peter J.; Voelkel-Johnson, Christina; Grant, Steven; Dent, Paul



Celecoxib induces p53PUMA pathway for apoptosis in human colorectal cancer cells  

Microsoft Academic Search

Celecoxib, a clinical non-steroidal anti-inflammatory drug, displays anticarcinogenic and chemopreventive activities in human colorectal cancers, although the mechanisms of apoptosis by celecoxib are poorly understood. The existence of functional p53 but not securin in colorectal cancer cells was higher on the induction of cytotoxicity than the p53-mutational colorectal cancer cells following celecoxib treatment. The p53-wild type HCT116 cells were more

Huei-Fang Liu; Po-Wen Hsiao; Jui-I Chao



The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia.  


Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels. PMID:11238001

Rancourt, R C; Keng, P C; Helt, C E; O'Reilly, M A



Hyaluronic acid-functionalized mesoporous silica nanoparticles for efficient photodynamic therapy of cancer cells.  


Mesoporous silica nanoparticles (MSN) for photodynamic therapy (PDT) were coated with poly-(L-lysine) and hyaluronic acid (HA) by using the layer-by-layer method. HA is able to target cancer cells over-expressing the corresponding CD44 receptor. MSN functionalized with HA (MSN-HA) were more efficient than MSN without the targeting moiety when PDT was performed at low fluence (14 Jcm(-2)) and low dosage of MSN (20 ?gmL(-1)) on HCT 116 colorectal cancer cells, known to over-express the CD44 receptor. Incubation of HCT-116 cancer cells with an excess of HA impaired the PDT effect with MSN-HA thus demonstrating that an active endocytosis mechanism was involved in the uptake of MSN-HA by these cells. PMID:22959805

Gary-Bobo, Magali; Brevet, David; Benkirane-Jessel, Nadia; Raehm, Laurence; Maillard, Philippe; Garcia, Marcel; Durand, Jean-Olivier



Krüppel-like factor 4 prevents centrosome amplification following ?-irradiation-induced DNA damage  

Microsoft Academic Search

Centrosome duplication is a carefully controlled process in the cell cycle. Previous studies indicate that the tumor suppressor, p53, regulates centrosome duplication. Here, we present evidence for the involvement of the mammalian Krüppel-like transcription factor, KLF4, in preventing centrosome amplification following DNA damage caused by ?-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+\\/+), displayed

Hong S Yoon; Amr M Ghaleb; Mandayam O Nandan; Irfan M Hisamuddin; William Brian Dalton; Vincent W Yang



Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks  

Microsoft Academic Search

To elucidate the relationship between intragenic DNA methylation and chromatin marks, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC) and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO). Analysis of H3K36me3 profiles indicated that this intragenic mark of active genes is associated with

Maria A. Hahn; Xiwei Wu; Arthur X. Li; Torsten Hahn; Gerd P. Pfeifer; Robert Feil



Wogonin induced G1 cell cycle arrest by regulating Wnt/?-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells.  


Wogonin, a naturally occurring mono-flavonoid, has been reported to have tumor therapeutic potential and good selectivity both in vitro and in vivo. Herein, we investigated the anti-proliferation effects and associated mechanisms of wogonin in human colorectal cancer in vitro. The flow-cytometric analysis showed that wogonin induced a G1 phase cell cycle arrest in HCT116 cells in a concentration- and time-dependent manner. Meanwhile, the cell cycle-related proteins, such as cyclin A, E, D1, and CDK2, 4 were down-regulated in wogonin-induced G1 cell cycle arrest. Furthermore, we showed that the anti-proliferation and G1 arrest effect of wogonin on HCT116 cells was associated with deregulation of Wnt/?-catenin signaling pathway. Wogonin-treated cells showed decreased intracellular levels of Wnt proteins, and activated degradation complex to phosphorylated and targeted ?-catenin for proteasomal degradation. Wogonin inhibited ?-catenin-mediated transcription by interfering in the transcriptional activity of TCF/Lef, and repressing the kinase activity of CDK8 which has been considered as an oncogene involving in the development of colorectal cancers. Moreover, CDK8 siRNA-transfected HCT116 cells showed similar results to wogonin treated cells. Thus, our data suggested that wogonin induced anti-proliferation and G1 arrest via Wnt/?-catenin signaling pathway and it can be developed as a therapeutic agent against human colorectal cancer. PMID:23907061

He, Licheng; Lu, Na; Dai, Qinsheng; Zhao, Yue; Zhao, Li; Wang, Hu; Li, Zhiyu; You, Qidong; Guo, Qinglong



c-Jun N-terminal kinase 1 is required for cordycepin-mediated induction of G2\\/M cell-cycle arrest via p21WAF1 expression in human colon cancer cells  

Microsoft Academic Search

Cordycepin (3?-deoxyadenosine) has many anti-cancer properties. However, neither its molecular mechanism nor its molecular targets are well understood. In the present study, we investigated novel molecular mechanisms for the anti-tumor effects of cordycepin in human colon cancer HCT116 cells. After treatment of cells with cordycepin, dose-dependent cell growth inhibition was observed at an IC50 value of 200?M. Cordycepin treatment resulted

Se-Jung Lee; Gi-Seong Moon; Kyung-Hwan Jung; Wun-Jae Kim; Sung-Kwon Moon



Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo  

PubMed Central

Human LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is the 14th member of the tumor necrosis factor (TNF) superfamily and is therefore also known as TNFSF14. LIGHT has been proven to be a multifunctional molecule affecting cell proliferation, differentiation and a number of other biological processes, in particular, cell growth inhibition. However, the expression and molecular mechanisms of the LIGHT gene in human colorectal carcinoma cells remain largely unclear. In the present study, the LIGHT gene was overexpressed using a lentiviral expression vector in HCT116 human colorectal carcinoma cells in vitro and in vivo, in order to explore the mechanism by which the LIGHT gene inhibits cell growth and suppresses tumor formation. The results showed that the recombinant lentivirus with LIGHT overexpression inhibited the proliferative capacity of the HCT116 cells and significantly decreased the xenografted tumor volumes in nude mice. Furthermore, LIGHT treatment effectively initiated increased caspase-3 and decreased Bcl-2 activities in the HCT116 cells. This study provides a basis for the improved understanding of the role and molecular mechanisms of the LIGHT gene in human colorectal carcinoma cells and may facilitate further functional studies of LIGHT.




Antitumor activity of 2-hydroxycinnamaldehyde for human colon cancer cells through suppression of ?-catenin signaling.  


The antiproliferative and antitumor activities of 2-hydroxycinnamaldehyde (1), a phenylpropanoid isolated from the bark of Cinnamomum cassia, were investigated using human colorectal cancer cells. Compound 1 exhibited antiproliferative effects in HCT116 colon cancer cells, accompanied by modulation of the Wnt/?-catenin cell signaling pathway. This substance was found also to inhibit ?-catenin/T-cell factor (TCF) transcriptional activity in HEK293 cells and HCT116 colon cancer cells. Further mechanistic investigations in human colon cancer cells with aberrantly activated Wnt/?-catenin signaling showed that 1 significantly suppressed the binding of ?-catenin/TCF complexes to their specific genomic targets in the nucleus and led to the down-regulation of Wnt target genes such as c-myc and cyclin D1. In an in vivo xenograft model, the intraperitoneal administration of 1 (10 or 20 mg/kg body weight, three times/week) for four weeks suppressed tumor growth in athymic nude mice implanted with HCT116 colon cancer cells significantly, without any apparent toxicity. In an ex vivo biochemical analysis of the tumors, compound 1 was also found to suppress Wnt target genes associated with tumor growth including ?-catenin, c-myc, cyclin D1, and survivin. The suppression of the Wnt/?-catenin signaling pathway is a plausible mechanism of action underlying the antiproliferative and antitumor activity of 1 in human colorectal cancer cells. PMID:23855266

Lee, Min Ai; Park, Hyen Joo; Chung, Hwa-Jin; Kim, Won Kyung; Lee, Sang Kook



The PTEN Phosphatase Controls Intestinal Epithelial Cell Polarity and Barrier Function: Role in Colorectal Cancer Progression  

Microsoft Academic Search

BackgroundThe PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.Methodology\\/Principal FindingsCaco-2\\/15, HCT116

Marie-Josée Langlois; Sébastien Bergeron; Gérald Bernatchez; François Boudreau; Caroline Saucier; Nathalie Perreault; Julie C. Carrier; Nathalie Rivard; Hang Thi Thu Nguyen



Colonic luminal contents induce cyclooxygenase 2 transcription in human colon carcinoma cells  

Microsoft Academic Search

Background & Aims: Evidence is accumulating that inhibitors of cyclooxygenase (COX)-2 activity are useful for preventing human colon cancer. Therefore, it is important to determine whether agents in the colonic luminal contents can influence the transcriptional regulation of COX-2 in colonic cells.Methods: Transient transfections were performed, using a human COX-2 promoter-luciferase construct, in HCT 116 cells, and the effects of

Bjorn Glinghammar; Joseph Rafter



The Interaction between Two Radiosensitizers: 5Iododeoxyuridine and Caffeine  

Microsoft Academic Search

Iododeoxyuridine (IUdR) and caffeine are recognized as potential radiosensitizers with different mechanisms of interaction with ionizing radiation (IR). To assess the interaction of these two types of radiosensitizers, we com- pared treatment responses to these drugs alone and in combination with IR in two p53-proficient and p53-deficient pairs of human colon cancer cell lines (HCT116 versus HCT116 p53? \\/? and

Yuji Seo; Jane E. Schupp; Kazuhiko Yamane; Tomas Radivoyevitch; Timothy J. Kinsella



Induction of apoptosis and suppression of ERCC1 expression by the potent amonafide analogue 8-c in human colorectal carcinoma cells.  


Previous studies have reported that 8-c [6-(2-(2-(dimethylamino)ethylamino)ethylamino)-2-octyl-1H-benzo[de]isoquinoline-1,3(2H)-dione], a novel amonafide analogue, was generated as a new anticancer candidate. However, little is known about its activity in chemoresistant cells. In this study, the antitumor effects of 8-c on the multi-drug-resistant human colorectal carcinoma cancer cell lines HCT-116/L-OHP and HCT-8/VCR have been investigated for the first time. 8-c showed similar concentration-dependent inhibitory activities against multi-drug-resistant cells and corresponding parental cell lines by the MTT assay after 48 h of treatment. 8-c treatment resulted in the induction of apoptosis, as evidenced by fluorescent staining analysis, comet assay data, and the increase in the number of apoptotic cells as detected by flow cytometry. Western blot, qPCR, and siRNA techniques were used to elucidate the molecular mechanism. Our study suggested that the apoptotic effect of 8-c can be attributed to the upregulation of p53, caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) and the downregulation of Bcl-2. Furthermore, ERCC1 is essential for nucleotide excision repair. ERCC1 expression was correlated with sensitivity to chemotherapy in various colon cancer cell lines. It is intriguing that decreases in ERCC1 protein and mRNA levels were also observed in the HCT-116/L-OHP and HCT-8/VCR cells after exposure to 8-c. Further transient transfection of multi-drug-resistant cells with ERCC1 siRNA enhanced 8-c-induced cytotoxicity. In contrast, epidermal growth factor-induced increase in ERCC1 protein levels was shown to rescue cell viability upon 8-c treatment. These findings suggest that 8-c has a strong potential to be developed as a new antitumor agent for the treatment of multi-drug-resistant colon cancer cells, and is worthy of further studies. PMID:23426174

Wang, Ziyuan; Liang, Xin; Cheng, Zhuoan; Xu, Yufang; Yin, Peihao; Zhu, Huirong; Li, Qi; Qian, Xuhong; Liu, Jianwen



PAMAM-Camptothecin Conjugate Inhibits Proliferation and Induces Nuclear Fragmentation in Colorectal Carcinoma Cells  

PubMed Central

Purpose To synthesize and characterize a poly (amido amine) dendrimer-camptothecin (PAMAM-CPT) conjugate and evaluate its activity on human colorectal carcinoma cells (HCT-116). Methods The attachment of CPT to amine-terminated PAMAM was facilitated through a succinic acid-glycine linker. The conjugate was characterized for absence of small molecular weight impurities, size and drug content. Stability of the conjugate in PBS and growth media and its in vitro activity on HCT-116 were studied. Cell cycle arrest and nuclear fragmentation upon PAMAM-CPT treatment were investigated. Results The conjugate was stable under physiological pH (7.4) in PBS and in growth media (with 10% FBS) with minimal release of 4% and 6% drug, respectively, at 48 h. PAMAM-CPT inhibited proliferation of HCT-116 cells with an IC50 value of 1.6±0.3 ?M. The conjugate induced signs of cell cycle arrest with up to 68% of cells blocked in the G2 phase. Confocal images of cells treated with PAMAM-CPT suggest nuclear fragmentation and formation of apoptotic bodies. Conclusions Results show that the PAMAM-CPT conjugate was active against colorectal cancer cells in vitro, inhibiting their growth and inducing nuclear fragmentation. Coupled with the ability to target macromolecular therapeutics to tumors, this conjugate shows promise for cancer chemotherapy.

Thiagarajan, Giridhar; Ray, Abhijit; Malugin, Alexander; Ghandehari, Hamidreza



Proteasome inhibitors MG-132 and bortezomib induce AKR1C1, AKR1C3, AKR1B1, and AKR1B10 in human colon cancer cell lines SW-480 and HT-29.  


Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1-1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1-1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the phenomenon of multidrug resistance upon cancer chemotherapy. PMID:21215737

Ebert, Bettina; Kisiela, Michael; Wsól, Vladimir; Maser, Edmund



Impact of p53 Knockout and Topotecan Treatment on Gene Expression Profiles in Human Colon Carcinoma Cells: A Pharmacogenomic Study  

Microsoft Academic Search

To uncover transcriptional stress responses related to p53, we used cDNA microarrays (National Cancer Institute Oncochips comprising 6500 different genes) to characterize the gene expression profiles of wild-type p53 HCT-116 cells and an isogenic p53 knockout counterpart after treat- ment with topotecan, a specific topoisomerase I inhibitor. The use of the p53 knockout cells had the advantage over p53-overexpressing systems

Sayed S. Daoud; Peter J. Munson; William Reinhold; Lynn Young; Vinay V. Prabhu; Qiang Yu; Jihyun LaRose; Kurt W. Kohn; John N. Weinstein; Yves Pommier



Dual-Color Imaging of Nuclear-Cytoplasmic Dynamics, Viability, and Proliferation of Cancer Cells in the Portal Vein Area  

Microsoft Academic Search

We used dual-color in vivo cellular imaging to visualize trafficking, nuclear-cytoplasmic dynamics, and the viability of cancer cells after their injection into the portal vein of mice. For these studies, we used dual-color fluorescent cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and retroviral red fluorescent protein (RFP) in the cytoplasm. Human HCT-116-GFP-RFP

Kazuhiko Tsuji; Kensuke Yamauchi; Meng Yang; Ping Jiang; Michael Bouvet; Hitoshi Endo; Yoshikatsu Kanai; Koji Yamashita; Abdool R. Moossa


Synthesis and inhibitory activities against colon cancer cell growth and proteasome of alkylresorcinols.  


We have identified alkylresorcinols (ARs) as the major active components in wheat bran against human colon cancer cell growth (HCT-116 and HT-29) using a bioassay-guided approach. To further study the structure-activity relationships, 15 ARs and their intermediates (1-15) were synthesized expediently by the modified Wittig reaction in aqueous media, and six 5-alkylpyrogallols and their analogues (16-21) were prepared by the general Grignard reaction. The synthetic AR analogues were evaluated for activities against the growth of human colon cancer cells HCT-116 and HT-29 and the chymotrypsin-like activity of the human 20S proteasome. Our results found that (1) AR C13:0 and C15:0 (13 and 14) had the greatest inhibitory effects in human colon cancer cells HCT-116 and HT-29, while decreasing or increasing the side chain lengths diminished the activities; (2) two free meta-hydroxyl groups at C-1 and C-3 on the aromatic ring of the AR analogues greatly contributed to their antitumor activity; (3) the introduction of a third hydroxyl group at C-2 (20 and 21) into the aromatic ring of the AR analogues yielded no significant enhancement in activity against HCT-116 cells and decimated the effects against HT-29 cells, but dramatically increased the activity against the chymotrypsin-like activity of the human 20S proteasome; and (4) AR C11:0 (12) was found to have the greatest effect in a series of AR C9:0-C17:0 against the chymotrypsin-like activity of the human 20S proteasome. PMID:22897570

Zhu, Yingdong; Soroka, Dominique N; Sang, Shengmin



Encapsulation of selenium in chitosan nanoparticles improves selenium availability and protects cells from selenium-induced DNA damage response.  


Selenium, an essential mineral, plays important roles in optimizing human health. Chitosan (CS) is an effective, naturally oriented material for synthesizing nanoparticles with preferable properties such as biocompatibility, biodegradation and resistance to certain enzymes. We have recently shown that cellular exposure to selenium compounds activates ataxia-telangiectasia mutated (ATM)-dependent DNA damage responses, a tumorigenesis barrier. To test whether nanoencapsulation of selenium modulates the cellular response to selenium compounds, the HCT 116 cancerous and the MRC-5 normal cells were treated with Na(2)SeO(3) and methylseleninic acid (MSeA) encapsulated in CS/polyphosphate nanoparticles. Analyses of cellular selenium levels demonstrate that (1) the nanoencapsulation enhances selenium levels in cells after exposure to Na(2)SeO(3) and MSeA (1-10 ?M); (2) cells retained more selenium when treated with Na(2)SeO(3) than with MSeA; (3) selenium levels are greater in HCT 116 than in MRC-5 cells after Na(2)SeO(3), but not MSeA, exposure. Survival analysis shows that CS encapsulation desensitizes HCT 116 and MRC-5 cells to Na(2)SeO(3) or MSeA exposure. Immunofluorescent analysis demonstrates that CS encapsulation attenuates the selenium-induced ATM phosphorylation on Ser-1981, and the extent is greater in HCT 116 than in MRC-5 cells. Our results reveal features of selenium nanoencapsulation in CS, including increased selenium retention in cells and decreased cellular sensitivity and DNA damage response to selenium exposure. PMID:21292467

Zhang, Shu; Luo, Yangchao; Zeng, Huawei; Wang, Qin; Tian, Fei; Song, Jiuzhou; Cheng, Wen-Hsing



Momordica charantia Extract Induces Apoptosis in Human Cancer Cells through Caspase- and Mitochondria-Dependent Pathways.  


Plants are an invaluable source of potential new anti-cancer drugs. Momordica charantia is one of these plants with both edible and medical value and reported to exhibit anticancer activity. To explore the potential effectiveness of Momordica charantia, methanol extract of Momordica charantia (MCME) was used to evaluate the cytotoxic activity on four human cancer cell lines, Hone-1 nasopharyngeal carcinoma cells, AGS gastric adenocarcinoma cells, HCT-116 colorectal carcinoma cells, and CL1-0 lung adenocarcinoma cells, in this study. MCME showed cytotoxic activity towards all cancer cells tested, with the approximate IC(50) ranging from 0.25 to 0.35?mg/mL at 24 h. MCME induced cell death was found to be time-dependent in these cells. Apoptosis was demonstrated by DAPI staining and DNA fragmentation analysis using agarose gel electrophoresis. MCME activated caspase-3 and enhanced the cleavage of downstream DFF45 and PARP, subsequently leading to DNA fragmentation and nuclear condensation. The apoptogenic protein, Bax, was increased, whereas Bcl-2 was decreased after treating for 24?h in all cancer cells, indicating the involvement of mitochondrial pathway in MCME-induced cell death. These findings indicate that MCME has cytotoxic effects on human cancer cells and exhibits promising anti-cancer activity by triggering apoptosis through the regulation of caspases and mitochondria. PMID:23091557

Li, Chia-Jung; Tsang, Shih-Fang; Tsai, Chun-Hao; Tsai, Hsin-Yi; Chyuan, Jong-Ho; Hsu, Hsue-Yin



Momordica charantia Extract Induces Apoptosis in Human Cancer Cells through Caspase- and Mitochondria-Dependent Pathways  

PubMed Central

Plants are an invaluable source of potential new anti-cancer drugs. Momordica charantia is one of these plants with both edible and medical value and reported to exhibit anticancer activity. To explore the potential effectiveness of Momordica charantia, methanol extract of Momordica charantia (MCME) was used to evaluate the cytotoxic activity on four human cancer cell lines, Hone-1 nasopharyngeal carcinoma cells, AGS gastric adenocarcinoma cells, HCT-116 colorectal carcinoma cells, and CL1-0 lung adenocarcinoma cells, in this study. MCME showed cytotoxic activity towards all cancer cells tested, with the approximate IC50 ranging from 0.25 to 0.35?mg/mL at 24 h. MCME induced cell death was found to be time-dependent in these cells. Apoptosis was demonstrated by DAPI staining and DNA fragmentation analysis using agarose gel electrophoresis. MCME activated caspase-3 and enhanced the cleavage of downstream DFF45 and PARP, subsequently leading to DNA fragmentation and nuclear condensation. The apoptogenic protein, Bax, was increased, whereas Bcl-2 was decreased after treating for 24?h in all cancer cells, indicating the involvement of mitochondrial pathway in MCME-induced cell death. These findings indicate that MCME has cytotoxic effects on human cancer cells and exhibits promising anti-cancer activity by triggering apoptosis through the regulation of caspases and mitochondria.

Li, Chia-Jung; Tsang, Shih-Fang; Tsai, Chun-Hao; Tsai, Hsin-Yi; Chyuan, Jong-Ho; Hsu, Hsue-Yin



An efficient method to produce clonal colonies of cancer cells using laser enabled analysis and processing (LEAP)  

NASA Astrophysics Data System (ADS)

Many in vitro studies require a pure clonal population of cells that derive from a single cell. Traditionally this task has been performed using the inefficient manual process of ultimate limiting dilution. We have developed a novel clonal dilution technique using the Laser Enabled Analysis and Processing (LEAPTM) instrument (Cyntellect Inc. San Diego, CA). The LEAP instrument performs automated fluorescence imaging and real time image analysis to identify and measure fluorescence and morphological parameters of cells. The LEAP instrument also features a laser that can be used to manipulate targeted cells. To perform clonal dilution, cells are seeded at a low density (~10 cells/well) into each well of a 384 well plate and viably stained. The LEAP instrument will then image each well and automatically target all of the cells that are present. Then one cell will be chosen to keep (at random or based on a variety of metrics) and the others will be eliminated by laser ablation. We have successfully used this technique to produce single cell clones of HCT116 cells, a heterogeneous colorectal cancer model, in 84 percent of wells (originally containing 5 +/- 2.1 cells/well). This is a marked improvement over the traditional technique of ultimate limiting dilution which produces a clone in only 33 percent of wells. The ability to efficiently produce clonal colonies has great utility in the isolation of subpopulations of cancer cells and purification of transformed cell lines.

Zordan, Michael; Fatig, Ray; Reece, Lisa; Davisson, V. Jo; Leary, James



Silencing of PTK7 in Colon Cancer Cells: Caspase10Dependent Apoptosis via Mitochondrial Pathway  

Microsoft Academic Search

Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference

Ling Meng; Kwame Sefah; Meghan B. O'Donoghue; Guizhi Zhu; Dihua Shangguan; Afshan Noorali; Yan Chen; Lei Zhou; Weihong Tan; Zhongjun Zhou



Contribution of HIF-1 and drug penetrance to oxaliplatin resistance in hypoxic colorectal cancer cells  

Microsoft Academic Search

Background:Hypoxia is as an indicator of poor treatment outcome. Consistently, hypoxic HCT116 colorectal cancer cells are resistant to oxaliplatin, although the mechanistic basis is unclear. This study sought to investigate the relative contribution of HIF-1 (hypoxia-inducible factor-1)-mediated gene expression and drug penetrance to oxaliplatin resistance using three-dimensional spheroids.Methods:Hypoxia-inducible factor-1? function was suppressed by the stable expression of a dominant-negative form

D L Roberts; K J Williams; R L Cowen; M Barathova; A J Eustace; S Brittain-Dissont; M J Tilby; D G Pearson; C J Ottley; I J Stratford; C Dive



Metabolism of [6]-shogaol in mice and in cancer cells.  


Ginger has received extensive attention because of its antioxidant, anti-inflammatory, and antitumor activities. However, the metabolic fate of its major components is still unclear. In the present study, the metabolism of [6]-shogaol, one of the major active components in ginger, was examined for the first time in mice and in cancer cells. Thirteen metabolites were detected and identified, seven of which were purified from fecal samples collected from [6]-shogaol-treated mice. Their structures were elucidated as 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 5-methoxy-1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M7), 3',4'-dihydroxyphenyl-decan-3-one (M8), 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-ol (M9), 5-methylthio-1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M10), 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), and 5-methylthio-1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-ol (M12) on the basis of detailed analysis of their (1)H, (13)C, and two-dimensional NMR data. The rest of the metabolites were identified as 5-cysteinyl-M6 (M1), 5-cysteinyl-[6]-shogaol (M2), 5-cysteinylglycinyl-M6 (M3), 5-N-acetylcysteinyl-M6 (M4), 5-N-acetylcysteinyl-[6]-shogaol (M5), and 5-glutathiol-[6]-shogaol (M13) by analysis of the MS(n) (n = 1-3) spectra and comparison to authentic standards. Among the metabolites, M1 through M5, M10, M12, and M13 were identified as the thiol conjugates of [6]-shogaol and its metabolite M6. M9 and M11 were identified as the major metabolites in four different cancer cell lines (HCT-116, HT-29, H-1299, and CL-13), and M13 was detected as a major metabolite in HCT-116 human colon cancer cells. We further showed that M9 and M11 are bioactive compounds that can inhibit cancer cell growth and induce apoptosis in human cancer cells. Our results suggest that 1) [6]-shogaol is extensively metabolized in these two models, 2) its metabolites are bioactive compounds, and 3) the mercapturic acid pathway is one of the major biotransformation pathways of [6]-shogaol. PMID:22246389

Chen, Huadong; Lv, Lishuang; Soroka, Dominique; Warin, Renaud F; Parks, Tiffany A; Hu, Yuhui; Zhu, Yingdong; Chen, Xiaoxin; Sang, Shengmin



Maple polyphenols, ginnalins A-C, induce S- and G2/M-cell cycle arrest in colon and breast cancer cells mediated by decreasing cyclins A and D1 levels.  


Polyphenols are bioactive compounds found in plant foods. Ginnalins A-C are polyphenols present in the sap and other parts of the sugar and red maple species which are used to produce maple syrup. Here we evaluated the antiproliferative effects of ginnalins A-C on colon (HCT-116) and breast (MCF-7) tumourigenic and non-tumourigenic colon (CCD-18Co) cells and investigated whether these effects were mediated through cell cycle arrest and/or apoptosis. Ginnalins A-C were twofold more effective against the tumourigenic than non-tumourigenic cells. Among the polyphenols, ginnalin A (84%, HCT-116; 49%, MCF-7) was more effective than ginnalins B and C (50%, HCT-116; 30%, MCF-7) at 50 ?M concentrations. Ginnalin A did not induce apoptosis of the cancer cells but arrested cell cycle (in the S- and G(2)/M-phases) and decreased cyclins A and D1 protein levels. These results suggest that maple polyphenols may have potential cancer chemopreventive effects mediated through cell cycle arrest. PMID:23122108

González-Sarrías, Antonio; Ma, Hang; Edmonds, Maxwell E; Seeram, Navindra P



Anti-tumor activity of ESX1 on cancer cells harboring oncogenic K-ras mutation  

SciTech Connect

Human ESX1 is a 65-kilodalton (kDa) paired-like homeoprotein that is proteolytically processed into N-terminal 45-kDa and C-terminal 20-kDa fragments. The N-terminal ESX1 fragment, which contains the homeodomain, localizes to the nucleus and represses mRNA transcription from the K-ras gene. When we inoculated human colorectal carcinoma HCT116 constitutive expressing N-terminal region of ESX1 (N-ESX1) into nude mice, transfectant cells uniformly showed decreased tumor-forming activity compared with that of the parental cells. Furthermore, pretreatment of HCT116 carcinoma cells with a fusion protein consisting of N-ESX1 and the protein-transduction domain derived from the human immunodeficiency virus type-1 TAT protein gave rise to a dramatic reduction in the tumorigenicity of HCT116 cells in nude mice. Our results provide first in vivo evidence for the molecular targeting therapeutic application of the K-ras repressor ESX1, especially TAT-mediated transduction of N-ESX1, in the treatment of human cancers having oncogenic K-ras mutations.

Nakajima, Junta; Ishikawa, Susumu [Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815 (Japan); Hamada, Jun-Ichi [Division of Cancer-related Genes, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815 (Japan); Yanagihara, Masatomo [Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815 (Japan); Koike, Takao [Department of Medicine II, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638 (Japan); Hatakeyama, Masanori [Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815 (Japan)], E-mail:



Knockdown of PAK4 or PAK1 inhibits the proliferation of mutant KRAS colon cancer cells independently of RAF/MEK/ERK and PI3K/AKT signaling.  


The p21-activated kinase (PAK) serine/threonine kinases are important effectors of the small GTPases Rac and Cdc42, and play significant roles in controlling cell growth, motility, and transformation. Knockdown of PAK4 or PAK1 inhibited the proliferation of mutant KRAS or BRAF colon cancer cells in vitro. Dependence on PAK4 or PAK1 protein for colon cancer cell proliferation was independent of PAK4 or PAK1 protein expression levels. Mutant KRAS HCT116 colorectal cells were the most sensitive to PAK4 or PAK1 knockdown resulting in the potent inhibition of anchorage-dependent and -independent proliferation as well as the formation and proliferation of HCT116 colon cancer spheroids. This inhibition of proliferation did not correlate with inhibition of RAF/MEK/ERK or PI3K/AKT signaling. In HCT116 cells, knockdown of PAK4 or PAK1 caused changes to the actin cytoskeleton resulting in reduced basal spread and cell elongation and increased cell rounding. These cytoskeletal rearrangements seemed to be independent of LIMK/cofilin/paxillin phosphorylation. PAK4 or PAK1 knockdown initially induced growth arrest in HCT116 cells followed by cell death at later time points. Inhibition of the antiapoptotic proteins Bcl-2 and Bcl-X(L) with the pharmacologic inhibitor ABT-737 increased effector caspase activation and apoptosis, and reduced cell survival with PAK4 or PAK1 knockdown. These results support a role for the PAKs in the proliferation of mutant KRAS-driven colorectal carcinoma cells via pathways not involving RAF/MEK/ERK and PI3K/AKT signaling. PMID:23233484

Tabusa, Hana; Brooks, Teresa; Massey, Andrew J



Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice  

NASA Astrophysics Data System (ADS)

We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

Hoffman, Robert M.



Antiproliferative activity and induction of apoptosis in human colon cancer cells treated in vitro with constituents of a product derived from Pistacia lentiscus L. var. chia.  


In this report, we demonstrate that a 50% ethanol extract of the plant-derived product, Chios mastic gum (CMG), contains compounds which inhibit proliferation and induce death of HCT116 human colon cancer cells in vitro. CMG-treatment induces cell arrest at G(1), detachment of the cells from the substrate, activation of pro-caspases-8, -9 and -3, and causes several morphological changes typical of apoptosis in cell organelles. These events, furthermore, are time- and dose-dependent, but p53- and p21-independent. Apoptosis induction by CMG is not inhibited in HCT116 cell clones expressing high levels of the anti-apoptotic protein, Bcl-2, or dominant-negative FADD, thereby indicating that CMG induces cell death via a yet-to-be identified pathway, unrelated to the death receptor- and mitochondrion-dependent pathways. The findings presented here suggest that CMG (a) induces an anoikis form of cell death in HCT116 colon cancer cells that includes events associated with caspase-dependent pathways; and (b) might be developed into a chemotherapeutic agent for the treatment of human colon and other cancers. PMID:16713222

Balan, K V; Prince, J; Han, Z; Dimas, K; Cladaras, M; Wyche, J H; Sitaras, N M; Pantazis, P



MSH3-Deficiency Initiates EMAST without Oncogenic Transformation of Human Colon Epithelial Cells  

PubMed Central

Background/Aim Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. Methods HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay. Results Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10?4) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. Conclusions MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.

Campregher, Christoph; Schmid, Gerald; Ferk, Franziska; Knasmuller, Siegfried; Khare, Vineeta; Kortum, Benedikt; Dammann, Kyle; Lang, Michaela; Scharl, Theresa; Spittler, Andreas; Roig, Andres I.; Shay, Jerry W.; Gerner, Christopher; Gasche, Christoph



p53 interferes with microtubule-stabilizing agent-induced apoptosis in prostate and colorectal cancer cells.  


Taxanes are microtubule-stabilizing agents that have anticancer activity against several types of human solid tumors. Although the primary mechanism of action of these drugs is well understood, the signaling pathways that confer resistance to these agents in certain types of cancer remain poorly understood. In particular, the association of p53 with the mechanism(s) of taxane-mediated cell death is still controversial. In this study, we showed that p53 has a profound inhibitory effect on docetaxel (Doc)-induced apoptosis in prostate and colorectal cancer cells and that caspases play a critical role in this process. Doc induced prostate cancer cell apoptosis at high levels in p53-null PC3 cells, at intermediate levels in p53-mutant DU145 cells and at low levels in p53 wild-type LNCaP cells. While transient overexpression of p53 in PC3 cells suppressed Doc-induced apoptosis, knockdown of p53 in LNCaP cells increased apoptosis. This finding was further confirmed using an isogenic pair of colorectal cancer cell lines, HCT-116 p53-/- and p53+/+, indicating that p53 inhibits induction of apoptosis by Doc. To our knowledge, this is the first report describing that chemical or genetic knockout of p53 enhances the susceptibility of both prostate and colorectal cancer cells to Doc-induced apoptosis. These results may suggest an approach to stratify patients for regimens involving Doc. PMID:23563592

Kim, Ji Young; Chung, Jin-Yong; Lee, Seung Gee; Kim, Yoon-Jae; Park, Ji-Eun; Yun, Jeanho; Park, Young Chul; Kim, Byeong Gee; Yoo, Young Hyun; Kim, Jong-Min



Effect of differentiation agents on C-MYC expression in transformed fibroblasts and in human colon carcinoma cells  

SciTech Connect

Previous work in this laboratory indicated that growth of methylcholanthrene-transformed AKR-2B fibroblasts (AKR-MCA), as well as certain human colon carcinoma lines on retinoic acid (RA) or N,N-dimethylformamide (DMF) resulted in a more differentiated phenotype. Moreover, morphological transformation of AKR-2B cells by transforming growth factor-..beta.. was blocked by simultaneous addition of DMF. In the present work Northern transfer analysis of total cellular RNA was utilized to determine the effect of these differentiation agents (DA) on c-myc expression. Expression of the c-myc proto-oncogene was elevated 2 to 4 fold in AKR-MCA cells relative to untransformed AKR-2B cells. 1.0% DMF resulted in a decrease in c-myc expression in AKR-MCA cells to a level at or below that in AKR-MCA cells. RA treatment (1.0 of AKR-MCA cells reduced c-myc expression to a level intermediate between AKR-MCA and AKR-2B cells. Similarly, growth of human colon carcinoma cells (HCT 116 and MOSER) on 0.5% DMF or 1.0 RA resulted in a greater reduction of c-myc expression by DMF than by RA. These DA's may act by inhibition of oncogene-mediated cell responsiveness to positive autocrine growth factors, or alternatively, by stimulation of oncogene-mediated responsiveness to growth inhibitory factors.

Mulder, K.; Rickling, S.; Levine, A.; Brattain, M.



Synthetic Lethality by Lentiviral Short Hairpin RNA Silencing of Thymidylate Kinase and Doxorubicin in Colon Cancer Cells Regardless of the p53 Status  

Microsoft Academic Search

Intracellular supply of dTTP is a highly regulated process and has been a key target for chemotherapeutic drug development. Thymidylate kinase (TMPK) is the key enzyme for dTTP formation in both de novo and salvage pathways. In this study,we used lentiviral-based small hairpin RNA to silence TMPK expression in p53(+\\/+) and p53(\\/) HCT-116 colon cancer cells. This approach was sufficient

Chun-Mei Hu; Zee-Fen Chang



KLF4 gene expression is inhibited by the notch signaling pathway that controls goblet cell differentiation in mouse gastrointestinal tract.  


In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a gamma-secretase inhibitor (compound E, 10 micromol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract. PMID:19109406

Zheng, Hai; Pritchard, D Mark; Yang, Xiangdong; Bennett, Elaine; Liu, Gang; Liu, Chunming; Ai, Walden



REST-dependent expression of TRF2 renders non-neuronal cancer cells resistant to DNA damage during oxidative stress.  


REST is a neuronal gene silencing factor ubiquitously expressed in non-neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non-neuronal cancers. Conversely, recent findings on REST-dependent tumorigenesis in non-neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non-neuronal cancers. We observed abundant expression of REST in various types of non-neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non-neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere-binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down-regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST-dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress-resistant REST mutants found in specific human cancers, may account for REST-dependent tumorigenesis. PMID:22821339

Kwon, Jung-Hee; Shin, Ji Hye; Kim, Eung-Sam; Lee, Namgyu; Park, Jin Young; Koo, Bonik Samuel; Hong, Sun Mi; Park, Chang Wook; Choi, Kwan Yong



Serum-nutrient starvation induces cell death mediated by Bax and Puma that is counteracted by p21 and unmasked by Bcl-x(L) inhibition.  


The cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) is a multifunctional protein known to promote cell cycle arrest and survival in response to p53-dependent and p53 independent stimuli. We herein investigated whether and how it might contribute to the survival of cancer cells that are in low-nutrient conditions during tumour growth, by culturing isogenic human colorectal cancer cell lines (HCT116) and breast cancer cell lines in a medium deprived in amino acids and serum. We show that such starvation enhances, independently from p53, the expression of p21 and that of the pro-apoptotic BH3-only protein Puma. Under these conditions, p21 prevents Puma and its downstream effector Bax from triggering the mitochondrial apoptotic pathway. This anti-apoptotic effect is exerted from the cytosol but it is unrelated to the ability of p21 to interfere with the effector caspase 3. The survival function of p21 is, however, overcome by RNA interference mediated Bcl-x(L) depletion, or by the pharmacological inhibitor ABT-737. Thus, an insufficient supply in nutrients may not have an overt effect on cancer cell viability due to p21 induction, but it primes these cells to die, and sensitizes them to the deleterious effects of Bcl-x(L) inhibitors regardless of their p53 status. PMID:21887277

Braun, Frédérique; Bertin-Ciftci, Joséphine; Gallouet, Anne-Sophie; Millour, Julie; Juin, Philippe



Ovarian granulosa cell lines  

Microsoft Academic Search

The ovary is a complex endocrine gland responsible for production of sex steroids and is the source of fertilizable ova for reproduction. It also produces various growth factors, transcription factors and cytokines that assist in the complex signaling pathways of folliculogenesis. The ovary possesses two primary steroidogenic cell types. The theca cells (and to a lesser extent, the stroma) are

Jon C. Havelock; William E. Rainey; Bruce R. Carr



Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3sigma and CDKN1A (p21) knockout cell lines.  


Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from approximately 15 h to approximately 3- 5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from approximately 18 h to approximately 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i.e. metabolic activity. Thus mitotic catastrophe itself is not a direct mode of death. Instead, apoptosis during interphase of both uninucleated and polyploid cells was the primary mode of death observed in the four cell types. Knocking out either CDKN1A or 14-3-3sigma increased the amount of cell death at 96 h, from 52% to approximately 70%, with an even greater increase to 90% when both genes were knocked out. Thus, in addition to effects of CDKN1A and 14-3-3sigma expression on transient cell cycle delay, CDKN1A has both an anti-proliferative and anti-apoptosis function, while 14-3-3sigma has only an anti-apoptosis function. Finally, the large alterations in the amounts of cell death did not correlate overall with the small alterations in clonogenic survival (dose-modifying ratios of 1.05-1.13); however, knocking out CDKN1A resulted in a decrease in arrested cells and an increase in survival, while knocking out 14-3-3sigma resulted in an increase in apoptosis and a decrease in survival. PMID:15332997

Chu, Kenneth; Teele, Noella; Dewey, Michael W; Albright, Norman; Dewey, William C



Patulin induces colorectal cancer cells apoptosis through EGR-1 dependent ATF3 up-regulation  

PubMed Central

Patulin is a fungal mycotoxin of Aspergilus and Penicillium that is commonly found in rotting fruits and exerts its potential toxic effect mainly by reactive oxygen species (ROS) generation. However, the effect of patulin on cancer cells as well as its intracellular mechanism has been controversial and not clearly defined yet. In this study, patulin was found to induce G1/S accumulation and cell growth arrest accompanied by caspase-3 activation, PARP cleavage and ATF3 expression in human colon cancer cell line HCT116. Ser/Thr phosphorylation of a transcription factor, EGR-1, was increased while its expression did not change upon patulin treatment to the cells. Knockdown of ATF3 and EGR-1 using their respective siRNAs showed EGR-1 dependent ATF3 expression. Moreover, treatment of the cells with antioxidants N-acetylcysteine (NAC) and glutathione (GSH) revealed that patulin induced ATF3 expression and apoptosis were dependent on ROS generation. ATF3 expression was also increased by patulin in other colorectal cancer cell types, Caco2 and SW620. Collectively, our data present a new anti-cancer molecular mechanism of patulin, suggesting EGR-1 and ATF3 as critical targets for the development of anti-cancer chemotherapeutics. In this regard, patulin could be a candidate for the treatment of colorectal cancers.

Kwon, Osong; Soung, Nak Kyun; Thimmegowda, N.R.; Jeong, Sook Jung; Jang, Jae Hyuk; Moon, Dong-Oh; Chung, Jong Kyeong; Lee, Kyung Sang; Kwon, Yong Tae; Erikson, Raymond Leo; Ahn, Jong Seog; Kim, Bo Yeon



Patulin induces colorectal cancer cells apoptosis through EGR-1 dependent ATF3 up-regulation.  


Patulin is a fungal mycotoxin of Aspergilus and Penicillium that is commonly found in rotting fruits and exerts its potential toxic effect mainly by reactive oxygen species (ROS) generation. However, the effect of patulin on cancer cells as well as its intracellular mechanism has been controversial and not clearly defined yet. In this study, patulin was found to induce G1/S accumulation and cell growth arrest accompanied by caspase-3 activation, PARP cleavage and ATF3 expression in human colon cancer cell line HCT116. Ser/Thr phosphorylation of a transcription factor, EGR-1, was increased while its expression did not change upon patulin treatment to the cells. Knockdown of ATF3 and EGR-1 using their respective siRNAs showed EGR-1 dependent ATF3 expression. Moreover, treatment of the cells with antioxidants N-acetylcysteine (NAC) and glutathione (GSH) revealed that patulin induced ATF3 expression and apoptosis were dependent on ROS generation. ATF3 expression was also increased by patulin in other colorectal cancer cell types, Caco2 and SW620. Collectively, our data present a new anti-cancer molecular mechanism of patulin, suggesting EGR-1 and ATF3 as critical targets for the development of anti-cancer chemotherapeutics. In this regard, patulin could be a candidate for the treatment of colorectal cancers. PMID:22230687

Kwon, Osong; Soung, Nak Kyun; Thimmegowda, N R; Jeong, Sook Jung; Jang, Jae Hyuk; Moon, Dong-Oh; Chung, Jong Kyeong; Lee, Kyung Sang; Kwon, Yong Tae; Erikson, Raymond Leo; Ahn, Jong Seog; Kim, Bo Yeon



Adrenocortical Cell Lines  

Microsoft Academic Search

\\u000a Initially, primary cultures of adrenocortical cells have traditionally been utilized to study the mechanisms controlling adrenocortical\\u000a steroidogenesis. However, the eventual onset of senescence in culture creates a recurring need for the costly and difficult\\u000a isolation of fresh cultures, and subsequently increases the risk of contamination. For these reasons, the use of primary cultures\\u000a has been increasingly replaced by immortalized cell

Jeniel Parmar; Anita Kulharya; William Rainey


Synthetic Lethal Targeting of PTEN-Deficient Cancer Cells Using Selective Disruption of Polynucleotide Kinase/Phosphatase.  


A recent screen of 6,961 siRNAs to discover possible synthetic lethal partners of the DNA repair protein polynucleotide kinase/phosphatase (PNKP) led to the identification of the potent tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Here, we have confirmed the PNKP/PTEN synthetic lethal partnership in a variety of different cell lines including the PC3 prostate cancer cell line, which is naturally deficient in PTEN. We provide evidence that codepletion of PTEN and PNKP induces apoptosis. In HCT116 colon cancer cells, the loss of PTEN is accompanied by an increased background level of DNA double-strand breaks, which accumulate in the presence of an inhibitor of PNKP DNA 3'-phosphatase activity. Complementation of PC3 cells with several well-characterized mutated PTEN cDNAs indicated that the critical function of PTEN required to prevent toxicity induced by an inhibitor of PNKP is most likely associated with its cytoplasmic lipid phosphatase activity. Finally, we show that modest inhibition of PNKP in a PTEN knockout background enhances cellular radiosensitivity, suggesting that such a "synthetic sickness" approach involving the combination of PNKP inhibition with radiotherapy may be applicable to PTEN-deficient tumors. Mol Cancer Ther; 12(10); 2135-44. ©2013 AACR. PMID:23883586

Mereniuk, Todd R; El Gendy, Mohamed A M; Mendes-Pereira, Ana M; Lord, Christopher J; Ghosh, Sunita; Foley, Edan; Ashworth, Alan; Weinfeld, Michael



Plant cell lines in cell morphogenesis research.  


Plant organs and tissues consist of many various cell types, often in different phases of their development. Such complex structures do not allow direct studies on behavior of individual cells. In contrast, populations of in vitro-cultured plant cells represent valuable tool for studying processes on a single-cell level, including cell morphogenesis. Here we describe characteristics of well-established model tobacco and Arabidopsis cell lines and provide detailed protocol on their cultivation, characterization, and genetic transformation. PMID:24132432

Seifertová, Daniela; Klíma, Petr; Pa?ezová, Markéta; Petrášek, Jan; Zažímalová, Eva; Opatrný, Zden?k



Thyroid cell lines in research on goitrogenesis.  


Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

Gerber, H; Peter, H J; Asmis, L; Studer, H



Inhibition of N -linked glycosylation by tunicamycin induces E-cadherin-mediated cell–cell adhesion and inhibits cell proliferation in undifferentiated human colon cancer cells  

Microsoft Academic Search

Purpose  Aberrant protein glycosylation and disassembly of E-cadherin-mediated cell–cell adhesion are characteristics of epithelial\\u000a cancer. However, the relationship between these two events in colorectal cancer remains to be defined. In this study, we analyzed\\u000a whether N-glycan expression is crucial for the loss of E-cadherin-mediated cell–cell adhesion in human colorectal cancer cells.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Differentiated Caco-2 and undifferentiated HCT-116 colon cancer cells were used

Julio Cesar Madureira de Freitas Junior; Bárbara Du Rocher D’Aguiar Silva; Waldemir Fernandes de Souza; Wallace Martins de Araújo; Eliana Saul Furquim Werneck Abdelhay; José Andrés Morgado-Díaz



Effects of omega-3 and omega-6 fatty acids on IGF-I receptor signalling in colorectal cancer cells.  


The insulin-like growth factor (IGF) system plays a critical role in normal growth and development as well as in malignant states. Most of the biological activities of the IGFs are mediated by the IGF-IR, which is over-expressed in most tumours and cancer cell lines. Fatty acids have critical roles in both systemic physiological processes (e.g. metabolism) and cellular events (e.g. proliferation, apoptosis, signal transduction, and gene expression). Alpha-linolenic acid (ALA) and linoleic acid (LA) are essential fatty acids of the omega-3 and omega-6 families, respectively. The aim of this study was to investigate the potential interactions between fatty acids and the IGF signal transduction pathways, and to evaluate the impact of this interplay on colon cancer cells survival and proliferation. Results of Western blot analyses revealed that ALA and LA enhanced the ligand-induced IGF-IR phosphorylation and, in addition, increased receptor phosphorylation in an IGF-I independent manner. Furthermore, fatty acid treatment led to phosphorylation of downstream signalling molecules, including Akt and Erk. In addition, FACS analysis and apoptosis measurements indicated that ALA and LA have a potential mitogenic effect on HCT116 cells, as reflected by the number of cells in S phase and by a reduction of PARP cleavage, implying a reduction in apoptotic activity. In summary, our results provide evidence that omega-3 and omega-6 fatty acids modulate IGF-I action in colon cancer cells. PMID:19480565

Seti, Hila; Leikin-Frenkel, Alicia; Werner, Haim



Concurrent expression of C4.4A and Tenascin-C in tumor cells relates to poor prognosis of esophageal squamous cell carcinoma.  


C4.4A is a glycolipid-anchored membrane protein expressed in several human malignancies. We recently found that C4.4A expression was associated with poor prognosis of esophageal squamous carcinoma cells (ESCCs), but the underlying mechanism is unknown. To uncover this, we performed PCR array analysis using the HCT116 cell line, a positive control for C4.4A expression and we found that Tenascin-C (TNC) among the many adhesion molecules and extracellular matrix proteins was the best candidate for C4.4A molecule induction. Based on in vitro studies using the TE8 esophageal cancer cells, we examined by immunohistochemistry TNC expression in 111 ESCCs. We found that the TNC-positive group (24.3%) had significantly poorer prognosis than the TNC-negative group in 5-year overall survival. We also found there was a significant correlation between TNC and C4.4A in ESCC tissues (P=0.007). Finally, we found that only the double-positive group for C4.4A and TNC had a significantly worse prognosis (P=0.005). Our data suggest that TNC expression in ESCC may in part explain why C4.4A is associated with a poor prognosis of ESCC since TNC can promote invasion and metastasis. PMID:23708783

Ohtsuka, Masahisa; Yamamoto, Hirofumi; Oshiro, Ryota; Takahashi, Hidekazu; Masuzawa, Toru; Uemura, Mamoru; Haraguchi, Naotsugu; Nishimura, Junichi; Hata, Taishi; Yamasaki, Makoto; Takemasa, Ichiro; Miyata, Hiroshi; Mizushima, Tsunekazu; Takiguchi, Shuji; Doki, Yuichiro; Mori, Masaki



Platinum(II/IV) complexes containing ethylenediamine-N,N'-di-2/3-propionate ester ligands induced caspase-dependent apoptosis in cisplatin-resistant colon cancer cells.  


Several new R(2)eddp (R = i-Pr, i-Bu; eddp = ethylenediamine-N,N'-di-3-propionate) esters and corresponding platinum(ii) and platinum(iv) complexes of the general formula [PtCl(n)(R(2)edda-type)] (n = 2, 4) were synthesized and characterized by spectroscopic methods (IR, (1)H and (13)C NMR) and elemental analysis. The crystal structure of platinum(iv) complex [PtCl(4){(c-Pe)(2)eddip}] (3a) was resolved and is given herein. Ligand precursors, platinum(ii), and platinum(iv) complexes were tested against eight tumor cell lines (CT26CL25, HTC116, SW620, PC3, LNCaP, U251, A375, and B16). Selectivity in the action of those compounds between tumor and two normal primary cells (fibroblasts and keratinocytes) are discussed. A structure-activity relationship of these compounds is discussed. Furthermore, cell cycle distribution, induction of necrosis, apoptosis, autophagy, anoikis, caspase activation, ROS, and RNS are presented on the cisplatin-resistant colon carcinoma HCT116 cell line. PMID:22820831

Kalu?erovi?, Goran N; Mijatovi?, Sanja A; Zmejkovski, Bojana B; Bulatovi?, Mirna Z; Gómez-Ruiz, Santiago; Moji?, Marija K; Steinborn, Dirk; Miljkovi?, Djordje M; Schmidt, Harry; Stoši?-Gruji?i?, Stanislava D; Sabo, Tibor J; Maksimovi?-Ivani?, Danijela D



Assessing the anti-tumour properties of Iraqi propolis in vitro and in vivo.  


The study was designed to evaluate anti-tumour properties of Iraqi propolis collected from Mosul region (M) on HL-60 and HCT-116 cell lines and on HCT-116 in vivo. M induced an inhibitory effect against the proliferation of HL-60 and colony potential of HCT-116 cells. The apoptosis in HL-60 cells was associated with down-regulation of Bcl-2 and activation of Bax, while in HCT-116 cells, necrotic features were observed; size of cells was dramatically increased by swelling of cytoplasm and loss of membrane integrity, cell rupture and release of cellular contents. Analysis of BrdU/DNA cell cycle in both cell lines showed that M induced cell cycle perturbations in both BrdU positive and BrdU negative cells. The exposure of HL-60 to M caused ?-H2AX in a dose dependent manner and was associated with induction of apoptosis. The experiments in HCT-116 tumor-bearing mice showed that oral administration of propolis at doses that caused no detectable toxicity was associated with a decrease in mitotic cells and an increase in endoreduplications, increased p53 and decreased Ki-67 expression of cells in tumor sections. This study provides the rationale to investigate the potential beneficial effect of propolis in the diet of patients receiving anti-cancer therapies. PMID:22306915

Sulaiman, Ghassan M; Ad'hiah, Ali H; Al-Sammarrae, Khulood W; Bagnati, Renzo; Frapolli, Roberta; Bello, Ezia; Uboldi, Sarah; Romano, Michela; Panini, Nicolò; Scanziani, Eugenio; Pezzolato, Marzia; Erba, Eugenio; D'Incalci, Maurizio




PubMed Central

Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose deprivation-induced cytotoxicity. To determine if oxidative stress mediated by O2•? and hydroperoxides contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, oxidation of dihydroethidine (DHE; for O2•?) and 5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (CDCFH2; for hydroperoxides) were measured in human colon and breast cancer cells (HT29, HCT116, SW480, MB231) and compared to normal human cells (FHC, 33Co, HMEC). Cancer cells showed significant increases in DHE (2–20 fold) and CDCFH2 (1.8–10 fold) oxidation, relative to normal cells that were more pronounced in the presence of the mitochondrial electron transport chain blocker, antimycin A. Furthermore, HCT116 and MB231 cells were more susceptible to glucose deprivation-induced cytotoxicity and oxidative stress, relative to 33Co and HMEC. HT-29 cells were also more susceptible to 2-deoxyglucose-(2DG)-induced cytotoxicity, relative to FHC. Over expression of manganese superoxide dismutase and mitochondrially targeted catalase significantly protected HCT116 and MB231 cells from glucose deprivation-induced cytotoxicity and oxidative stress, as well as protecting HT-29 cells from 2DG-induced cytotoxicity. These results show cancer cells (relative to normal cells) demonstrate increased steady-state levels of reactive oxygen species (ROS, i.e. O2•? and H2O2) that contribute to differential susceptibility to glucose deprivation-induced cytotoxicity and oxidative stress. These studies support the hypotheses that cancer cells increase glucose metabolism to compensate for excess metabolic production of ROS as well as that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.

Aykin-Burns, Nukhet; Ahmad, Iman M.; Zhu, Yueming; Oberley, Larry W.; Spitz, Douglas R.



Confocal spectral imaging by microspectrofluorometry using two-photon excitation: application to the study of anticancer drugs within single living cancer cells  

NASA Astrophysics Data System (ADS)

The use of the two-photon excitation (TPE) is believed to be prominent for fluorometric studies with cells. We evaluated the advantages and limitations of the two-photon technique compared to the single photon one when it used to detect potent anticancer drugs, camptothecins (CPTs), within single living cancer cells. The technique we used was confocal microspectrofluorometry amplified with possibility of the spectral imaging analysis. We have previously reported the use of the florescence emission of CPTs to study them qualitatively and quantitatively, namely, to follow the status of their hydrolyzable lactone moiety. However, the intracellular investigation of CPTs using microspectrofluorometry with single photon UV excitation (SPE) is hindered by significant interference of their fluorescence emission with cellular autofluorescence. We attempted to overcome these problems using the two-photon excitation. The intracellular single-photon- and two-photon-excited emission spectra from treated and control cells (HCT-116 line) were recorded using a spectral imaging approach. The obtained data demonstrate that, apart from intrinsically increased three- dimensional resolution, the two-photon approach was advantageous over the single-photon method with respect to selective fluorometric detection of intracellular CPTs. Nevertheless, much attention should be paid to avoid any excessive irradiation of the cells with UV and even NIR light.

Chourpa, Igor; Pereira, Manuela; Millot, Jean-Marc; Morjani, Hamid; Manfait, Michel



Multimodal tissue imaging: using coregistered optical tomography data to estimate tissue autofluorescence intensity change due to scattering and absorption by neoplastic epithelial cells.  


ABSTRACT. Autofluorescence (AF) imaging provides valuable information about the structural and chemical states of tissue that can be used for early cancer detection. Optical scattering and absorption of excitation and emission light by the epithelium can significantly affect observed tissue AF intensity. Determining the effect of epithelial attenuation on the AF intensity could lead to a more accurate interpretation of AF intensity. We propose to use optical coherence tomography coregistered with AF imaging to characterize the AF attenuation due to the epithelium. We present imaging results from three vital tissue models, each consisting of a three-dimensional tissue culture grown from one of three epithelial cell lines (HCT116, OVCAR8, and MCF7) and immobilized on a fluorescence substrate. The AF loss profiles in the tissue layer show two different regimes, each approximately linearly decreasing with thickness. For thin cell cultures (<300???m), the AF signal changes as AF(t)/AF(0)=1-1.3t (t is the thickness in millimeter). For thick cell cultures (>400???m), the AF loss profiles have different intercepts but similar slopes. The data presented here can be used to estimate AF loss due to a change in the epithelial layer thickness and potentially to reduce AF bronchoscopy false positives due to inflammation and non-neoplastic epithelial thickening. PMID:24108573

Pahlevaninezhad, Hamid; Cecic, Ivana; Lee, Anthony M D; Kyle, Alastair H; Lam, Stephen; Macaulay, Calum; Lane, Pierre M



Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F?F?-ATP synthase.  


We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53(+/+) cells simultaneously show increased O? consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F?F?-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F?F?-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria. PMID:23966169

Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent



PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab.  


Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR-overexpressing advanced colorectal cancer, recent studies have shown a lack of association between EGFR overexpression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in vitro and sensitive and resistant lines were identified. In sensitive cell lines, cetuximab induced a G(0)-G(1) arrest without inducing apoptosis. Notably, cetuximab-sensitive but not cetuximab-resistant cell lines were preferentially responsive to EGF-stimulated growth. Whereas neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR showed that cell lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type (WT)/PTEN-expressing cell lines (14 +/- 5.0% versus 38.5 +/- 6.4% growth inhibition, mean +/- SE; P = 0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared with PIK3CA WT controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss (10.8 +/- 4.3% versus 38.8 +/- 5.9% growth inhibition, respectively; P = 0.002), indicating that constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy. PMID:18339877

Jhawer, Minaxi; Goel, Sanjay; Wilson, Andrew J; Montagna, Cristina; Ling, Yi-He; Byun, Do-Sun; Nasser, Shannon; Arango, Diego; Shin, Joongho; Klampfer, Lidija; Augenlicht, Leonard H; Perez-Soler, Roman; Soler, Roman Perez; Mariadason, John M



Synthesis and biological evaluation of novel anthranilamide derivatives as anticancer agents.  


A new series of anthranilamide derivatives were synthesized and evaluated for their antiproliferative activities against human colon carcinoma cell lines (HCT 116) and human breast adenocarcinoma cell lines (MDA-MB-231) in vitro. The bioassay results indicated that compounds 7a-7d, 11a, and 11b with flexible linkers showed promising antiproliferative activity against both cell lines. Among the compounds synthesized, 7c showed the most significant antiproliferative activity. Flow cytometric analysis indicated that 7c inhibited HCT 116 and MDA-MB-231 cell growth by inducing apoptosis in a dose-dependent manner and suppressed HCT 116 cell proliferation by G1 and S phase arrest. Compound 7c may serve as a lead candidate in the development of novel anticancer agents. PMID:24071576

Liu, J Z; Liang, W; Wang, Y Y; Zhao, G S



Human Adrenocortical Carcinoma Cell Lines  

PubMed Central

Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies.

Wang, Tao; Rainey, William E.



Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells.  

PubMed Central

BACKGROUND: Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells. MATERIALS AND METHODS: Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay. RESULTS: Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media. CONCLUSIONS: Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth. Images Fig. 9 Fig. 11

Stepan, V. M.; Sawada, M.; Todisco, A.; Dickinson, C. J.



Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.  


Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation. PMID:12062184

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi



Metabolites of Ginger Component [6]-Shogaol Remain Bioactive in Cancer Cells and Have Low Toxicity in Normal Cells: Chemical Synthesis and Biological Evaluation  

PubMed Central

Our previous study found that [6]-shogaol, a major bioactive component in ginger, is extensively metabolized in cancer cells and in mice. It is unclear whether these metabolites retain bioactivity. The aim of the current study is to synthesize the major metabolites of [6]-shogaol and evaluate their inhibition of growth and induction of apoptosis in human cancer cells. Twelve metabolites of [6]-shogaol (M1, M2, and M4–M13) were successfully synthesized using simple and easily accessible chemical methods. Growth inhibition assays showed that most metabolites of [6]-shogaol had measurable activities against human cancer cells HCT-116 and H-1299. In particular, metabolite M2 greatly retained the biological activities of [6]-shogaol, with an IC50 of 24.43 µM in HCT-116 human colon cancer cells and an IC50 of 25.82 µM in H-1299 human lung cancer cells. Also exhibiting a relatively high potency was thiol-conjugate M13, with IC50 values of 45.47 and 47.77 µM toward HCT-116 and H-1299 cells, respectively. The toxicity evaluation of the synthetic metabolites (M1, M2, and M4–M13) against human normal fibroblast colon cells CCD-18Co and human normal lung cells IMR-90 demonstrated a detoxifying metabolic biotransformation of [6]-shogaol. The most active metabolite M2 had almost no toxicity to CCD-18Co and IMR-90 normal cells with IC50s of 99.18 and 98.30 µM, respectively. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay indicated that apoptosis was triggered by metabolites M2, M13, and its two diastereomers M13-1 and M13-2. There was no significant difference between the apoptotic effect of [6]-shogaol and the effect of M2 and M13 after 6 hour treatment.

Zhu, Yingdong; Chen, Huadong; Sang, Shengmin



Metabolites of ginger component [6]-shogaol remain bioactive in cancer cells and have low toxicity in normal cells: chemical synthesis and biological evaluation.  


Our previous study found that [6]-shogaol, a major bioactive component in ginger, is extensively metabolized in cancer cells and in mice. It is unclear whether these metabolites retain bioactivity. The aim of the current study is to synthesize the major metabolites of [6]-shogaol and evaluate their inhibition of growth and induction of apoptosis in human cancer cells. Twelve metabolites of [6]-shogaol (M1, M2, and M4-M13) were successfully synthesized using simple and easily accessible chemical methods. Growth inhibition assays showed that most metabolites of [6]-shogaol had measurable activities against human cancer cells HCT-116 and H-1299. In particular, metabolite M2 greatly retained the biological activities of [6]-shogaol, with an IC(50) of 24.43 µM in HCT-116 human colon cancer cells and an IC(50) of 25.82 µM in H-1299 human lung cancer cells. Also exhibiting a relatively high potency was thiol-conjugate M13, with IC(50) values of 45.47 and 47.77 µM toward HCT-116 and H-1299 cells, respectively. The toxicity evaluation of the synthetic metabolites (M1, M2, and M4-M13) against human normal fibroblast colon cells CCD-18Co and human normal lung cells IMR-90 demonstrated a detoxifying metabolic biotransformation of [6]-shogaol. The most active metabolite M2 had almost no toxicity to CCD-18Co and IMR-90 normal cells with IC(50)s of 99.18 and 98.30 µM, respectively. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay indicated that apoptosis was triggered by metabolites M2, M13, and its two diastereomers M13-1 and M13-2. There was no significant difference between the apoptotic effect of [6]-shogaol and the effect of M2 and M13 after 6 hour treatment. PMID:23382939

Zhu, Yingdong; Warin, Renaud F; Soroka, Dominique N; Chen, Huadong; Sang, Shengmin



Combined effects of storage and processing on the bioactive compounds and pro-apoptotic properties of color-fleshed potatoes in human colon cancer cells.  


Potatoes can be stored for up to 1 year before being processed and consumed. The objective of this study was to determine the extent to which fresh and stored color-fleshed potatoes retain their anticancer properties after baking and chipping compared with unprocessed potatoes. We utilized white-, yellow-, and purple-fleshed potato clones and tested their phenolic and anthocyanin content, antioxidant activity, metabolite profile, and antiproliferative and pro-apoptotic properties. When compared with unprocessed samples, baking or chipping led to significant losses in the phenolic and anthocyanin content and antioxidant activity of the potatoes. However, with storage, total phenolic and anthocyanin content and antioxidant activity increased in baked samples while in the chipped samples they remained constant. Ethanolic extracts of baked and chipped samples suppressed proliferation and elevated apoptosis (p < 0.05) in HCT-116 (p53 wild-type; ras mutated) and HT-29 (p53 mutated; ras wild-type) human colon cancer cell lines. Antiproliferative and pro-apoptotic properties of baked potatoes were similar to that of fresh potatoes, while chipping caused a significant suppression. Phenolic content and antioxidant activity of purple-fleshed potatoes, after baking, were comparable with those of anthocyanin-rich berries. Hence, purple-fleshed potatoes can be a healthier choice for consumers as they possess greater levels of bioactive compounds and anticancer properties even after processing as compared with their white- and yellow-fleshed counterparts. PMID:23039105

Madiwale, Gaurav P; Reddivari, Lavanya; Stone, Martha; Holm, David G; Vanamala, Jairam



Transmission line analysis of MRAM cell  

Microsoft Academic Search

A test configuration of magnetic random access memory (MRAM) unit cell was modeled and its three-dimensional finite element method (FEM) analysis was utilized to calculate S-parameters. Transmission line analysis was performed for word line, bit line and readout signal path. Line width was varied from 0.5 to 2 ?m, line thickness from 0.1 to 1 ?m, and line length from

S. Jo



Development and characterization of insect cell lines  

Microsoft Academic Search

With the wide availability of insect cell culture media, it can generally be considered a routine process to develop new cell lines. Exceptions to this statement do exist, of course. Difficulties may arise when attempting to culture a specific cell type. For example, while there are a few cell lines from insect fat body and at least one from the

Dwight E. Lynn



Metabolic Profiling of Hypoxic Cells Revealed a Catabolic Signature Required for Cell Survival  

PubMed Central

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography–mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

Frezza, Christian; Zheng, Liang; Tennant, Daniel A.; Papkovsky, Dmitri B.; Hedley, Barbara A.; Kalna, Gabriela; Watson, David G.; Gottlieb, Eyal



Biosynthetic O-Methylation Protects Cladoniamides from Self-destruction.  


Bisindole cladoniamides, nanomolar inhibitors of colon cancer cell line HCT-116, contain a rare, indolotryptoline substructure. In this report, the structures of xenocladoniamides A-E (9-13) are described. Compounds 9-13 are generated from a cladoniamide heterologous production system where O-methyltransferase gene claM3 has been inactivated. The results suggest that O-methylation, installed by enzyme ClaM3, is critical to maintaining the structural integrity of the indolotryptoline scaffold. Xenocladoniamides D and E are modestly cytotoxic against colon cancer cell line HCT-116. PMID:23639001

Du, Yi-Ling; Ding, Tong; Ryan, Katherine S



Dicer 1, ribonuclease type III modulates a reprogramming effect in colorectal cancer cells.  


Complete cell reprogramming can be achieved by the introduction of specific transcription factors, Oct4 [also known as POU class 5 homeobox 1 (Pou5f1)]; sex-determining region Y (SRY)-box 2 (Sox2); Kruppel-like factor 4 (Klf4); and myelocytomatosis viral oncogene homolog (c-Myc), into terminally differentiated mouse somatic fibroblasts. This reprogramming process may be accelerated or suppressed by various factors, including microRNAs (miRNAs). Introduction of these transcription factors or miRNAs considerably modifies the malignant phenotype of cancer cells. We studied the effect of introducing these transcription factors into two distinct colorectal cancer (CRC) cell lines, HCT116 and DLD-1, in the presence and absence of Dicer 1, ribonuclease type III (Dicer1), a critical miRNA processing enzyme. We assessed cell reprogramming based on the number of cells exhibiting alkaline phosphatase staining and an increase in embryonic stem cell-like gene expression, indicating the return of cells to an immature state. Dicer1-deficient CRC cells showed a reduced number of alkaline phosphatase-positive reprogrammed cells than wild-type (WT) cells. Before reprogramming, endogenous expression of an immature carbohydrate epitope, TRA-1-60, was high in Dicer1-deficient CRC cells, whereas after reprogramming, the expression of this epitope was increased in Dicer1-sufficient more than in Dicer1-deficient CRC cells. Our data demonstrate the critical role of miRNAs in the reprogramming process and determination of a differentiated phenotype of CRC cells. PMID:22446887

Dewi, Dyah Laksmi; Ishii, Hideshi; Haraguchi, Naotsugu; Nishikawa, Shimpei; Kano, Yoshihiro; Fukusumi, Takahito; Ozaki, Miyuki; Saito, Toshiyuki; Sakai, Daisuke; Satoh, Taroh; Doki, Yuichiro; Mori, Masaki



A new phenanthrene derivative and two diarylheptanoids from the roots of Brassica rapa ssp. campestris inhibit the growth of cancer cell lines and LDL-oxidation.  


Brassica rapa ssp. campestris (Brassicaceae) is a conical, deep purple, edible root vegetable commonly known as a turnip. We initiated phytochemical and pharmacological studies to search for biological active compounds from the roots of B. rapa ssp. campestris. We isolated a novel phenanthrene derivative, 6-methoxy-1-[10-methoxy-7-(3-methylbut-2-enyl)phenanthren-3-yl]undecane-2,4-dione, named brassicaphenanthrene A (3) along with two known diarylheptanoid compounds, 6-paradol (1) and trans-6-shogaol (2), through the repeated silica gel (SiO2), octadecyl silica gel, and Sephadex LH-20 column chromatography. The chemical structures of the compounds were determined by spectroscopic data analyses including nuclear magnetic resonance, mass spectrometry, ultraviolet spectroscopy, and infra-red spectroscopy. All compounds exhibited high inhibitory activity against the growth of human cancer lines, HCT-116, MCF-7, and HeLa, with IC50 values ranging from 15.0 to 35.0 ?M and against LDL-oxidation with IC50 values ranging from 2.9 to 7.1 ?M. PMID:23435947

Wu, Qian; Cho, Jin-Gyeong; Yoo, Ki-Hyun; Jeong, Tae-Sook; Park, Ji-Hae; Kim, Su-Yeon; Kang, Ji-Hyun; Chung, In-Sik; Choi, Myung-Sook; Lee, Kyung-Tae; Chung, Hae-Gon; Bang, Myun-Ho; Baek, Nam-In



RNA Interference Against Peroxisome Proliferator-Activated Receptor ? Gene Promotes Proliferation of Human Colorectal Cancer Cells  

Microsoft Academic Search

Purpose  This study was designed to investigate the effects of peroxisome proliferator-activated receptor ? (PPAR ?) on the proliferation\\u000a and apoptosis of human colorectal cancer cells.\\u000a \\u000a \\u000a \\u000a Methods  For RNA interfering (RNAi), HCT-116 cells were transfected with short hairpin RNA (shRNA)-expressing plasmids against PPAR\\u000a ? or negative control vectors, and the stably transfected cells were selected with G418. The efficacy of RNAi was

L. Yang; Z.-G. Zhou; X.-L. Zheng; L. Wang; Y.-Y. Yu; B. Zhou; J. Gu; Y. Li



Design, synthesis and biological evaluation of indeno[1,2-d]thiazole derivatives as potent histone deacetylase inhibitors.  


Novel indeno[1,2-d]thiazole hydroxamic acids were designed, synthesized, and evaluated for histone deacetylases (HDACs) inhibition and antiproliferative activities on tumor cell lines. Most of the tested compounds exhibited HDAC inhibition and antiproliferative activity against both MCF7 and HCT116 cells with GI50 values in the sub-micromolar range. Among them, compound 6o showed good inhibitory activity against pan-HDAC with IC50 value of 0.14 ?M and significant growth inhibition on MCF7 and HCT116 cells with GI50 values of 0.869 and 0.535 ?M, respectively. PMID:23639537

Zhou, Ming; Ning, Chengqing; Liu, Ruihuan; He, Yujun; Yu, Niefang



Molecular Characterization of Putative Chordoma Cell Lines  

PubMed Central

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

Bruderlein, Silke; Sommer, Joshua B.; Meltzer, Paul S.; Li, Sufeng; Osada, Takuya; Ng, David; Moller, Peter; Alcorta, David A.; Kelley, Michael J.



Resveratrol 3-O-d-glucuronide and resveratrol 4'-O-d-glucuronide inhibit colon cancer cell growth: Evidence for a role of A3 adenosine receptors, cyclin D1 depletion, and G1 cell cycle arrest.  


SCOPE: Resveratrol is a plant-derived polyphenol with chemotherapeutic properties in animal cancer models and many biochemical effects in vitro. Its bioavailability is low and raises the possibility that the metabolites of resveratrol have biological effects. Here we investigate the actions of resveratrol 3-O-d-glucuronide, resveratrol 4'-O-d-glucuronide, and resveratrol 3-O-d-sulfate on the growth of colon cancer cells in vitro. METHODS AND RESULTS: The growth of Caco-2, HCT-116, and CCL-228 cells was measured using the neutral red and MTT assays. Resveratrol and each metabolite inhibited cell growth with IC50 values of 9.8-31 ?M. Resveratrol caused S phase arrest in all three cell lines. Resveratrol 3-O-d-glucuronide and resveratrol 4'-O-d-glucuronide caused G1 arrest in CCL-228 and Caco-2 cells. Resveratrol 3-O-d-sulfate had no effect on cell cycle. Growth inhibition was reversed by an inhibitor of AMP-activated protein kinase (compound C) or an adenosine A3 receptor antagonist (MRS1191). The A3 receptor agonist 2Cl-IB-MECA inhibited growth and A3 receptors were detected in all cell lines. The resveratrol glucuronides also reduced cyclin D1 levels but at higher concentrations than in growth experiments and generally did not increase phosphorylated AMP-activated protein kinase. CONCLUSION: Resveratrol glucuronides inhibit cell growth by G1 arrest and cyclin D1 depletion, and our results strongly suggest a role for A3 adenosine receptors in this inhibition. PMID:23650147

Polycarpou, Elena; Meira, Lisiane B; Carrington, Simon; Tyrrell, Elizabeth; Modjtahedi, Helmout; Carew, Mark A



CellLineMiner: a knowledge portal for human cell lines  

PubMed Central

Experimental models of human tissues and disease phenotypes frequently rely upon immortalized cell lines, which are easily accessible and simple to use due to their infinite capability of cell division. For decades, cell lines have been used to investigate cellular mechanisms of disease and the efficacy of drugs, most prominently for human cancers. However, the large body of knowledge with respect to human cell lines exists primarily in an unstructured fashion, that is, as free text in the scientific literature. Here we present CellLineMiner, a novel text mining-based web database that provides a comprehensive view of human cell line knowledge. The application offers a simple search in all indexed cell lines, accompanied by a rapid display of all identified literature associations. The CellLineMiner is intended to serve as a knowledge resource companion to the cellular model systems used in biomedical research. Availability CellLineMiner is accessible at

Nakken, Sigve; Johansen, Morten; Fillebeen, Julien; Berge, Ole Petter; Kirker?d, Harald; Jenssen, Tor-Kristian; Hovig, Eivind



Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies  

PubMed Central

The urokinase plasminogen activator receptor (uPAR) plays a critical role in urokinase-mediated plasminogen activation and thereby in the process leading to invasion and metastasis. Soluble urokinase receptor (suPAR) is released from tumours, and in cancer patients the blood level of soluble receptor is increased. Using an enzyme-linked, immunosorbent assay (ELISA)-specific for the human urokinase receptor, release of soluble receptor was measured in cultures of human breast carcinoma cells, in tumour extracts and in plasma from mice with xenografted human tumours. Soluble human urokinase receptor (shuPAR) was released into culture supernatant during the growth of the human breast cancer cell line MDA-MB-231 BAG, and the level of shuPAR in conditioned medium determined by ELISA was a linear function of both viable cell number and time of incubation. Western blotting showed that the form of shuPAR measured by ELISA in conditioned medium consisted virtually exclusively of the three-domain full-length protein, while uPAR in cell lysates consisted of full-length uPAR as well as the domains (2+3) cleavage product. shuPAR was also released into the plasma of nude mice during growth of MDA-MB-231 BAG, MDA-MB-435 BAG and HCT 116 cells as subcutaneously xenografted tumours. Western blotting demonstrated that the shuPAR released from the xenografted human tumours into plasma consisted of the three-domain full-length protein, despite the finding of some cleaved uPAR in detergent extracts of tumour tissue. The levels of shuPAR determined by ELISA in the plasma of host mice during the growth of xenografted cell lines were highly correlated with tumour volume. © 1999 Cancer Research Campaign

Holst-Hansen, C; Hamers, M J A G; Johannessen, B E; Brunner, N; Stephens, R W



Fucoidan present in brown algae induces apoptosis of human colon cancer cells  

PubMed Central

Background Fucoidan is a sulfated polysaccharide found in brown algae; it has been shown to exhibit a number of biological effects, including anti-tumor effects. In this study, we evaluated the effects of fucoidan on apoptosis in HT-29 and HCT116 human colon cancer cells. Methods HT-29 and HCT116 cells were cultured with various concentrations of fucoidan (0 - 20 ?g/mL). Apoptosis was assayed via Hoechst staining and Annexin V staining followed by flow cytometric analysis. Western blot analyses and JC-1 staining were conducted to determine the levels of apoptosis-regulating proteins and mitochondrial membrane permeability, respectively. Results Fucoidan induced substantial reductions in viable cell numbers and apoptosis of HT-29 and HCT116 cells in a dose-dependent manner. In HT-29 cells, fucoidan also increased the levels of cleaved caspases-8, -9, -7, and -3, and cleaved poly (ADP-ribose) polymerase (PARP) levels. The levels of the X-linked inhibitor of apoptosis protein and survivin were attenuated in the fucoidan-treated cells. Fucoidan was also shown to enhance mitochondrial membrane permeability, as well as the cytochrome c and Smac/Diablo release from the mitochondria. Fucoidan increased the levels of the Bak and truncated Bid proteins, but reduced the levels of Mcl-1. Additionally, fucoidan increased the levels of the tumor necrosis factor-related apoptosis-inducing ligand, Fas and death receptor 5 proteins. The caspase-8 and -9 inhibitors Z-IETD-FMK and Z-LEHD-FMK induced a reduction in fucoidan-mediated apoptosis. Caspase-8 inhibitor inhibited the fucoidan-induced cleavage of Bid, caspases-9 and -3, and PARP. Conclusion The findings of this study indicate that fucoidan induces apoptosis in HT-29 and HCT116 human colon cancer cells, and that this phenomenon is mediated via both the death receptor-mediated and mitochondria-mediated apoptotic pathways. These results suggest that fucoidan may prove useful in the development of a colon cancer-preventive protocol.



Tezacitabine enhances the DNA-directed effects of fluoropyrimidines in human colon cancer cells and tumor xenografts.  


Tezacitabine is a nucleoside analogue characterized by a dual mechanism of action. Following intracellular phosphorylation, the tezacitabine diphosphate irreversibly inhibits ribonucleotide reductase, while the tezacitabine triphosphate can be incorporated into DNA during replication or repair, resulting in DNA chain termination. In the present study we have investigated the effect of the combination of tezacitabine and 5-fluorouracil (5-FU) or 5-fluoro-2'-deoxyuridine (FUdR) on HCT 116 human colon carcinoma cells and xenografts. We used response surface analysis (RSA) and clonogenic assay to evaluate combination effects of tezacitabine and 5-FU. Tezacitabine is antagonistic when combined with 5-FU in the RSA assay and does not effect the clonogenicity of HCT 116 cells when compared with cells treated with 5-FU alone. However, when combined sequentially with FUdR, tezacitabine leads to potentiation of cell killing in the clonogenic assay, additivity in the RSA assay, and increased apoptosis when compared to FUdR alone, suggesting that cytotoxicity of fluoropyrimidines such as FUdR that have more DNA-directed effects can be potentiated by tezacitabine. We also report that oral administration of the fluoropyrimidine capecitabine, an oral prodrug of 5-FU, in combination with tezacitabine shows statistically significant additivity in the HCT 116 xenograft model. This interaction may be explained by the finding that tezacitabine elevates activity of thymidine phosphorylase (TP), the enzyme required for activation of the capecitabine prodrug in tumors. Our results provide evidence that tezacitabine enhances the DNA-directed effects of fluoropyrimidines in human colon cancer cells and may modulate the antitumor activity of fluoropyrimidines. PMID:17046720

Taverna, Pietro; Rendahl, Katherine; Jekic-McMullen, Dragana; Shao, Yi; Aardalen, Kim; Salangsang, Fernando; Doyle, Laura; Moler, Eddie; Hibner, Barbara



Cell culturing of human and murine microglia cell lines.  


Despite the fact that microglia cells were first described almost a century ago, microglia-derived immortalized cell lines have only been established in the last two decades. One should be aware of their limitations but also of their advantages. Cell lines offer a potentially powerful tool to investigate some functional aspects of microglia. Cell culturing of human and murine microglia cell lines will be described in this chapter. It includes a presentation of equipment needed, cell culture medium and supplements, cell culture monitoring, and a protocol describing the steps for subculturing of microglia cell lines. PMID:23813364

Rodhe, Johanna



Securin Enhances the Anti-Cancer Effects of 6-Methoxy-3-(3?,4?,5?-Trimethoxy-Benzoyl)-1H-Indole (BPR0L075) in Human Colorectal Cancer Cells  

PubMed Central

BPR0L075 [6-methoxy-3-(3?,4?,5?-trimethoxy-benzoyl)-1H-indole] is a novel anti-microtubule drug with anti-tumor and anti-angiogenic activities in vitro and in vivo. Securin is required for genome stability, and is expressed abundantly in most cancer cells, promoting cell proliferation and tumorigenesis. In this study, we found that BPR0L075 efficiently induced cell death of HCT116 human colorectal cancer cells that have higher expression levels of securin. The cytotoxicity of BPR0L075 was attenuated in isogenic securin-null HCT116 cells. BPR0L075 induced DNA damage response, G2/M arrest, and activation of the spindle assembly checkpoint in HCT116 cells. Interestingly, BPR0L075 induced phosphorylation of securin. BPR0L075 withdrawal resulted in degradation of securin, mitotic exit, and mitotic catastrophe, which were attenuated in securin-null cells. Inhibition of cdc2 decreased securin phosphorylation, G2/M arrest and cell death induced by BPR0L075. Moreover, BPR0L075 caused cell death through a caspase-independent mechanism and activation of JNK and p38 MAPK pathways. These findings provided evidence for the first time that BPR0L075 treatment is beneficial for the treatment of human colorectal tumors with higher levels of securin. Thus, we suggest that the expression levels of securin may be a predictive factor for application in anti-cancer therapy with BPR0L075 in human cancer cells.

Yang, Pei-Ming; Chen, Chiung-Tong; Chao, Jung-Chi; Lin, Ming-Der; Chiu, Shu-Jun



MicroRNA-21 induces stemness by downregulating transforming growth factor beta receptor 2 (TGF?R2) in colon cancer cells  

PubMed Central

Although microRNA-21 (miR-21) is emerging as an oncogene and has been shown to target several tumor suppressor genes, including programmed cell death 4 (PDCD4), its precise mechanism of action on cancer stem cells (CSCs) is unclear. Herein, we report that FOLFOX-resistant HCT-116 and HT-29 cells that are enriched in CSCs show a 3- to 7-fold upregulation of pre- and mature miR-21 and downregulation of PDCD4. Likewise, overexpression of miR-21 in HCT-116 cells, achieved through stable transfection, led to the downregulation of PDCD4 and transforming growth factor beta receptor 2 (TGF?R2). In contrast, the levels of ?-catenin, TCF/LEF activity and the expression of c-Myc, Cyclin-D, which are increased in CSCs, are also augmented in miR-21 overexpressing colon cancer cells, accompanied by an increased sphere forming ability in vitro and tumor formation in SCID mice. Downregulation of TGF?R2 could be attributed to decreased expression of the receptor as evidenced by reduction in the activity of the luciferase gene construct comprising TGF?R2-3? untranslated region (UTR) sequence that binds to miR-21. Moreover, we observed that downregulation of miR-21 enhances luciferase-TGF?R2-3? UTR activity suggesting TGF?R2 as being one of the direct targets of miR-21. Further support is provided by the observation that transfection of TGF?R2 in HCT-116 cells attenuates TCF/LEF luciferase activity, accompanied by decreased expression of ?-catenin, c-Myc and Cyclin-D1. Our current data suggest that miR-21 plays an important role in regulating stemness by modulating TGF?R2 signaling in colon cancer cells.

Yu, Yingjie; Kanwar, Shailender S.; Patel, Bhaumik B.; Oh, Phil-Sun; Nautiyal, Jyoti; Sarkar, Fazlul H.; Majumdar, Adhip P.N.



Overexpression of miR22 reverses paclitaxel-induced chemoresistance through activation of PTEN signaling in p53 -mutated colon cancer cells  

Microsoft Academic Search

Chemoresistance is a key cause of treatment failure in colon cancer. MiR-22 is a tumor-suppressing microRNA. To explore whether\\u000a miR-22 is an important player in the development of chemoresistance in colon cancer, we overexpressed miR-22 and subsequently\\u000a tested its role in cell proliferation, apoptosis, survival, and associated signaling in p53-mutated HT-29 and HCT-15 cells, and p53 wild-type HCT-116 cells. We

Jian Li; Yangde Zhang; Jingfeng Zhao; Fangren Kong; Yuxiang Chen


Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer  

PubMed Central

Introduction We evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients. Methods EPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years. Results CK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis). Conclusions Full-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.

Alix-Panabieres, Catherine; Vendrell, Jean-Pierre; Slijper, Monique; Pelle, Olivier; Barbotte, Eric; Mercier, Gregoire; Jacot, William; Fabbro, Michel; Pantel, Klaus



PAMAM-Camptothecin Conjugate Inhibits Proliferation and Induces Nuclear Fragmentation in Colorectal Carcinoma Cells  

Microsoft Academic Search

Purpose  To synthesize and characterize a poly (amido amine) dendrimer-camptothecin (PAMAM-CPT) conjugate and evaluate its activity\\u000a on human colorectal carcinoma cells (HCT-116).\\u000a \\u000a \\u000a \\u000a \\u000a Methods  The attachment of CPT to amine-terminated PAMAM was facilitated through a succinic acid-glycine linker. The conjugate was\\u000a characterized for absence of small molecular weight impurities, size and drug content. Stability of the conjugate in PBS and\\u000a growth media and

Giridhar Thiagarajan; Abhijit Ray; Alexander Malugin; Hamidreza Ghandehari



Apoptotic-like death occurs through a caspase-independent route in colon carcinoma cells undergoing mitotic catastrophe.  


We have examined the relationship between chemotherapy-induced mitotic catastrophe and cell death by apoptosis in both wild-type and p53(-/-) HCT116 human colon carcinoma cells treated with nanomolar concentrations of paclitaxel (PTX), a drug that acts on tubulin altering the normal development of mitosis. After treatment, HCT116 cells entered mitosis regardless of the presence of functional p53, which resulted in changes in the distribution of cells in the different phases of the cell cycle, and in cell death. In the presence of PTX, the percentage of polyploid cells observed was higher in p53-deficient cells, indicating that mitotic slippage was favored compared to wild-type cells, with the presence of large multinucleate cells. PTX caused mitotic catastrophe and about 50-60% cells that were entering an aberrant mitosis died through an apoptotic-like pathway characterized by the presence of phosphatidylserine in the outer cell membrane, which occurred in the absence of significant activation of caspases. Lack of p53 facilitated endoreduplication and polyploidy in PTX-treated cells, but cells were still killed with similar efficacy through the same apoptotic-like mechanism in the absence of caspase activity. PMID:22885806

Llovera, Laia; Mansilla, Sylvia; Portugal, José



Red blood cell production from immortalized progenitor cell line  

Microsoft Academic Search

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If immortalized erythroid progenitor\\u000a cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell\\u000a lines have not been established. We have developed a robust method to establish immortalized erythroid progenitor cell lines\\u000a following the induction of hematopoietic

Yukio Nakamura; Takashi Hiroyama; Kenichi Miharada; Ryo Kurita



Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling  

Microsoft Academic Search

Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model.Compound 8e exhibited the potential growth inhibition

Hwa-Jin Chung; Hee-Kyung Rhee; Sang Kook Lee; Hea-Young Park Choo



Serine/threonine kinase 17A is a novel p53 target gene and modulator of cisplatin toxicity and reactive oxygen species in testicular cancer cells.  


Testicular cancer is highly curable with cisplatin-based therapy, and testicular cancer-derived human embryonal carcinoma (EC) cells undergo a p53-dominant transcriptional response to cisplatin. In this study, we have discovered that a poorly characterized member of the death-associated protein family of serine/threonine kinases, STK17A (also called DRAK1), is a novel p53 target gene. Cisplatin-mediated induction of STK17A in the EC cell line NT2/D1 was prevented with p53 siRNA. Furthermore, STK17A was induced with cisplatin in HCT116 and MCF10A cells but to a much lesser extent in isogenic p53-suppressed cells. A functional p53 response element that binds endogenous p53 in a cisplatin-dependent manner was identified 5 kb upstream of the first coding exon of STK17A. STK17A is not present in the mouse genome, but the closely related gene STK17B is induced with cisplatin in mouse NIH3T3 cells, although this induction is p53-independent. Interestingly, in human cells containing both STK17A and STK17B, only STK17A is induced with cisplatin. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with the up-regulation of detoxifying and antioxidant genes, including metallothioneins MT1H, MT1M, and MT1X that have previously been implicated in cisplatin resistance. In addition, knockdown of STK17A resulted in decreased cellular reactive oxygen species, whereas STK17A overexpression increased reactive oxygen species. In summary, we have identified STK17A as a novel direct target of p53 and a modulator of cisplatin toxicity and reactive oxygen species in testicular cancer cells. PMID:21489989

Mao, Pingping; Hever, Mary P; Niemaszyk, Lynne M; Haghkerdar, Jessica M; Yanco, Esty G; Desai, Damayanti; Beyrouthy, Maroun J; Kerley-Hamilton, Joanna S; Freemantle, Sarah J; Spinella, Michael J



The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site-specific cancer risk modification  

PubMed Central

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer while it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

Sohn, Kyoung-Jin; Jang, Hyeran; Campan, Mihaela; Weisenberger, Daniel J.; Dickhout, Jeffrey; Wang, Yi-Cheng; Cho, Robert C.; Yates, Zoe; Lucock, Mark; Chiang, En-Pei; Austin, Richard C.; Choi, Sang-Woon; Laird, Peter W.; Kim, Young-In



Mechanism of Alternariol monomethyl ether-induced mitochondrial apoptosis in human colon carcinoma cells.  


Alternariol monomethyl ether (AME) is a major mycotoxin produced by fungi of the genus Alternaria and a common contaminant of food products such as fruits and cereals worldwide. AME can cause serious health problems for animals as well as for humans. In this study, human colon carcinoma cells (HCT116) were used to explore the mechanisms of cell death induced by AME. Exposure of HCT116 cells to AME resulted in significant cytotoxicity manifested by a loss in cell viability mainly mediated by activation of apoptotic process. AME activated the mitochondrial apoptotic pathway evidenced by the opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (??m) downstream generation of O(2)(-), cytochrome c release and caspase 9 and 3 activation. Experiments conducted on isolated organelles indicated that AME does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Moreover, no difference was observed in Bax-KO cells in comparison to parental cells, suggesting that the pro-apoptotic protein Bax is not involved in AME-induced mitochondrial apoptosis. Our findings demonstrate for the first time that AME induces cell death in human colon carcinoma cells by activating the mitochondrial pathway of apoptosis. PMID:22001388

Bensassi, Fatma; Gallerne, Cindy; el Dein, Ossama Sharaf; Hajlaoui, Mohamed Rabeh; Bacha, Hassen; Lemaire, Christophe



MCM-2 is a therapeutic target of Trichostatin A in colon cancer cells.  


Histone deacetylase (HDAC) inhibitors have recently emerged as a new class of anti-cancer agents. Trichostatin A (TSA), a classical HDAC inhibitor, has been demonstrated to induce cell cycle arrest, promote cell apoptosis, and inhibit metastasis. However, the molecular mechanism underlying TSA function has not been fully elucidated. In the current study, we found that TSA treatment induced altered expression of cell cycle-associated genes in HCT116 cells by RT-PCR array. Among the 84 genes related to cell cycle control, 34 genes were significantly altered by TSA treatment, with 7 genes upregulated and 27 genes downregulated. Interestingly, gene expression of minichromosome maintenance protein-2 (MCM-2) was significantly downregulated by TSA treatment. This was confirmed by quantitative RT-PCR and Western blotting. Moreover, silencing of MCM-2 by siRNA led to cell cycle arrest and apoptosis in HCT116 cells. In addition, TSA caused an increase of phosphorylated JNK, which was involved in downregulation of MCM-2. Together, our results suggest that MCM-2 is a noval therapeutic target of TSA in colon cancer cells. PMID:23770000

Liu, Yangbo; He, Gang; Wang, Yan; Guan, Xinging; Pang, Xueli; Zhang, Bo



Cell Lines Secreting Uteroglobin In vitro.  

National Technical Information Service (NTIS)

The invention is two immortal cell lines that secrete uteroglobin in vitro when stimulated by a steroid hormone. The invention includes a method for screening the steroid stimulating property of a compound.

A. B. Mukherjee J. Y. Chou



Ecdysone action on insect cell lines  

Microsoft Academic Search

Summary  Cell lines derived from embryos of the tobacco hornworm,Manduca sexta (L.), showed a marked morphological response to treatment with physiological doses of ?-ecdysone. The response of these cell\\u000a lines with ?-ecdysone indicated that the penta-ol (?-ecdysone) must be converted to the hexa-ol (?-ecdysone) form before the\\u000a morphological response can appear. Liquid chromatographic analysis of the spent medium confirmed that the

Edwin P. Marks; G. Mark Holman



Lactobacillus gasseri SF1183 affects intestinal epithelial cell survival and growth.  


It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host's intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis. PMID:23894414

Di Luccia, Blanda; Manzo, Nicola; Baccigalupi, Loredana; Calabrò, Viola; Crescenzi, Elvira; Ricca, Ezio; Pollice, Alessandra



Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies  

PubMed Central

The large molecular size of antibodies is considered one major factor preventing them from becoming more efficient therapeutically. It is well established that all camelids have unique antibodies circulating in their blood called heavy-chain antibodies (HcAbs). Unlike antibodies from other species, these HcAbs contain a single variable domain and two constant domains (CH2 and CH3). HcAbs are a novel type of immunoglobulin-like, antigen binding protein with beneficial pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. Since the antigen-binding site of dromedary HcAb is comprised in one single domain, it was referred to as nanobody. In the present work, the different IgG subclasses from immunized camel (Camelus dromedairus) were purified employing their different affinity for protein A column (PA) and protein G column (PG). Characterization of IgG subclasses was done by using 12% SDS–PAGE under reducing conditions. Protein bands were visualized after staining with Coomassie Brilliant Blue, showing two bands at 50 kDa and 30 kDa in case of IgG1 while IgG2 and IgG3 produce only one band at 46 kDa and 43 kDa respectively. The induction of apoptosis by either conventional or nanobodies was evaluated on two different cell lines, Colon and Hepatic cancer cell (HCT116 and HepG2), using the comet assay. Induced apoptosis were confirmed by visualizing DNA fragmentation bands on 2% agarose gel, and the gel was photographed under UV light. This study demonstrates the successful targeting of human cancer colon cell lines by nanobodies in vitro. It may open perspectives for their future use as tumor target vehicle, due to their small size, soluble behavior and they interact with epitopes that are less antigenic for conventional antibodies.

Shaker, Ghada H.; Melake, Nahla A.



Acquired irinotecan resistance is accompanied by stable modifications of cell cycle dynamics independent of MSI status.  


Irinotecan is a major anticancer agent specifically targeting DNA topoisomerase I. Its cytotoxicity is mediated via a two-step process involving accumulation of reversible DNA?topoisomerase I complexes associated with transient DNA single-strand breaks which subsequently are converted into permanent DNA double-strand breaks by the replication fork during S phase. Irinotecan may be selectively active for treatment of colorectal cancers that show microsatellite instability (MSI) due to deficiencies in mismatch repair enzymes, compared to tumors that are microsatellite stable but show chromosome instability (CIN). Although the clinical activity of irinotecan is principally limited by acquired drug resistance, surprisingly little is known about the influence of prolonged irinotecan exposure on the cell cycle dynamics. We have developed two colon cancer cell lines resistant to SN-38, the active metabolite of irinotecan, one derived from HT-29 (CIN), the other from HCT-116 (MSI). We here show that besides classical resistance mechanisms, SN-38 resistance is accompanied by an increased generation doubling time, a decreased S phase fraction and an increased G2 fraction in vitro as in tumor xenografts for both CIN and MSI models. As a consequence, SN-38-resistant cells and tumors show cross-resistance to the S-phase selective agent 5-fluorouracil. The resistance is accompanied by increased basal levels of ?-H2AX and phospho-Chk2 without notable changes in the levels of phospho-Chk1. Taken together, our results show that prolonged irinotecan exposure is accompanied by stable modifications of cell cycle dynamics which could have profound impact on tumor sensitivity to a wide range of antitumor agents and may influence tumor progression in patients. PMID:23546019

Petitprez, Amélie; Poindessous, Virginie; Ouaret, Djamila; Regairaz, Marie; Bastian, Gérard; Guérin, Eric; Escargueil, Alexandre E; Larsen, Annette K



Global dissociation of HuR-mRNA complexes promotes cell survival after ionizing radiation  

PubMed Central

Ionizing radiation (IR) triggers adaptive changes in gene expression. Here, we show that survival after IR strongly depends on the checkpoint kinase Chk2 acting upon its substrate HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. Microarray analysis showed that in human HCT116 colorectal carcinoma cells (WT), IR-activated Chk2 triggered the dissociation of virtually all of HuR-bound mRNAs, since IR did not dissociate HuR target mRNAs in Chk2-null (CHK2?/?) HCT116 cells. Accordingly, several HuR-interacting mRNAs encoding apoptosis- and proliferation-related proteins (TJP1, Mdm2, TP53BP2, Bax, K-Ras) dissociated from HuR in WT cells, but remained bound and showed altered post-transcriptional regulation in CHK2?/? cells. Use of HuR mutants that were not phosphorylatable by Chk2 (HuR(3A)) and HuR mutants mimicking constitutive phosphorylation by Chk2 (HuR(3D)) revealed that dissociation of HuR target transcripts enhanced cell survival. We propose that the release of HuR-bound mRNAs via an IR-Chk2-HuR regulatory axis improves cell outcome following IR.

Masuda, Kiyoshi; Abdelmohsen, Kotb; Kim, Mihee M; Srikantan, Subramanya; Lee, Eun Kyung; Tominaga, Kumiko; Selimyan, Roza; Martindale, Jennifer L; Yang, Xiaoling; Lehrmann, Elin; Zhang, Yongqing; Becker, Kevin G; Wang, Jian-Ying; Kim, Hyeon Ho; Gorospe, Myriam



Caveolin-1-Mediated Expression and Secretion of Kallikrein 6 in Colon Cancer Cells1  

PubMed Central

Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6) has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1), the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion.

Henkhaus, Rebecca S; Roy, Upal Kunal Basu; Cavallo-Medved, Dora; Sloane, Bonnie F; Gerner, Eugene W; Ignatenko, Natalia A



Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling.  


Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O2. A decrease in iO2 (intracellular O2) to 0-10 ?M, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1? and HIF-2?, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO2 nor HIF-? stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-? stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments. PMID:22943412

Zhdanov, Alexander V; Dmitriev, Ruslan I; Papkovsky, Dmitri B



Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling  

PubMed Central

Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O2. A decrease in iO2 (intracellular O2) to 0–10 ?M, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1? and HIF-2?, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO2 nor HIF-? stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-? stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.

Zhdanov, Alexander V.; Dmitriev, Ruslan I.; Papkovsky, Dmitri B.



Suppression of Src/ERK and GSK-3/?-catenin signaling by pinosylvin inhibits the growth of human colorectal cancer cells.  


Pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, has exhibited a potential cancer chemopreventive activity. However, the growth inhibitory activity against cancer cells and the underlying molecular mechanisms remain to be elucidated. Therefore, the anti-proliferative activity of pinosylvin was investigated in human colorectal HCT 116 cancer cells. Pinosylvin inhibited the proliferation of HCT 116 cells by arresting transition of cell cycle from G1 to S phase along with the downregulation of cyclin D1, cyclin E, cyclin A, cyclin dependent kinase 2 (CDK2), CDK4, c-Myc, and retinoblastoma protein (pRb), and the upregulation of p21(WAF1/CIP1) and p53. Pinosylvin was also found to attenuate the activation of proteins involved in focal adhesion kinase (FAK)/c-Src/extracellular signal-regulated kinase (ERK) signaling, and phosphoinositide 3-kinase (PI3K)/Akt/ glycogen synthase kinase 3? (GSK-3?) signaling pathway. Subsequently, pinosylvin suppressed the nuclear translocation of ?-catenin, one of downstream molecules of PI3K/Akt/GSK-3? signaling, and these events led to the sequential downregulation of ?-catenin-mediated transcription of target genes including BMP4, ID2, survivin, cyclin D1, MMP7, and c-Myc. These findings demonstrate that the anti-proliferative activity of pinosylvin might be associated with the cell cycle arrest and downregulation of cell proliferation regulating signaling pathways in human colorectal cancer cells. PMID:23333577

Park, Eun-Jung; Chung, Hwa-Jin; Park, Hyen Joo; Kim, Gi Dae; Ahn, Yong-Hyun; Lee, Sang Kook



Impact of Phytolacca americana Extracts on Gene Expression of Colon Cancer Cells.  


Native Americans have used Phytolacca americana to treat breast ailments, gastrointestinal disorders, rashes, and inflammation. Some anti-cancer and anti-viral research has been reported on this perennial herb, but none has been published concerning the effects of its extracts on cancer cell genes. In this study, changes in gene expression at the transcription level were evaluated in HCT-116 colon cancer cells after exposure to P. americana ethanol extract and its water fraction using the Human Cancer Pathway Finder PCR Array. Of the genes significantly affected in HCT-116 cells exposed to the ethanol extract at 3200?µg/ml, changes in expression of MYC, PLAU, and TEK may benefit the treatment of colon cancer. Exposing the cells to 1600?µg/ml of the water fraction resulted in several gene changes that may also be beneficial in the treatment of colon cancer: NME4, TEK, and THBS1. A few genes on this array that are known to play a specific role in colon cancer had activities changed in a way that may be detrimental in the treatment of colon cancer. Further studies should be performed to understand how these changes would impact colon cancer treatment. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23553997

Maness, L; Goktepe, I; Chen, H; Ahmedna, M; Sang, S



Anti-proliferative effects of a new docosapentaenoic acid monoacylglyceride in colorectal carcinoma cells.  


N-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to inhibit the induction and progression of many tumor types. However, the anticancer effect of n-3 PUFA monoglyceride on colorectal cancer has yet to be assessed. The aim of the present study was to determine the anti-tumorigenic effects of docosahexaenoic acid monoglyceride (MAG-DHA), eicosapentaenoic acid monoglyceride (MAG-EPA) and docosapentaenoic acid (22:5n-3) monoglyceride (MAG-DPA) in colorectal carcinoma cells. Our results demonstrate that MAG-DHA, MAG-EPA and MAG-DPA all decreased cell proliferation and induced apoptosis in HCT116 cells, with MAG-DPA having the higher anti-proliferative and pro-apoptotic effects in vitro. In a HCT116 xenograft mouse model, oral administration of MAG-DPA significantly inhibited tumor growth. Furthermore, MAG-DPA treatments decreased NF?B activation leading to a reduction in Bcl-2, CyclinD1, c-myc, COX-2, MMP9 and VEGF expression levels in tumor tissue sections. Altogether, these data provide new evidence regarding the mode of action of MAG-DPA in colorectal cancer cells. PMID:23932824

Morin, Caroline; Rousseau, Eric; Fortin, Samuel



Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines  

PubMed Central

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB ( is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (, that allows to link authentication data to actual cell lines.

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara



Fish cell lines: Establishment and characterization of nine cell lines from salmonids  

Microsoft Academic Search

Summary  Nine permanent cell lines have been established from five species of salmonids native to America's Pacific Northwest. With\\u000a the exception of a hepatoma from an adult trout, the lines were derived from normal tissues of embryonic or juvenile fish.\\u000a Cells were routinely grown in Eagle's minimum essential medium with 10% fetal bovine serum. Optimum growth temperatures for\\u000a these lines ranged

C. N. Lannan; J. R. Winton; J. L. Fryer



Disruption of microRNA biogenesis confers resistance to ER stress-induced cell death upstream of the mitochondrion.  


Global downregulation of microRNAs (miRNAs) is a common feature of human tumors and has been shown to enhance cancer progression. Several components of the miRNA biogenesis machinery (XPO5, DICER and TRBP) have been shown to act as haploinsufficient tumor suppressors. How the deregulation of miRNA biogenesis promotes tumor development is not clearly understood. Here we show that loss of miRNA biogenesis increased resistance to endoplasmic reticulum (ER) stress-induced cell death. We observed that HCT116 cells with a DICER hypomorphic mutation (Exn5/Exn5) or where DICER or DROSHA were knocked down were resistant to ER stress-induced cell death. Extensive analysis revealed little difference in the unfolded protein response (UPR) of WT compared to Exn5/Exn5 HCT116 cells upon ER stress treatment. However, analysis of the intrinsic apoptotic pathway showed that resistance occurred upstream of the mitochondria. In particular, BAX activation and dissipation of mitochondrial membrane potential was attenuated, and there was altered expression of BCL-2 family proteins. These observations demonstrate a key role for miRNAs as critical modulators of the ER stress response. In our model, downregulation of miRNA biogenesis delays ER stress-induced apoptosis. This suggests that disrupted miRNA biogenesis may contribute to cancer progression by inhibiting ER stress-induced cell death. PMID:23977393

Cawley, Karen; Logue, Susan E; Gorman, Adrienne M; Zeng, Qingping; Patterson, John; Gupta, Sanjeev; Samali, Afshin



The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells  

PubMed Central

BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor ?B and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD.

Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; Garcia, Noemi Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nunez, Gabriel; McDonald, Christine



Caudal Homeobox Protein Cdx-2 Cooperates with Wnt Pathway to Regulate Claudin-1 Expression in Colon Cancer Cells  

PubMed Central

Dysregulation of tight junctions (TJs) is often associated with human diseases including carcinogenesis and recent studies support role of TJ integral proteins in the regulation of Epithelial-to-Mesenchymal Transition (EMT). In this regard, expression of claudin-1, a key constituent of TJs, is highly increased in colon cancer and is causally associated with the tumor growth and progression. However, mechanism/s underlying regulation of claudin-1 expression in intestinal epithelial cells remains poorly understood. In our studies, we have identified putative binding sites for intestinal transcription factors Cdx1, -2 and GATA4 in the 5?-flanking region of the claudin-1 gene. Our further studies using full length and/or deletion mutant constructs in two different human colon cancer cell lines, SW480 and HCT116, showed key role of Cdx1, Cdx2 and GATA4 in the regulation of claudin-1 mRNA expression. However, overexpression of Cdx2 had the most potent effect upon claudin-1 mRNA expression and promoter activity. Also, in colon cancer patient samples, we observed a significant and parallel correlation between claudin-1 and Cdx2 expressions. Chromatin immunoprecipitation (ChIP) assay confirmed the Cdx2 binding with claudin-1 promoter in vivo. Using Cdx2 deletion mutant constructs, we further mapped the Cdx2 C-terminus domain to be important in the regulation of claudin-1 promoter activity. Interestingly, co-expression of activated ?-catenin further induced the Cdx2-dependent upregulation of claudin-1 promoter activity while expression of the dominant negative (dn)-TCF-4 abrogated this activation. Taken together, we conclude that homeodomain transcription factors Cdx1, Cdx2 and GATA4 regulate claudin-1 gene expression in human colon cancer cells. Moreover, a functional crosstalk between Wnt-signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins and GATA-proteins is demonstrated in the regulation of claudin-1 promoter-activation.

Bhat, Ajaz A.; Sharma, Ashok; Pope, Jillian; Krishnan, Moorthy; Washington, Mary K.; Singh, Amar B.; Dhawan, Punita



The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells  

PubMed Central

Colorectal cancer (CRC) is a major cause of tumor-related morbidity and mortality worldwide. Recent research suggests that pharmacological intervention using dietary factors that activate the redox sensitive Nrf2/Keap1-ARE signaling pathway may represent a promising strategy for chemoprevention of human cancer including CRC. In our search for dietary Nrf2 activators with potential chemopreventive activity targeting CRC, we have focused our studies on trans-cinnamic aldehyde (cinnamaldeyde, CA), the key flavor compound in cinnamon essential oil. Here we demonstrate that CA and an ethanolic extract (CE) prepared from Cinnamomum cassia bark, standardized for CA content by GC-MS analysis, display equipotent activity as inducers of Nrf2 transcriptional activity. In human colon cancer cells (HCT116, HT29) and non-immortalized primary fetal colon cells (FHC), CA- and CE-treatment upregulated cellular protein levels of Nrf2 and established Nrf2 targets involved in the antioxidant response including heme oxygenase 1 (HO-1) and ?-glutamylcysteine synthetase (?-GCS, catalytic subunit). CA- and CE-pretreatment strongly upregulated cellular glutathione levels and protected HCT116 cells against hydrogen peroxide-induced genotoxicity and arsenic-induced oxidative insult. Taken together our data demonstrate that the cinnamon-derived food factor CA is a potent activator of the Nrf2-orchestrated antioxidant response in cultured human epithelial colon cells. CA may therefore represent an underappreciated chemopreventive dietary factor targeting colorectal carcinogenesis.

Wondrak, Georg T.; Villeneuve, Nicole F.; Lamore, Sarah D.; Bause, Alexandra S.; Jiang, Tao; Zhang, Donna D.



DNA methyltransferase 1 is essential for initiation of the colon cancers.  


DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs. PMID:23064049

Morita, Rena; Hirohashi, Yoshihiko; Suzuki, Hiromu; Takahashi, Akari; Tamura, Yasuaki; Kanaseki, Takayuki; Asanuma, Hiroko; Inoda, Satoko; Kondo, Toru; Hashino, Satoshi; Hasegawa, Tadashi; Tokino, Takashi; Toyota, Minoru; Asaka, Masahiro; Torigoe, Toshihiko; Sato, Noriyuki



Peptidomic analysis of human cell lines  

PubMed Central

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.



Fish cell lines: Establishment and characterization of three new cell lines from grass carp ( Ctenopharyngodon idella )  

Microsoft Academic Search

Summary  Three new cell lines were established from tissues of the grass carp,Ctenopharyngodon idella. Derived from the fin, snout, and swim bladder of two apparently healthy diploid fry, these cell lines have been designated\\u000a GCF, GCS-2, and GCSB, respectively. The cells grew at temperatures between 24° and 36° C with optimal growth at 32° C and\\u000a have been subcultured more than

Yuanan Lu; C. N. Lannan; J. S. Rohovec; J. L. Fryer



The clinical relevance of cancer cell lines.  


Although advances in genomics during the last decade have opened new avenues for translational research and allowed the direct evaluation of clinical samples, there is still a need for reliable preclinical models to test therapeutic strategies. Human cancer-derived cell lines are the most widely used models to study the biology of cancer and to test hypotheses to improve the efficacy of cancer treatment. Since the development of the first cancer cell line, the clinical relevance of these models has been continuously questioned. Based upon recent studies that have fueled the debate, we review the major events in the development of the in vitro models and the emergence of new technologies that have revealed important issues and limitations concerning human cancer cell lines as models. All cancer cell lines do not have equal value as tumor models. Some have been successful, whereas others have failed. However, the success stories should not obscure the growing body of data that motivates us to develop new in vitro preclinical models that would substantially increase the success rate of new in vitro-assessed cancer treatments. PMID:23434901

Gillet, Jean-Pierre; Varma, Sudhir; Gottesman, Michael M



Characterization of a new megakaryocytic cell line: the Dami cell  

Microsoft Academic Search

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryo- blastic leukemia. The Dami cells grow primarily in suspen- sion with a doubling time of 24 to 30 hours. By light and electron microscopy. the Dami cells range in size from 1 2 to 120 ?tm in diameter and have lobulated nuclei

SM Greenberg; DS Rosenthal; TA Greeley; R Tantravahi; RI Handin



Interaction of over-the-counter drugs with curcumin: influence on stability and bioactivities in intestinal cells.  


Curcumin, a major constituent in rhizomes of Curcuma longa L., has shown various biological activities. It has widely been used as a food additive to provide potential health benefits. In the present study, we investigated changes in chemical stability and cytotoxic properties of curcumin and commonly consumed over-the-counter (OTC) drugs including ibuprofen, acetylsalicylic acid (Asp), and acetaminophen (AAP), through their interactions. Stability of curcumin was significantly improved in phosphate-buffered saline or 0.01 N HCl containing each OTC drug; Asp showed the most prominent effect. Stability of Asp or AAP during 24 h incubation with curcumin was not changed significantly. Cytotoxic effects of curcumin were enhanced in the presence of the OTC drugs on INT 407 normal intestinal and HCT 116 colon cancer cells. Relative cytotoxicity of curcumin (>10 ?M) under the drug-treated conditions was significantly higher. Cellular uptake of curcumin in HCT 116 cells increased significantly when incubated with Asp or AAP. Intracellular thiol levels of the cells treated with curcumin were further reduced in the presence of the OTC drugs. The present study provides information that commonly consumed OTC drugs affect chemical stability of curcumin in physiological conditions, and certain bioactivities of curcumin can be altered through their interactions. PMID:23025432

Choi, Hyun A; Kim, Mi-Ri; Park, Kyung A; Hong, Jungil



Baicalein, an active component of Scutellaria baicalensis Georgi, induces apoptosis in human colon cancer cells and prevents AOM/DSS-induced colon cancer in mice.  


Flavonoids have been demonstrated to provide health benefits in humans. Baicalein (5,6,7-trihydroxyflavone) is a phenolic flavonoid compound derived mainly from the root of Scutellaria baicalensis Georgi, a medicinal plant traditionally used in oriental medicine. Baicalein is widely used in Korean and Chinese herbal medicines as anti-inflammatory and anticancer therapy. However, the molecular mechanisms of its activity remain poorly understood and warrant further investigation. This study was performed to investigate the anticancer effect of baicalein on HCT116 human colon cancer cells and the tumor preventing capacity of baicalein on colitis-associated cancer in mice. In in vivo experiments, we induced colon tumors in mice by azoxymethane (AOM) and dextran sulfate sodium (DSS) and evaluated the effects of baicalein on tumor growth. Baicalein treatment on HCT116 cells resulted in a concentration-dependent inhibition of cell growth and induction of apoptotic cell death. The induction of apoptosis was determined by morphological changes and cleavage of poly(ADP-ribose) polymerase. Baicalein also suppressed the activation of NF-?B through PPAR? activation. These results indicate that the anti-inflammatory effects of baicalein may be mediated through PPAR? activation. Finally, administration with baicalein significantly decreased the incidence of tumor formation with inflammation. Our findings suggest that baicalein is one of the candidates for the prevention of inflammation-associated colon carcinogenesis. PMID:24008356

Kim, Dong Hwan; Hossain, Mohammad Akbar; Kang, Yong Jung; Jang, Jung Yoon; Lee, Yu Jin; Im, Eunok; Yoon, Jeong-Hyun; Kim, Hyung Sik; Chung, Hae Young; Kim, Nam Deuk



Glioma Cell Lines: Role of Cancer Stem Cells  

Microsoft Academic Search

\\u000a In this chapter, we review the cancer stem cell hypothesis and discuss implications for this paradigm in considering whether\\u000a glioma cell lines contain bonafide cancer stem cells, the source material used for tissue culture, and experimental methods\\u000a used in preclinical research. We identify three key modifications to standard tissue culture protocols that allow for enrichment\\u000a of cancer stem cells that

John R. Ohlfest; Stacy A. Decker


New cell lines with chondrocytic phenotypes from human chondrosarcoma  

Microsoft Academic Search

In the present study, we investigated chondrocytic characterization for newly established human chondrosarcoma cell lines. A chondrosarcoma cell line, HCS-TG, was established by the implantation of grade-2 human chondrosarcoma into athymic mice. Cloning of HCS-TG cells from passage 17 was performed. After cell cloning, two clonal-cell lines (HCS-TG C3 and E2) with good proliferative activities were obtained. These cell lines

Ikuo Kudawara; Nobuhito Araki; Akira Myoui; Yoichi Kato; Atsumasa Uchida; Hideki Yoshikawa



Evaluation of anti-cancer activity of Acanthester planci extracts obtained by different methods of extraction.  


Acanthaster planci, the crown-of-thorns starfish, naturally endowed with the numerous toxic spines around the dorsal area of its body. Scientific investigations demonstrated several toxico-pharmacological efficacies of A. planci such as, myonecrotic activity, hemorrhagic activity, hemolytic activity, mouse lethality, phospholipase A2 (PLA2) activity, capillary permeability-increasing activity, edema-forming activity, anticoagulant activity and histamine-releasing activity from mast cells. The present study was performed to evaluate the cytotoxic activity of A. planci extracts obtained by different methods of extraction on MCF-7 and HCT-116, human breast and colon cancer cell lines, respectively. Results of the cell proliferation assay showed that PBS extract exhibited very potent cytotoxic activity against both MCF-7 and HCT-116 cell lines with IC(50) of 13.48 ?g/mL and 28.78 ?g/mL, respectively, while the extracts prepared by Bligh and Dyer method showed moderate cytotoxicity effect against MCF-7 and HCT-116 cell lines, for chloroform extract, IC(50) = 121.37 ?g/mL (MCF-7) and 77.65 ?g/mL (HCT-116), and for methanol extract, IC(50) = 46.11 ?g/mL (MCF-7) and 59.29 ?g/mL (HCT-116). However, the extracts prepared by sequential extraction procedure from dried starfish found to be ineffective. This study paves the way for further investigation on the peptide composition in the PBS extract of the starfish to discover potential chemotherapeutic agents. PMID:23009983

Mutee, Ahmed Faisal; Salhimi, Salizawati Muhamad; Ghazali, Farid Che; Aisha, Abdalrahim Fa; Lim, Chung Pin; Ibrahim, Kamarruddin; Asmawi, Mohd Zaini



Involvement of PI3K-AKT-mTOR pathway in protein kinase CKII inhibition-mediated senescence in human colon cancer cells.  


Cellular senescence is a tumor suppression mechanism. We previously reported that CKII downregulation induces senescence in human lung fibroblast IMR-90 and colon cancer HCT116 cells. In this study, potential longevity drugs, including rapamycin, vitamin C, and vitamin E, blocked CKII downregulation-mediated senescence through reduction of reactive oxygen species (ROS) production in HCT116 cells. Since rapamycin is a mammalian target of rapamycin (mTOR) inhibitor, we examined the roles of mTOR and its upstream regulators phosphatidylinositol 3-kinase (PI3K) and AKT in CKII inhibition-mediated senescence. CKII? knock-down or CKII inhibitor treatment strikingly increased phosphorylation of mTOR, p70S6K, an mTOR substrate, and AKT, whereas CKII? overexpression reduced this phosphorylation event. This result indicated that CKII inhibition activated the PI3K-AKT-mTOR pathway. Further, pharmacological inhibition of PI3K and AKT attenuated ROS production and senescence in CKII-downregulated cells. Taken together, these results demonstrate, for the first time, that the PI3K-AKT-mTOR-ROS pathway is necessary for CKII inhibition-mediated cellular senescence. PMID:23523798

Park, Ji Hye; Kim, Jin Joo; Bae, Young-Seuk



Single cell metabolic profiling of tumor mimics.  


Chemical cytometry employs modern analytical methods to study the differences in composition between single cells to better understand development, cellular differentiation, and disease. Metabolic cytometry is a form of chemical cytometry wherein cells are incubated with and allowed to metabolize fluorescently labeled small molecules. Capillary electrophoresis with laser-induced fluorescence detection is then used to characterize the extent of metabolism at the single cell level. To date, all metabolic cytometry experiments have used conventional two-dimensional cell cultures. HCT 116 spheroids are a three-dimensional cell culture system, morphologically and phenotypically similar to tumors. Here, intact HCT 116 multicellular spheroids were simultaneously incubated with three fluorescently labeled glycosphingolipid substrates, GM3-BODIPY-FL, GM1-BODIPY-TMR, and lactosylceramide-BODIPY-650/665. These substrates are spectrally distinct, and their use allows the simultaneous probing of metabolism at three different points in the glycolipid metabolic cascade. Beginning with intact spheroids, a serial trypsinization and trituration procedure was used to isolate single cells from spatially distinct regions of the spheroid. Cells from the distinct regions showed unique metabolic patterns. Treatment with the lysosomal inhibitor and potential chemotherapeutic chloroquine consistently decreased catabolism for all substrates. Nearly 200 cells were taken for analysis. Principal component analysis with a multivariate measure of precision was used to quantify cell-to-cell variability in glycosphingolipid metabolism as a function of cellular localization and chloroquine treatment. While cells from different regions exhibited differences in metabolism, the heterogeneity in metabolism did not differ significantly across the experimental conditions. PMID:24011091

Keithley, Richard B; Weaver, Eric M; Rosado, Andrea M; Metzinger, Mark P; Hummon, Amanda B; Dovichi, Norman J



Establishment and characterisation of two novel breast cancer cell lines  

Microsoft Academic Search

Two novel oestrogen receptor (ER) negative breast cancer cell lines, BCa-11 (familial) and BCa-15 (sporadic) were successfully established from primary tumours. Characterisation of these cell lines showed expression of epithelial specific antigen and cytokeratins confirming their epithelial lineage. Analysis of ultrastructure and anchorage independent growth confirmed the epithelial nature and transformed phenotype of these cells. Both cell lines showed loss

Sarangadhara Appala Raju Bagadi; Jatinder Kaur; Ranju Ralhan



Generation of Human Pulmonary Microvascular Endothelial Cell Lines  

Microsoft Academic Search

The limited lifespan of human microvascular endothelial cells in cell culture represents a major obstacle for the study of microvascular pathobiology. To date, no endothelial cell line is available that demonstrates all of the fundamental characteristics of microvascular endothelial cells. We have generated endothelial cell lines from human pulmonary microvascular endothelial cells (HPMEC) isolated from adult donors. HPMEC were cotransfected

Vera Krump-Konvalinkova; Fernando Bittinger; Ronald E Unger; Kirsten Peters; Hans-Anton Lehr; C James Kirkpatrick



CellLineNavigator: a workbench for cancer cell line analysis  

PubMed Central

The CellLineNavigator database, freely available at, is a web-based workbench for large scale comparisons of a large collection of diverse cell lines. It aims to support experimental design in the fields of genomics, systems biology and translational biomedical research. Currently, this compendium holds genome wide expression profiles of 317 different cancer cell lines, categorized into 57 different pathological states and 28 individual tissues. To enlarge the scope of CellLineNavigator, the database was furthermore closely linked to commonly used bioinformatics databases and knowledge repositories. To ensure easy data access and search ability, a simple data and an intuitive querying interface were implemented. It allows the user to explore and filter gene expression, focusing on pathological or physiological conditions. For a more complex search, the advanced query interface may be used to query for (i) differentially expressed genes; (ii) pathological or physiological conditions; or (iii) gene names or functional attributes, such as Kyoto Encyclopaedia of Genes and Genomes pathway maps. These queries may also be combined. Finally, CellLineNavigator allows additional advanced analysis of differentially regulated genes by a direct link to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources.

Krupp, Markus; Itzel, Timo; Maass, Thorsten; Hildebrandt, Andreas; Galle, Peter R.; Teufel, Andreas



Pancreastatin producing cell line from human pancreatic islet cell tumor.  


It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponentially growing cultures produced 5.7 fmol of pancreastatin/10(6) cells/hr. Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the tumor. Gel filtrations of culture medium and tumor extract identified heterogenous molecular forms of PST-LI which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin. PMID:2159299

Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Ikeda, Y; Kono, A



A bioinformatics analysis of the cell line nomenclature  

PubMed Central

Motivation: Cell lines are used extensively in biomedical research, but the nomenclature describing cell lines has not been standardized. The problems are both linguistic and experimental. Many ambiguous cell line names appear in the published literature. Users of the same cell line may refer to it in different ways, and cell lines may mutate or become contaminated without the knowledge of the user. As a first step towards rationalizing this nomenclature, we created a cell line knowledgebase (CLKB) with a well-structured collection of names and descriptive data for cell lines cultured in vitro. The objectives of this work are: (i) to assist users in extracting useful information from biomedical text and (ii) to highlight the importance of standardizing cell line names in biomedical research. This CLKB contains a broad collection of cell line names compiled from ATCC, Hyper CLDB and MeSH. In addition to names, the knowledgebase specifies relationships between cell lines. We analyze the use of cell line names in biomedical text. Issues include ambiguous names, polymorphisms in the use of names and the fact that some cell line names are also common English words. Linguistic patterns associated with the occurrence of cell line names are analyzed. Applying these patterns to find additional cell line names in the literature identifies only a small number of additional names. Annotation of microarray gene expression studies is used as a test case. The CLKB facilitates data exploration and comparison of different cell lines in support of clinical and experimental research. Availability: The web ontology file for this cell line collection can be downloaded at Contact: Supplementary information: Supplementary data are available at Bioinformatics online.

Sarntivijai, Sirarat; Ade, Alexander S.; Athey, Brian D.; States, David J.



On the Ontology Based Representation of Cell Lines  

PubMed Central

Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT) integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed.

Ganzinger, Matthias; He, Shan; Breuhahn, Kai; Knaup, Petra



Establishment and characterization of four human pancreatic carcinoma cell lines  

Microsoft Academic Search

We characterized four pancreatic carcinoma cell lines (designated SNU-213, SNU-324, SNU-410, and SNU-494) established from histopathologically varied primary or liver metastatic tumor samples of Korean patients. Three cell lines grew as adherent monolayers and one as adherent and floating cell clumps. All lines had: (1) relatively high viability; (2) an absence of mycoplasma or bacterial contamination; (3) genetic heterogeneity as

Ja-Lok Ku; Kyong-Ah Yoon; Woo-Ho Kim; Jin Jang; Kyung-Suk Suh; Sun-Whe Kim; Yong-Hyun Park; Jae-Gahb Park



Human Myeloma Cell Line Carrying a Philadelphia Chromosome  

Microsoft Academic Search

A new human plasmacytoma cell line (Karpas 707) has been established from a myeloma patient. The cultured cells are negative for Epstein-Barr viral nuclear antigen and free of mycoplasma. They are similar to plasma cells and secrete only lambda light chains. The cells are hypodiploid and contain the Philadelphia chromosome and other abnormalities. This cell line may be suitable for

Abraham Karpas; Patricia Fischer; David Swirsky



Role of Glial Cell Line-Derived Neurotrophic Factor in Germ-Line Stem Cell Fate  

PubMed Central

The overall goal of this study is to unravel the role(s) played by glial cell line-derived neurotrophic factor (GDNF) in the fate of spermatogonial stem cells. There is great interest in the biology of spermatogonial stem cells, or Asingle spermatogonia, because of their importance in the treatment of infertility, the development of contraceptives, and the understanding of the etiology of testicular cancer, particularly seminoma. In the mouse, spermatogonial stem cells express GFR?-1, the receptor for GDNF, and respond to this growth factor in vivo and in vitro. GDNF is produced by the adjacent Sertoli cells, which are part of the germ-line stem cell niche in vertebrates. We specifically isolated GFR?-1–positive spermatogonia using an immunomagnetic bead technique. We then stimulated the cells with 100 ng/mL of rGDNF for 10 hours; unstimulated cells served as negative controls. Microarray analysis, immunocytochemistry, and Western blotting revealed that Numb, a regulator of the Notch pathway, is upregulated by GDNF in spermatogonial stem cells. There are indications that in rats, mice, and humans, the Notch pathway promotes spermatogonial differentiation. We observed that an increase in Numb expression is concomitant with Notch degradation in these cells. Thus, through Numb, GDNF might inhibit differentiation and allows the maintenance of the stem cell pool in the mouse seminiferous epithelium.

Braydich-Stolle, Laura; Nolan, Courtney; Dym, Martin; Hofmann, Marie-Claude



Tetanus toxin as a marker for small-cell lung cancer cell lines  

Microsoft Academic Search

Tetanus toxin labeling of human lung cancer cell lines was investigated using direct and indirect immunofluorescence and immunohistochemical staining. Cells of characterized permanent cell lines, eight small-cell lung cancer (SCLC) cell lines of classic subtype, six SCLC cell lines of variant subtype and seven non-small-cell lung cancer (NSCLC) cell lines, were incubated with a saturating concentration of tetanus toxin. For

Jochen Heymanns; Kurt Neumann; Klaus Havemann



Detection Algorithm for the Validation of Human Cell Lines  

PubMed Central

Cell lines are an important tool in understanding all aspects of cancer growth, development, metastasis, and tumor cell death. There has been a dramatic increase in the number of cell lines and diversity of the cancers they represent; however, misidentification and cross-contamination of cell lines can lead to erroneous conclusions. One method that has gained favor for authenticating cell lines is the use of short tandem repeats (STR) to generate a unique DNA profile. The challenge in validating cell lines is the requirement to compare the large number of existing STR profiles against cell lines of interest, particularly when considering that the profiles of many cell lines have drifted over time and original samples are not available. We report here methods that analyze the variations and the proportional changes extracted from tetra-nucleotide repeat regions in the STR analysis. This technique allows a paired match between a target cell line and a reference database of cell lines to find cell lines that match within a user designated percentage cut-off quality matrix. Our method accounts for DNA instability and can suggest whether the target cell lines are misidentified or unstable.

Eltonsy, Nevine; Gabisi, Vivian; Li, Xuesong; Russe, K. Blair; Mills, Gordon B.; Stemke-Hale, Katherine



Road for understanding cancer stem cells: model cell lines.  


There is increasing evidence suggesting that stem cells are susceptive to carcinogenesis and, consequently, can be the origin of many cancers. Recently, the neoplastic potential of stem cells has been supported by many groups showing the existence of subpopulations with stem cell characteristics in tumor biopsies such as brain and breast. Evidence supporting the cancer stem cell hypothesis has gained impact due to progress in stem cell biology and development of new models to validate the self-renewal potential of stem cells. Recent evidence on the possible identification of cancer stem cells may offer an opportunity to use these cells as future therapeutic targets. Therefore, model systems in this field have become very important and useful. This review will focus on the state of knowledge on cancer stem cell research, including cell line models for cancer stem cells. The latter will, as models, help us both in the identification and characterization of cancer stem cells and in the further development of therapeutic strategies including tissue engineering. PMID:18034633

Serakinci, Nedime; Erzik, Can



Match criteria for human cell line authentication: where do we draw the line?  


Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ?80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines. PMID:23136038

Capes-Davis, Amanda; Reid, Yvonne A; Kline, Margaret C; Storts, Douglas R; Strauss, Ethan; Dirks, Wilhelm G; Drexler, Hans G; MacLeod, Roderick A F; Sykes, Gregory; Kohara, Arihiro; Nakamura, Yukio; Elmore, Eugene; Nims, Raymond W; Alston-Roberts, Christine; Barallon, Rita; Los, Georgyi V; Nardone, Roland M; Price, Paul J; Steuer, Anton; Thomson, Jim; Masters, John R W; Kerrigan, Liz



Expression and significance of hypoxia-inducible factor-1 alpha and MDR1/P-glycoprotein in human colon carcinoma tissue and cells  

PubMed Central

Purpose Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance (MDR1) gene/transporter P-glycoprotein (P-gp) has not been clearly investigated. This study aims at examining the expression levels of HIF-1? and MDR1/P-gp in human colon carcinoma tissues and cell lines (HCT-116, HT-29, LoVo, and SW480) and ascertaining whether HIF-1? plays an important role in tumor multidrug resistance with MDR1/P-gp. Methods The expression and distribution of HIF-1? and P-gp proteins were detected in human colon carcinoma tissues and cell lines by immunohistochemistry and immunocytochemistry using streptavidin/peroxidase (SP) and double-label immunofluorescence methods. HIF-1? and MDR1 mRNA expression levels in cell lines were analyzed using RT-PCR under normoxic and hypoxic conditions, respectively. Results The immunohistochemical method shows that HIF-1? and P-gp expression were not correlated with gender, age, location, and differentiation degree (P > 0.05). However, the expression of HIF-1? and P-gp at different Dukes’ stages and whether involved in lymphatic invasion shows a significant difference (P < 0.05). Correlation analysis displays that HIF-1? protein expression was correlated significantly with P-gp expression (P < 0.01). Double-label immunofluorescence demonstrates that coexpression of HIF-1? and P-gp does exist in human colon carcinoma tissues. The mRNA expression of HIF-1? and MDR1 was detected in the four human colon carcinoma cell lines under both normoxia and hypoxia. Optical density values representing mRNA expression levels of HIF-1? and MDR1 were found to be significantly higher in the same type cells under hypoxic conditions than that under normoxic conditions, respectively (P < 0.01). However, no significant differences of HIF-1? or MDR1 mRNA expression were found among these cell lines, which exposed under the same PaO2 cultural conditions (P > 0.05). And the immunocytochemistry results were corresponding with the analysis of mRNA expression. Conclusions These results suggest that hypoxia induce the expression of HIF-1? and MDR1/P-gp in colon carcinoma and HIF-1? expression may be associated with the gene MDR1 (P-gp) and interactively involved in the occurrence of tumor multidrug resistance.

Ding, Zhenyu; Yang, Li; Xie, Xiaodong; Xie, Fangwei; Pan, Feng; Li, Jianjun; He, Jianming



Distinct differentiation characteristics of individual human embryonic stem cell lines  

Microsoft Academic Search

BACKGROUND: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. RESULTS: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could

Milla Mikkola; Cia Olsson; Jaan Palgi; Jarkko Ustinov; Tiina Palomaki; Nina Horelli-Kuitunen; Sakari Knuutila; Karolina Lundin; Timo Otonkoski; Timo Tuuri



Human embryonic stem cell lines derived from the Chinese population  

Microsoft Academic Search

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1–81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed

Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG



Casein kinase-2 mediates cell survival through phosphorylation and degradation of inositol hexakisphosphate kinase-2  

PubMed Central

The inositol pyrophosphate, diphosphoinositol pentakisphosphate, regulates p53 and protein kinase Akt signaling, and its aberrant increase in cells has been implicated in apoptosis and insulin resistance. Inositol hexakisphosphate kinase-2 (IP6K2), one of the major inositol pyrophosphate synthesizing enzymes, mediates p53-linked apoptotic cell death. Casein kinase-2 (CK2) promotes cell survival and is upregulated in tumors. We show that CK2 mediated cell survival involves IP6K2 destabilization. CK2 physiologically phosphorylates IP6K2 at amino acid residues S347 and S356 contained within a PEST sequence, a consensus site for ubiquitination. HCT116 cells depleted of IP6K2 are resistant to cell death elicited by CK2 inhibitors. CK2 phosphorylation at the degradation motif of IP6K2 enhances its ubiquitination and subsequent degradation. IP6K2 mutants at the CK2 sites that are resistant to CK2 phosphorylation are metabolically stable.

Chakraborty, Anutosh; Werner, J. Kent; Koldobskiy, Michael A.; Mustafa, Asif K.; Juluri, Krishna R.; Pietropaoli, Joseph; Snowman, Adele M.; Snyder, Solomon H.



Establishment and characterization of human neuroblastoma cell lines.  


Three new tissue culture cell lines, CHP-100, CHP-126, and CHP-134, have been established from explant cultures of human neuroblastoma. The cell lines have been characterized with respect to morphology, chromosomes constitution, growth, neural enzyme content, and their ability to grow in nude mice. The cells grow as dense masses comprised of fibroblast-or neuroblast-like cells with small processes. The cell lines differ in their neural enzyme acitivity. The chromosomal content of the 3 cell lines is near diploid, and all are capable of forming tumors in nude mice. The morphological findings indicate that the cells in culture resemble those found in the tumor, and the enzyme activities are consistent with those of nervous tissue. This the morphological, biochemical, and tumorigenic properties confirm that the 3 cell lines are neoplastic cells of neural origin. PMID:10079

Schlesinger, H R; Gerson, J M; Moorhead, P S; Maguire, H; Hummeler, K



Cell line misidentification: the beginning of the end.  


Cell lines are used extensively in research and drug development as models of normal and cancer tissues. However, a substantial proportion of cell lines is mislabelled or replaced by cells derived from a different individual, tissue or species. The scientific community has failed to tackle this problem and consequently thousands of misleading and potentially erroneous papers have been published using cell lines that are incorrectly identified. Recent efforts to develop a standard for the authentication of human cell lines using short tandem repeat profiling is an important step to eradicate this problem. PMID:20448633



Characterization of a transformed rat retinal ganglion cell line  

Microsoft Academic Search

The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the ?2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells.

R. R. Krishnamoorthy; P. Agarwal; G. Prasanna; K. Vopat; W. Lambert; H. J. Sheedlo; I.-H. Pang; D. Shade; R. J. Wordinger; T. Yorio; A. F Clark; N. Agarwal



Growth properties and alloantigenic expression of murine lymphoblastoid cell lines  

PubMed Central

Murine lymphoblastoid cell lines were evaluated for their expression of Thy-1 and thymus leukemia (TL) differentiation alloantigens. Two culture conditions were shown to affect this expression. Cells grown in fetal bovine serum (FBS)-enriched medium expressed up to 15 times the amount of TL as cells grown in horse serum (HS)-enriched medium. Thy-1 expression was less affected by the type of serum used for culture. The phase of growth when the cells were harvested, was demonstrated to affect the expression of Thy-1. The expression of Thy- 1.2 for one cell line examined, L-251A, during logarithmic growth was threefold greater than cells collected during either lag or stationary growth. When culture conditions were standardized a ranking of the amount of Thy-1 and TL expressed by several cell lines was made. All cell lines, except one, L-1210, expressed Thy-1. There was a 450-fold difference in the expression of Thy-1 between the cell lines evaluated. Seven cell lines expressed TL-1,2,3 with a ninefold difference in the amount of expression. The L-251A cell line was cultured in a 14 liter fermentor for a 26 day period. During this time TL and Thy-1 expression did not vary significantly, demonstrating that lymphoblastoid cell lines can be cultured on a continuous basis and will continue to express their surface alloantigens.



Human embryonic stem cell lines with genetic disorders  

Microsoft Academic Search

A previous study described the establishment of human embryonic stem cell (ESC) lines from different sources of embryonic material, including morula, whole blastocyst and isolated inner cell mass. Using these methods, a repository of ESC lines has been established with different genetic abnormalities, which provides an unlimited source of disease cells in culture for undertaking research on the primary disturbances

Y Verlinsky; N Strelchenko; V Kukharenko; S Rechitsky; O Verlinsky; V Galat; A Kuliev



Capture of MicroRNA–Bound mRNAs Identifies the Tumor Suppressor miR34a as a Regulator of Growth Factor Signaling  

Microsoft Academic Search

A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines.

Ashish Lal; Marshall P. Thomas; Gabriel Altschuler; Francisco Navarro; Elizabeth ODay; Xiao Ling Li; Carla Concepcion; Yoon-Chi Han; Jerome Thiery; Danielle K. Rajani; Aaron Deutsch; Oliver Hofmann; Andrea Ventura; Winston Hide; Judy Lieberman



Characterization and Analysis of Human Chordoma Cell Lines  

PubMed Central

Study Design An experimental study to investigate the characterization of 3 chordoma cell lines. Objective To characterize chordoma cell lines and generate hypothesis for further chordoma studies. Summary of Background Data Three cultured human chordoma cell lines have been successfully generated; however, their characterization is incomplete. Complete characterization of chordoma cell lines is necessary for these reagents to be a useful preclinical model. Methods Three chordoma cell lines, CH 8, U-CH1, and GP 60, were cultured in different commercially available tissue culture media. They were also cultured in different environments, which included collagen substrate, various concentrations of glucose, and various levels of hypoxic conditions. The rate of cell proliferation was assessed by either MTT or numeration assay. A 3-dimensional (3D) cell culture model of these chordoma cell lines was also studied, and the expression of vimentin and cytokeratin was measured by immunofluorescence and Western blot. Additionally, the sensitivity of the 3 chordoma cell lines to 6 chemotherapeutic drugs was analyzed. Results CH 8, GP 60, and U-CH1 cells proliferate more actively in Iscove Modified Dulbecco Medium or Dulbecco modified Eagle Medium and less actively in RPMI medium. All 3 chordoma cell lines universally grow better in collagen substrate and survive in hypoxic conditions, whereas glucose concentration has no significant influence on their growth properties. Chordoma cell lines grew well in 3D culture systems and formed acini-like spheroids and retained the expression of vimentin and cytokeratin. MTT analysis indicates that all 3 chordoma cell lines are sensitive to doxorubicin, yondelis, zalypsis, and cisplatin. Conclusion We characterized 3 chordoma cell lines for differential growth properties in a variety of media and response to chemotherapeutic agents.

Yang, Cao; Hornicek, Francis J.; Wood, Kirkham B.; Schwab, Joseph H.; Choy, Edwin; Iafrate, John; Rosenberg, Andrew; Nielsen, G. Petur; Xavier, Ramnik J.; Mankin, Henry; Duan, Zhenfeng



Antiproliferative action of metformin in human lung cancer cell lines.  


The oral antidiabetic agent metformin has anticancer properties, probably via adenosine monophosphate-activated protein kinase activation. In the present study, growth inhibition was assessed by a clonogenic and by a cell survival assay, apoptosis induction was assessed by Hoechst staining and caspase activities and cell cycle alteration after exposure to metformin, and the interaction of metformin with cisplatin in vitro were elucidated in four human lung cancer cell lines representing squamous, adeno-, large cell and small cell carcinoma. Clonogenicity and cell proliferation were inhibited by metformin in all the cell lines examined. This inhibitory effect was not specific to cancer cells because it was also observed in a non-transformed human mesothelial cell line and in mouse fibroblast cell lines. Inhibition of clonogenicity was observed only when the cells were exposed to metformin for a long period, (10 days) and the surviving fraction, obtained after inhibiting proliferation by increasing the dose, reached a plateau at approximately 0.1-0.3, indicating the cytostatic characteristics of metformin. Metformin induced significant apoptosis only in the small cell carcinoma cell line. A tendency of cell cycle accumulation at the G0/G1 phase was observed in all four cell lines. Cisplatin, in a dose-dependent manner, severely antagonized the growth inhibitory effect of metformin, and even reversed the effect in three cell lines but not in the adenocarcinoma cell line. The present data obtained using various histological types of human lung cancer cell lines in vitro illustrate the cytostatic nature of metformin and its cytoprotective properties against cisplatin. PMID:22576795

Ashinuma, Hironori; Takiguchi, Yuichi; Kitazono, Satoru; Kitazono-Saitoh, Miyako; Kitamura, Atsushi; Chiba, Tetsuhiro; Tada, Yuji; Kurosu, Katsushi; Sakaida, Emiko; Sekine, Ikuo; Tanabe, Nobuhiro; Iwama, Atsushi; Yokosuka, Osamu; Tatsumi, Koichiro



Hyaluronan production regulation from porcine hyalocyte cell line by cytokines  

Microsoft Academic Search

The objective of this study were to establish a cell line derived from porcine hyalocytes and to investigate the regulation of hyaluronan (HA) synthesis in response to cytokines. After 50 passages of the cells derived from porcine vitreous tissue, a cell line was generated. The immortalized cells showed fibroblastic morphology. The cell doubling time was 56.9h. In the mRNA level,

Koichi Nishitsuka; Yoshiko Kashiwagi; Naoki Tojo; Chikako Kanno; Yoshinori Takahashi; Teiko Yamamoto; Paraskevi Heldin; Hidetoshi Yamashita



Establishment and therapeutic use of human embryonic stem cell lines  

Microsoft Academic Search

Embryonic stem (ES) cell lines, which are derived from the inner cell mass of blastocysts, proliferate indefinitely in vitro,\\u000a retaining their potency to differentiate into various cell types derived from all of the three embryonic germ layers: the\\u000a ectoderm, mesoderm and endoderm. Establishment of human ES cell lines in 1 998 has indicated the great potential of ES cells\\u000a for

Hirofumi Suemori



Clonal derivation and characterization of human embryonic stem cell lines  

Microsoft Academic Search

Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived

Nico Heins; Anders Lindahl; Ulrika Karlsson; Marie Rehnström; Gunilla Caisander; Katarina Emanuelsson; Charles Hanson; Henrik Semb; Petter Björquist; Peter Sartipy; Johan Hyllner



Comparative recombinant protein production of eight insect cell lines.  


A recombinant Autographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae and Estigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, the T. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than the S. frugiperda-derived Sf9 or Sf2l cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media. PMID:8314732

Davis, T R; Wickham, T J; McKenna, K A; Granados, R R; Shuler, M L; Wood, H A



Shortest path based splitting line finding for touching cells  

NASA Astrophysics Data System (ADS)

A shortest path based algorithm is proposed in this paper to find splitting lines for touching cells. Firstly, an initial splitting line is obtained through the distance transform of a marker image and the watershed algorithm. Then, the initial splitting line is separated into different line segments if necessary, and the start and end points of these line segments act as the start and end points of shortest path. Finally, the shortest path algorithm is used to find the splitting line between the start and end points, and the final result of touching cells splitting can be formed by the contour of the touching cells and the splitting lines. Experimental results show that the proposed algorithm is efficient for different types of touching cells.

Bai, Xiangzhi; Sun, Changming; Wang, Peng; Zhou, Fugen



Polyamine synthesis in maize cell lines  

SciTech Connect

Uptake of ({sup 14}C)putrescine, ({sup 14}C)arginine, and ({sup 14}C)ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.

Hiatt, A. (Scripps Clinic and Research Foundation, La Jolla, CA (USA))



Mortalin inhibitors sensitize K562 leukemia cells to complement-dependent cytotoxicity.  


Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy. PMID:19739077

Pilzer, David; Saar, Moran; Koya, Keizo; Fishelson, Zvi



Establishment and characterization of seven human breast cancer cell lines including two triple-negative cell lines.  


Permanently growing cell lines can be invaluable because of their usefulness in a variety of experimental situations. We report the characteristics of seven cell lines designated, SNU-306, SNU-334, SNU-1528, SNU-1553, SNU-1581, SNU-1958 and SNU-2372, which were established from three primary carcinomas, two pleural effusion, one pericardial effusion and one ascitic fluid samples obtained from seven Korean breast carcinoma patients. The histopathology of the primary tumors and their in vitro growth characteristics are described. DNA fingerprinting analysis and genetic alterations in the p53 and EGFR genes were conducted. The expression levels of the ER-?, PR, C-erbB2, E-cadherin, COX-2, MDR and MXR genes were investigated and sensitivity to anticancer drugs was screened. Growth was as adherent cells (four cell lines), floating aggregates (one cell line) and both (two cell lines). All lines were free of mycoplasma or bacteria and were proven unique by DNA fingerprinting analysis using 18 microsatellite markers. Estrogen receptor (ER) mRNA was highly expressed in five cell lines and low or undetectable in SNU-1958 and SNU-2372. Progesterone receptor (PR) mRNA was expressed only in the SNU-306. SNU-1958 and SNU-2372 were hormone receptor-negative and C-erbB2-negative (triple-negative). SNU-1528 had an in-frame deletion of 42 base pairs of p53 gene and showed over 20-fold resistance for taxol compared to the other cell lines. There were no mutation in the EGFR gene; COX-2 was expressed in four cell lines and MXR was expressed in two cell lines. These well-characterized seven breast cancer cell lines, which include two triple-negative cell lines, will be useful for the study of breast cancer biology. PMID:24141649

Ku, Ja-Lok; Park, Sung-Chan; Kim, Kyung-Hee; Jeon, You-Kyung; Kim, Sung-Hee; Shin, Young-Kyoung; Noh, Dong-Young; Im, Seock-Ah; Bang, Yung-Jue; Han, Wonshik; Kim, Woo Ho; Park, Jae-Gahb



Establishment of Mouse Embryonic Stem Cell-Derived Erythroid Progenitor Cell Lines Able to Produce Functional Red Blood Cells  

Microsoft Academic Search

BackgroundThe supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic

Takashi Hiroyama; Kenichi Miharada; Kazuhiro Sudo; Inaho Danjo; Naoko Aoki; Yukio Nakamura; Simon Williams



Authentication of the R06E Fruit Bat Cell Line  

PubMed Central

Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

Jordan, Ingo; Munster, Vincent J.; Sandig, Volker



Topic: Consideration of the Appropriateness of Cell Lines ...  

Center for Biologics Evaluation and Research (CBER)

Text Version... 9:20 History and Characterization of the A3.01 Cell Line Seung Ho Choo and its Tumorigenic Evaluation ... or Cells Derived from Human Tumors ... More results from


Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: utility and pitfalls.  


Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Hinson, Ashley R P; Jones, Rosanne; Crose, Lisa E S; Belyea, Brian C; Barr, Frederic G; Linardic, Corinne M



Differentiation of Embryonic Stem Cell Lines Generated from Adult Somatic Cells by Nuclear Transfer  

Microsoft Academic Search

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and

Teruhiko Wakayama; Viviane Tabar; Ivan Rodriguez; Anthony C. F. Perry; Lorenz Studer; Peter Mombaerts



Survey of Interferon Production and Sensitivity in Human Cell Lines  

PubMed Central

Seven presumed diploid and 11 established cell lines were studied for their ability to produce free interferon in response to a standardized Newcastle disease virus challenge. Interferon production was evaluated in both serum-containing and serum-free medium. The ability of these cell lines to respond to the application of a standard interferon preparation by becoming resistant to virus was also examined. The diploid lines were distinctly more efficient producers of interferon than were the established lines. They also evidenced a greater requirement for serum to produce their maximum titers, but some were able to produce good titers in serum-free medium. The diploid lines were uniformly more sensitive to the application of exogenous interferon than were the established cell lines and attained greater degrees of virus resistance, but all lines tested displayed measurable sensitivity to interferon.

Moehring, J. M.; Stinebring, W. R.; Merchant, D. J.



Caffeine markedly sensitizes human mesothelioma cell lines to pemetrexed  

Microsoft Academic Search

Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine\\u000a is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine\\u000a on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell\\u000a lines tested (H2052, H2373, H28 and

Sang Hee Min; I. David Goldman; Rongbao Zhao



The transcriptional diversity of 25 Drosophila cell lines  

SciTech Connect

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal discderived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. Wereport the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what those patterns reveal about the origins of the lines and the stability of spatial expression patterns. We also offer an initial analysis of previously unannotated transcripts in the cell lines.

Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu; Yang, Li; Zou, Yi; Eads, Brian D.; Carlson, Joseph W.; Landolin, Jane M.; Kapranov, Philipp; Dumais, Jacqueline; Samsonova, Anastasia; Choi, Jeong-Hyeon; Roberts, Johnny; Davis, Carrie A.; Tang, Haixu; van Baren, Marijke J.; Ghosh, Srinka; Dobin, Alexander; Bell, Kim; Lin, Wei; Langton, Laura; Duff, Michael O.; Tenney, Aaron E.; Zaleski, Chris; Brent, Michael R.; Hoskins, Roger A.; Kaufman, Thomas C.; Andrews, Justen; Graveley, Brenton R.; Perrimon, Norbert; Celniker, Susan E.; Gingeras, Thomas R.; Cherbas, Peter



Using Neuroblastoma Cell Lines to Examine Organophosphate Neurotoxicity.  

National Technical Information Service (NTIS)

The paper describes the initial characterization of neuroblastoma cell lines to address several aspects of organophosphate neurotoxicity. Several commercially available human and mouse cell lines (i.e., SY5Y, IMR-32, SK-N-MC, NB41A3) were evaluated for th...

B. Veronesi M. Ehrich



A comparison of the cell lines used in meningioma research  

Microsoft Academic Search

BackgroundImmortal cell lines and cell lines derived from operative specimens transplanted into animal models are used in meningioma research. We address 2 criticisms of the mouse xenograft flank tumor model: Why are tumor induction rates derived from operative specimens low and inconsistent? Are flank tumors meningiomas?

Brian T. Ragel; William T. Couldwell; David L. Gillespie; Merideth M. Wendland; Kum Whang; Randy L. Jensen



Development and characterization of rabbit proximal tubular epithelial cell lines  

Microsoft Academic Search

Development and characterization of rabbit proximal tubular epithelial cell lines. We have isolated rabbit kidney proximal tubular epithelial cell lines. The selection was based on their ability to form confluent monolayers on porous supports and to maintain receptor-mediated signal transduction and ion transport, characteristic of the proximal tubule. The isolation method consisted of several steps: (1) superficial cortical proximal tubule

Michael F Romero; Janice G Douglas; Richard L Eckert; Ulrich Hopfer; James W Jacobberger



Permissiveness of human hepatoma cell lines for HCV infection  

PubMed Central

Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). Results We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.



Production of Uniparental Embryonic Stem Cell Lines  

Microsoft Academic Search

\\u000a Embryonic stem cells, or induced pluripotent cells derived from somatic cells, can yield differentiated progeny with potential\\u000a applicability for tissue repair. This chapter describes the generation of embryonic stem cells from gamete-derived uniparental\\u000a embryos. These embryonic stem cells can be patient-derived and potentially histocompatible with the gamete donor. The production\\u000a of uniparental embryos followed by derivation of embryonic stem cells

Sigrid Eckardt; K. John McLaughlin


Cell-cell signaling interactions coordinate multiple cell behaviors that drive morphogenesis of the lateral line  

PubMed Central

The zebrafish sensory lateral line system has emerged as a powerful model for the mechanistic study of collective cell migration and morphogenesis. Recent work has uncovered the details of a signaling network involving the Wnt/?-catenin, Fgf and Delta-Notch pathways that patterns the migrating lateral line primordium into distinct regions. Cells within these regions exhibit different fundamental behaviors that together orchestrate normal lateral line morphogenesis. In this review, we summarize the signaling network that patterns the migrating lateral line primordium and describe how this patterning coordinates crucial morphogenic cell behaviors.

Aman, Andy



Establishment and culture of leukemia-lymphoma cell lines.  


The advent of continuous human leukemia-lymphoma cell lines as a rich resource of abundant, accessible, and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first leukemia-lymphoma cell lines were established in 1963 and since then large numbers of new cell lines have been described. The major advantages of continuous leukemia-lymphoma cell lines are the unlimited supply and worldwide availability of identical cell material and the infinite viable storability in liquid nitrogen. These cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and by specific genetic alterations. Here some of the more promising techniques for establishing new leukemia-lymphoma cell lines and the basic principles for culturing these cells are described. Several clinical and cell culture parameters might have some influence on the success rate, e.g., choice of culture medium and culture conditions, specimen site of the primary cells, and status of the patient at the time of sample collection. PMID:21516408

Drexler, Hans G



GREG cells, a dysferlin-deficient myogenic mouse cell line  

PubMed Central

The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; de Morree, Antoine; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua



Isolation of Amniotic Stem Cell Lines With Potential for Therapy  

Microsoft Academic Search

Stem cells capable of differentiating to multiple lineages may be valuable for therapy. We report the isolation of human and rodent amniotic fluid-derived stem (AFS) cells that express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders, double in 36 h and are not tumorigenic. Lines maintained for over 250 population doublings retained long telomeres and

Paolo De Coppi; Georg Bartsch; M Minhaj Siddiqui; Tao Xu; Cesar C. Santos; Laura Perin; Gustavo Mostoslavsky; Evan Y. Snyder; James J. Yoo; Mark E. Furth; Shay Soker; Anthony Atala



Human Embryonic Stem Cell Lines Derived from Discarded Embryos  

Microsoft Academic Search

ABSTRACT Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; how- ever, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth fac- tor, human recombinant leukemia inhibitory

Maisam Mitalipova; John Calhoun; Soojung Shin; David Wininger; Thomas Schulz; Scott Noggle; Alison Venable; Ian Lyons; Allan Robins; Steven Stice



A transcriptomic computational analysis of mastic oil-treated Lewis lung carcinomas reveals molecular mechanisms targeting tumor cell growth and survival  

PubMed Central

Background Mastic oil from Pistacia lentiscus variation chia, a blend of bioactive terpenes with recognized medicinal properties, has been recently shown to exert anti-tumor growth activity through inhibition of cancer cell proliferation, survival, angiogenesis and inflammatory response. However, no studies have addressed its mechanisms of action at genome-wide gene expression level. Methods To investigate molecular mechanisms triggered by mastic oil, Lewis Lung Carcinoma cells were treated with mastic oil or DMSO and RNA was collected at five distinct time points (3-48 h). Microarray expression profiling was performed using Illumina mouse-6 v1 beadchips, followed by computational analysis. For a number of selected genes, RT-PCR validation was performed in LLC cells as well as in three human cancer cell lines of different origin (A549, HCT116, K562). PTEN specific inhibition by a bisperovanadium compound was applied to validate its contribution to mastic oil-mediated anti-tumor growth effects. Results In this work we demonstrated that exposure of Lewis lung carcinomas to mastic oil caused a time-dependent alteration in the expression of 925 genes. GO analysis associated expression profiles with several biological processes and functions. Among them, modifications on cell cycle/proliferation, survival and NF-?B cascade in conjunction with concomitant regulation of genes encoding for PTEN, E2F7, HMOX1 (up-regulation) and NOD1 (down-regulation) indicated some important mechanistic links underlying the anti-proliferative, pro-apoptotic and anti-inflammatory effects of mastic oil. The expression profiles of Hmox1, Pten and E2f7 genes were similarly altered by mastic oil in the majority of test cancer cell lines. Inhibition of PTEN partially reversed mastic oil effects on tumor cell growth, indicating a multi-target mechanism of action. Finally, k-means clustering, organized the significant gene list in eight clusters demonstrating a similar expression profile. Promoter analysis in a representative cluster revealed shared putative cis-elements suggesting a common regulatory transcription mechanism. Conclusions Present results provide novel evidence on the molecular basis of tumor growth inhibition mediated by mastic oil and set a rational basis for application of genomics and bioinformatic methodologies in the screening of natural compounds with potential cancer chemopreventive activities.



Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins.  


We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals. PMID:23007278

Park, Mi Hee; Jo, Miran; Won, Dohee; Song, Ho Sueb; Song, Min Jong; Hong, Jin Tae



Respiratory epithelial cell lines exposed to anoxia produced inflammatory mediator  

PubMed Central

Human epithelial cell lines were utilized to examine the effects of anoxia on cellular growth and metabolism. Three normal human epithelial cells lines (A549, NHBE, and BEAS-2B) as well as a cystic fibrosis cell line (IB3-1) and its mutation corrected cell line (C38) were grown in the presence and absence of oxygen for varying periods of time. Interleukin-8 (IL-8) levels were measured by enzyme-linked immunosorbent assay technique. Cellular metabolism and proliferation were assayed by determining mitochondrial oxidative burst activity by tetrazolium compound reduction. The viability of cells was indirectly measured by lactate dehydrogenase release. A549, NHBE, and BEAS-2B cells cultured in the absence of oxygen showed a progressive decrease in metabolic activity and cell proliferation after one to three days. There was a concomitant increase in IL-8 production. Cell lines from cystic fibrosis (CF) patients did not show a similar detrimental effect of anoxia. However, the IL-8 level was significantly increased only in IB3-1 cells exposed to anoxia after two days. Anoxia appears to affect certain airway epithelial cell lines uniquely with decreased cellular proliferation and a concomitant increased production of a cytokine with neutrophilic chemotactic activity. The increased ability of the CF cell line to respond to anoxia with increased secretion of inflammatory cytokines may contribute to the inflammatory damage seen in CF bronchial airway. This study indicates the need to use different cell lines in in vitro studies investigating the role of epithelial cells in airway inflammation and the effects of environmental influences.

Shahriary, Cyrus M.; Nussbaum, Eliezer



Plant cell biology through the window of the highly synchronized tobacco BY2 cell line  

Microsoft Academic Search

Synchronous cell systems are highly desirable for investigating various aspects of plant cell biology. However, to date, the tobacco BY-2 cell line is the only plant cell line which can be synchronized to high levels. A cell synchrony starting from S phase is obtained after release of BY-2 cells from aphidicolin treatment, while that from M phase is available after

Toshiyuki Nagata; Fumi Kumagai



A potential rhodium cancer therapy: studies of a cytotoxic organorhodium(I) complex that binds DNA.  


Described is a novel organorhodium(I) complex that is cytotoxic to the colon cancer cell line HCT116 and alters cell migration, DNA replication, and DNA condensation. Most importantly, the mechanism observed is not seen for the parent organorhodium dimer complex [{RhCl(COD)}2], RhCl3, or the free ligand/proligands (COD and 1-(n)butyl-3-methylimidazolium chloride). Thus, the activity of this organorhodium complex is attributable to its unique structure. PMID:23541673

McConnell, Jeanette R; Rananaware, Dimple P; Ramsey, Deborah M; Buys, Kai N; Cole, Marcus L; McAlpine, Shelli R



Genotyping of 73 UM-SCC head and neck squamous cell carcinoma cell lines  

PubMed Central

Background We established multiple UM-SCC (University of Michigan Squamous Cell Carcinoma) cell lines. With time, these have been distributed to other labs all over the world. Recent scientific discussions have noted the need to confirm the origin and identity of cell lines in grant proposals and journal articles. We genotyped the UM-SCC cell lines in our collection to confirm their unique identity. Design Early passage UM-SCC cell lines were genotyped and photographed. Results Thus far, 73 unique head and neck UM-SCC cell lines (from 65 donors including 21 lines from 17 females) were genotyped. In 7 cases separate cell lines were established from the same donor. Conclusions These results will be posted on the U of M Head and Neck SPORE Tissue Core website for other investigators to confirm that the UM-SCC cells used in their laboratories have the correct features. Publications using UM-SCC cell lines should confirm the genotype.

Brenner, J. Chad; Graham, Martin P.; Kumar, Bhavna; Saunders, Lindsay M.; Kupfer, Robbi; Lyons, Robert H.; Bradford, Carol R.; Carey, Thomas E.



Establishment of Human Colon Cancer Cell Lines from Fresh Tumors versus Xenografts: Comparison of Success Rate and Cell Line Features  

Microsoft Academic Search

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%);

Virginie Dangles-Marie; Marc Pocard; Sophie Richon; Louis-Bastien Weiswald; Jean-Gabriel Judde; Jean-Louis Janneau; Nathalie Auger; Pierre Validire; Bernard Dutrillaux; Francoise Praz; Dominique Bellet; Marie-France Poupon; Departement de Biologie



Expression pattern of galectin-3 in neural tumor cell lines.  


Galectin-3 is a member of the galectin family of beta-galactoside-specific animal lectins. Here we show that galectin-3 is constitutively expressed in 15 out of 16 glioma cell lines tested, but not by normal or reactive astrocytes, oligodendrocytes, glial O-2A progenitor cells and the oligodendrocyte precursor cell line Oli-neu. Galectin-3 is also expressed by one oligodendroglioma cell line, but not by primitive neuroectodermal tumor and 4 neuroblastoma cell lines tested so far. In all galectin-3 expressing cell lines, the lectin is predominantly, if not exclusively, localized intracellularly and carries an active carbohydrate recognition domain (shown for C6 rat glioma cells). Moreover, in contrast to primary astrocytes, glioma cells do not or only weakly adhere to substratum-bound galectin-3, probably reflecting an unusual glycosylation pattern. Our findings indicate that the expression of galectin-3 selectively correlates with glial cell transformation in the central nervous system and could thus serve as a marker for glial tumor cell lines and glial tumors. PMID:10723067

Kuklinski, S; Pesheva, P; Heimann, C; Urschel, S; Gloor, S; Graeber, S; Herzog, V; Pietsch, T; Wiestler, O D; Probstmeier, R



Differential effects of bisphosphonates on breast cancer cell lines.  


Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential. PMID:16621245

Verdijk, R; Franke, H R; Wolbers, F; Vermes, I



Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage.  


The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G(2)/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G(2)/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G(2)/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53(-)/(-), HCT116p21(-)/(-), HT-29 and SW480). Flow cytometry confirmed that these lines underwent G(2)/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5-10 ?M). In all eight lines, the majority of this arrest occurred at the G(2)/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G(2)/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage. PMID:23125222

Blakemore, Louise M; Boes, Christoph; Cordell, Rebecca; Manson, Margaret M



Novel human bronchial epithelial cell lines for cystic fibrosis research  

PubMed Central

Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ?F508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 ?·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1?, TNF-?, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.

Fulcher, M. L.; Gabriel, S. E.; Olsen, J. C.; Tatreau, J. R.; Gentzsch, M.; Livanos, E.; Saavedra, M. T.; Salmon, P.; Randell, S. H.



Koetjapic acid, a natural triterpenoid, induces apoptosis in colon cancer cells.  


Deregulated cell signaling pathways result in cancer development. More than one signal transduction pathway is involved in colorectal cancer pathogenesis and progression. Koetjapic acid (KA) is a naturally occurring seco-A-ring oleanene triterpene isolated from the Sandoricum koetjape stem bark. We report the cellular and molecular mechanisms of anticancer activity of KA towards human colorectal cancer. The results showed that KA induces apoptosis in HCT 116 colorectal carcinoma cells by inducing the activation of extrinsic and intrinsic caspases. We confirmed that KA-induced apoptosis was mediated by DNA fragmentation, nuclear condensation and disruption in the mitochondrial membrane potential. Further studies on the effect of KA on cancer pathways show that the compound causes down-regulation of Wnt, HIF-1?, MAP/ERK/JNK and Myc/Max signaling pathways and up-regulates the NF-?B signaling pathway. The result of this study highlights the anticancer potential of KA against colorectal cancer. PMID:22134768

Nassar, Zeyad D; Aisha, Abdalrahim F A; Idris, Norshirin; Khadeer Ahamed, Mohamed B; Ismail, Zhari; Abu-Salah, Khalid M; Alrokayan, Salman A; Shah Abdul Majid, Amin Malik