HT-29 Human Colon Carcinoma Cells Research Articles

Characterization of cytotoxic actions of tricyclic antidepressants on human HT29 colon carcinoma cells

Preclinical studies have suggested that the long-term use of antidepressants may result in the initiation and/or promotion of tumor in the gastrointestinal tract. However, a possible relationship between the use of antidepressants and the production of colon cancer has not yet been confirmed, and hence requires to be further investigated. To address this issue, the effects of antidepressants on the proliferation of colorectal tumor cells were examined using human HT29 colon carcinoma cells, and tricyclic antidepressant, such as imipramine, desipramine and amitriptyline, were shown to reduce the cell viability in a manner dependent on the time exposing to these drugs. In addition to these drugs, a selective serotonin reuptake inhibitor fluoxetine, but not a monoamine oxidase inhibitor tranylcypromine, caused the reduction of cell viability, similar in extent to that caused by imipramine. Further studies showed that desipramine caused the apoptotic cell death, which could be prevented by neither catalase, reduced-form glutathione (GSH), nor N-acetylcysteine (NAC), without accompanying the disruption of mitochondrial membrane potential within the cells and the release of cytochrome c into the cell cytoplasm. Moreover, desipramine caused the arrest of cell-cycle progression at either G0/G1-phase or G2/M-phase, which might be depending upon the drug concentration. Thus, these results suggest that tricyclic antidepressants may be cytotoxic, and induce the non-oxidative apoptotic death of human HT29 colon carcinoma cells probably through a non-mitochondrial pathway associated with the cell-cycle progression.

Inhibition of Colon Cancer (HT-29) Cell Proliferation by a Triterpenoid Isolated from Azadirachta indica is Accompanied by Cell Cycle Arrest and Up-Regulation of p21

Nimbolide, a natural triterpenoid present in the edible parts of the neem tree ( Azadirachta indica), was found to be growth-inhibitory in human colon carcinoma HT-29 cells. Nimbolide treatment of cells at 2.5 - 10 microM resulted in moderate to very strong growth inhibition. Flow cytometric analysis of HT-29 cells showed that nimbolide treatment (2.5 microM, 12 h) caused a 6.5-fold increase in the number of cells (55.6 %) in the G2/M phase compared with the control cells (8.8 %). At 48 h, the cell population in the G2/M phase decreased to 18 %, while that in the G0/G1 phase increased to 52.3 %. Western blot analysis revealed that nimbolide-mediated G2/M arrest was accompanied by the up-regulation of p21, cyclin D2, Chk2; and down-regulation of cyclin A, cyclin E, Cdk2, Rad17. At G0/G1 cell cycle arrest, modulation in the expression of the cell cycle regulatory molecules was also observed. We found that nimbolide-induced growth inhibition and cell cycle arrest were not associated with cellular differentiation. Quantification of cells with respect to the expression of phosphatidylserine in the outer cell membrane showed an increase in apoptotic cells by about 13 % after 48 h of nimbolide treatment.

Dihydroxyphenylethanol induces apoptosis by activating serine/threonine protein phosphatase PP2A and promotes the endoplasmic reticulum stress response in human colon carcinoma cells

The search for effective chemopreventive compounds is a major challenge facing research into preventing the progression of cancer cells. The naturally occurring polyphenol antioxidants look very promising, but their mechanism of action still remains poorly understood. Here, we show that 2-(3,4-dihydroxyphenyl)ethanol (DPE), a phenol antioxidant derived from olive oil, induces growth arrest and apoptosis in human colon carcinoma HT-29 cells. The mechanisms involve prolonged stress of the endoplasmic reticulum (ER) leading to the activation of the two main branches of the unfolded protein response (UPR), including the Ire1/XBP-1/GRP78/Bip and PERK/eIF2alpha arms. DPE treatment led to overexpression of the pro-apoptotic factor CHOP/GADD153 and persistent activation of the Jun-NH2-terminal kinase/activator protein-1 signaling pathway. DPE concomitantly modulated the extracellular signal-regulated kinase 1/2 and Akt/PKB pro-survival factors by altering their phosphorylation status as well as inhibiting tumor necrosis factor-alpha-induced nuclear factor-kappaB activation by inactivating the phosphorylation of nuclear factor inhibitor-kappaB kinase. These findings prompted us to investigate the possible involvement of phosphatases in DPE-mediated action. Using phosphatase inhibitors and RNA interference to silence the Ser/Thr phosphatase 2A (PP2A) prevented DPE-induced cell death. These findings demonstrate that DPE specifically activates PP2A, which plays a key initiating role in various pathways that lead to apoptosis in colon cancer cells.

Low and High Tenascin-Expressing Tumors Are Efficiently Targeted by ST2146 Monoclonal Antibody

ST2146biot is a biotinylated anti-tenascin monoclonal antibody (mAb) to be used for Pretargeted Antibody Guided Radioimmunotherapy (PAGRIT) of solid tumors. In vivo biodistribution studies of (125)I-labeled ST2146biot were done in nude mice transplanted with human HT-29 colon carcinoma and/or human U-118MG glioblastoma cells characterized for low and high tenascin expression, respectively. In vitro results show that ST2146 retains immunoreactivity upon biotinylation, in contrast to other anti-tenascin mAbs. In vivo biodistribution of ST2146 shows specific tumor accumulation up to 10 days after the i.v. injection, with no relevant differences between biotinylated and nonbiotinylated ST2146. A dose of 4 microg/mouse saturates the low tenascin-expressing human colon carcinoma HT-29, whereas the high tenascin-expressing human glioblastoma U-118MG seems to be saturated at a ST2146biot dose between 320 and 640 microg/mouse. The percentage of injected dose per gram of tumor ranges from 10% to 30%, corresponding to an amount of ST2146biot/g of tumor of approximately 400 ng/g and >200 microg/g for HT-29 and U-118MG, respectively. Tumor to normal organs uptake ratios are between 15 and 60, confirming high tumor selectivity of ST2146biot despite its cross-reactivity with the tenascin expressed at low level in the normal mouse organs. The ST2146biot localization data are substantially confirmed even when both low and high tenascin-expressing tumors are implanted in the same animal. To our knowledge, the absolute amount of ST2146biot, specifically localized in xenotransplanted human tumors, is the highest thus far described and supports the clinical use of this mAb in PAGRIT(R).

Nitrobenzylcarbamate prodrugs of cytotoxic acridines for potential use with nitroreductase gene-directed enzyme prodrug therapy

The synthesis, solvolytic behaviour and cytotoxicity of novel 4-nitrobenzyl carbamates and carbonates derived from 3-amino-4-hydroxymethylacridine 1 are described. Compounds 2 and 6 are both substrates for Escherichia coli nitroreductase and the highly active lead structure 1 is liberated upon incubation of the two compounds in the presence of NTR and its cofactor NADH. Additionally, the cytostatic activity of 2 and 6 against human HT29 colon carcinoma cell lines is decreased 80-fold and 360-fold, respectively, indicating their suitability and potency as prodrugs for either gene-directed enzyme prodrug therapy or antibody-directed enzyme prodrug therapy.

Effects of inositol hexaphosphate on proliferation of HT-29 human colon carcinoma cell line

To investigate the effects of inositol hexaphosphate (IP(6)) on proliferation of HT-29 human colon carcinoma cell line. Cells were exposed to various concentrations (0, 1.8, 3.3, 5.0, 8.0, 13.0 mmol/L) of IP(6) for a certain period of time. Its effect on growth of HT-29 cells was measured by MTT assay. The expressions of cell cycle regulators treated with IP(6) for 2 d were detected by immunocytochemistry. IP(6) inhibited the HT-29 cell growth in a dose- and time-dependent manner. Analysis of cell cycle regulator expression revealed that IP(6) reduced the abnormal expression of P53 and PCNA and induced the expression of P21. IP(6) has potent inhibitory effect on proliferation of HT-29 cells by modulating the expression of special cell cycle regulators.

Apoptosis of human colon carcinoma HT-29 cells induced by ceramide

To investigate the effect of exogenous ceramide-induced apoptosis on human colon carcinoma HT-29 cells. Light microscope, transmission electron microscope and fluorescence microscope were used to observe the morphology change of apoptosis in HT-29 cells. Agarose gel electrophoresis was performed to detect the DNA fragment. Mitochondrial function was detected by MTT assay. mRNA expression of Bcl-2 family gene members was determined by reverse transcription polymerase chain reaction (RT-PCR) assay. After C2-ceramide treatment, typical characteristics of apoptosis, such as nuclear chromatin breakage, apoptotic body and DNA ladder, could be observed. After exposure to 50 micromol/L C2-ceramide for 12 and 24 h, cell apoptosis was 64.1% and 81.3% respectively, which had a time-and dose-effect relationship. Mitochondrial function started to decrease from 6 h after exposure to ceramide. Meanwhile, ceramide up-regulated or down-regulated the mRNA expression of Bcl-2 family gene members. Ceramide induces apoptosis of human colon carcinoma HT-29 cells by affecting the expression of Bcl-2 family gene members and impacting the mitochondrial function.

Spatial and temporal heparanase expression in colon mucosa throughout the adenoma-carcinoma sequence

Heparanase is a mammalian endo-β-D-glucuronidase that cleaves heparan sulfate side chains at a limited number of sites. Such enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix and to facilitate cell invasion associated with tumor metastasis, angiogenesis and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase upregulation has been documented in an increasing number of primary human tumors, correlating with poor postoperative survival and increased tumor vascularity. Here, we employed anti-heparanase 733 polyclonal antibody that preferentially recognizes the 50 kDa active heparanase subunit over the 65 kDa proenzyme, as well as anti-heparanase 92.4 monoclonal antibody that recognizes both the latent and the active enzyme, to follow heparanase expression, processing and localization throughout the adenoma–carcinoma transition of the colon epithelium. Normal (nondysplastic) mucosa of the large bowel near epithelial neoplasms, as well as areas of mild dysplasia in adenomas, exhibited a strong reactivity with antibody 733 that became even stronger in foci of moderate dysplasia. Interestingly, although reactivity with antibody 733 was markedly reduced in severe dysplasia and in colorectal carcinoma, response to antibody 92.4 exhibited the opposite trend and staining intensities increased in parallel with tumor stage, the highest being in carcinoma cells. Involvement of latent heparanase (detected by 92.4, but not by 733 antibody) in tumor progression was suggested by activation of the Akt/PKB signal transduction pathway upon heparanase overexpression or exogenous addition to HT29 human colon carcinoma cells. These results suggest that heparanase expression is induced during colon carcinogenesis, and that its processing, conformation and localization are tightly regulated during the course of colon adenoma–carcinoma progression.

Selective Cytotoxic Activity of Valinomycin against HT-29 Human Colon Carcinoma Cells via Down-Regulation of GRP78

Glucose deprivation is a fundamental feature of poorly vascularized solid tumors and leads to activation of the molecular chaperone GRP78, which is associated with the unfolded protein response (UPR), a stress-signaling pathway, in tumor cells. We recently isolated an active compound, M126, that inhibits transcription from a GRP78 promoter reporter construct. M126 was identified as valinomycin by various spectroscopic methods. We found that valinomycin prevents UPR-induced protein expression, such as GRP78 and GRP94. The GRPs-inhibitory action of valinomycin severe hypoglycemic and results in selective cell death of the stressed cancer cells. Our findings demonstrate that GRP78 may be an excellent target for the use of cancer chemotherapy in the treatment of solid tumors.

Indole-3-carbinol inhibits the growth of human colon carcinoma cells but enhances the tumor multiplicity and volume of azoxymethane-induced rat colon carcinogenesis

Indole-3-carbinol (I3C) is a naturally occurring phytochemical which exerts a broad range of biological activities. The purpose of this study was to examine the effects of I3C on colon carcinogenesis, cell proliferation, cell-cycle progression and apoptosis, and on the levels of expression of several cell-cycle control molecules. We used a long-term rat model by using azoxymethane (AOM) to induce tumors (adenomas and adenocarcinomas) in the colon. In the present study, we found that after AOM injection, the treatment of male F344 rats with 0.01 and 0.05% I3C caused a significant increase in the tumor multiplicity of adenocarcinomas by 2.2- (P<0.05 for 0.01% I3C) and 2.1-fold (P<0.0002 for 0.05% I3C) respectively, when compared to the control rats. In addition, the tumor multiplicity of adenoma plus adenocarcinoma and the volume of adenocarcinoma were also increased by 2.0- (P<0.00001) and 2.1-fold (P<0.05) respectively, compared to the control. I3C significantly increased the proliferating cell nuclear antigen labeling index (PCNA LI) (P<0.008) and decreased the apoptotic index (P<0.05) of the colon adenocarcinoma. In contrast, in HCT 116 and HT29 human colon carcinoma cells, I3C inhibited growth and induced G1-phase cell-cycle arrest and apoptosis. Furthermore, I3C caused approximately a 2- to 4-fold increase in the cellular levels of p27KIP1 and p21CIP1 mRNA. These results suggest that I3C inhibits the growth of human colon carcinoma cells, at least in part, by inducing p27KIP1 and p21CIP1-mediated G1 cell-cycle arrest but dietary I3C promotes AOM-induced rat colon carcinogenesis by inhibiting the apoptosis of colon tumors. Therefore, the present study may provide further evidence for the ambivalent modulatory activity of I3C and this information may be useful when including I3C in cancer chemoprevention and/or extensive clinical therapy trials.

Aberrant splicing of cables gene, a CDK regulator, in human cancers

Cables is a novel cell cycle regulatory protein that interacts with cdk2, cdk3, and cdk5. Cables inhibits cdk2 activity by enhancing cdk2 tyrosine 15 phosphorylation by Wee1, which consequently leads to inhibition of cell growth. Loss of Cables expression was found in many human cancers, especially colon and endometrial cancer. However, the role of the Cables gene in cancer development remains unclear. This study was undertaken to analyze transcripts of Cables gene in endometrial and colon cancers. The analysis of RT-PCR products of the Cables gene revealed shortened products in each sample along with the product of the expected size. Sequence analysis indicated that these shortened products represented 8 intragenic deletions in Cables mRNA transcripts. Analysis of DNA from the same tumor sample failed to show genomic rearrangements corresponding to the transcripts containing deletions, suggesting that the deletions are the result of RNA splicing. Sequence analysis demonstrated that 5 of the deletions resulted from alternative splicing (splicing at the exon/intron boundary consensus sites), whereas the remaining 3 deletions resulted from aberrant splicing (splicing at sites not considered to be exon/intron boundary sites). All 3 aberrant splicing products were only detected in tumor tissues. Ectopic expression of one of the aberrant splicing products, which was detected in both endometrial and colon carcinomas, resulted in increased cell growth rate in human colon carcinoma HT-29 cells, suggesting a role as a dominant negative mutant.

TRAIL (TNF‐Related Apoptosis‐Inducing Ligand) Induces Necrosis‐Like Cell Death in Tumor Cells at Acidic Extracellular pH

How tumor microenvironment, more specifically low extracellular pH (6.5), alters cell response to TNF-related apoptosis-inducing ligand (TRAIL)-based cancer therapy has yet to be determined. The aim of the current work was to test the effect of acidic extracellular pH on TRAIL-induced cell death in human HT29 colon carcinoma and HepG2 hepatocarcinoma cell lines as well as in human primary hepatocytes. We found an increase in TRAIL sensitivity at low extracellular pH, which is partially inhibited by Bcl-2 expression in HT29 cells. At low extracellular pH, TRAIL induced a new form of cell death, sharing necrotic and apoptotic features in tumor cells. By contrast, human primary hepatocytes were resistant to TRAIL-induced cell death even at acidic extracellular pH.

P53-independent G1 cell cycle arrest of human colon carcinoma cells HT-29 by sulforaphane is associated with induction of p21CIP1 and inhibition of expression of cyclin D1

Isothiocyanate sulforaphane (SFN) is a potent cancer chemopreventive agent. We investigated the mechanisms underlying the anti-proliferative effects of SFN in the human colon carcinoma cell line, HT-29. We demonstrate that SFN inhibits the growth of HT-29 cells in a dose- and time-dependent manner. Treatment of serum-stimulated HT-29 cells with SFN suppressed the re-initiation of cell cycle by inducing a G(1) phase cell cycle arrest. At high doses (>25 microM), SFN dramatically induces the expression of p21(CIP1) while significantly inhibits the expression of the G(1) phase cell cycle regulatory genes such as cyclin D1, cyclin A, and c-myc. This regulation can be detected at both the mRNA and protein levels as early as 4 h post-treatment of SFN at 50 microM. Additionally, SFN activates MAPKs pathways, including ERK, JNK and p38. Exposure of HT-29 cells with both SFN and an antioxidant, either NAC or GSH, completely blocked the SFN-mediated activation of these MAPK signaling cascades, regulation of cyclin D1and p21(CIP1) gene expression, and G(1)phase cell cycle arrest. This finding suggests that SFN-induced oxidative stress plays a role in these observed effects. Furthermore, the activation of the ERK and p38 pathways by SFN is involved in the upregulation of p21(CIP1) and cyclin D1, whereas the activation of the JNK pathway plays a contradictory role and may be partially involved in the downregulation of cyclin D1. Because cyclin D1 and p21(CIP1) play opposing roles in G(1) phase cell cycle progression regulation, blocking the activation of each MAPK pathway with specific MAPK inhibitors, is unable to rescue the SFN-induced G(1) phase cell cycle arrest in HT-29 cells.

Characterization of FimH Adhesins Expressed by Salmonella enterica Serovar Gallinarum Biovars Gallinarum and Pullorum: Reconstitution of Mannose-Binding Properties by Single Amino Acid Substitution

Recombinant FimH adhesins of type 1 fimbriae from Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, in contrast to those of Salmonella enterica serovar Typhimurium, did not bind to high-mannose oligosaccharides or to human colon carcinoma HT-29 cells. However, mutated FimH proteins from biovar Gallinarum and biovar Pullorum, in which the isoleucine at position 78 was replaced by the threonine found in S. enterica serovar Typhimurium, bound well to glycoproteins carrying high-mannose oligosaccharides and colon carcinoma cells. The loss of sugar-binding properties by biovar Gallinarum and biovar Pullorum FimH adhesins, which are a part of the type 1 fimbriae, is most probably the result of a single T78I mutation, as was proven by site-directed mutagenesis of FimH proteins.

Surface modification of gold and quantum dot nanoparticles with chitosan for bioapplications

Gold (Au) and quantum dot (QD) nanoparticles, which have been extensively used in many fields, were encapsulated with a natural polymer, chitosan, to improve their biocompatibility. Characterization was performed using ultraviolet-visible, dynamic light scattering, atomic force microscopy, and transmission electron microscope analyses. It was found that a Au/chitosan ratio of 1:1 and smaller produced chitosan-encapsulated Au nanoparticles of a sufficiently small size, and this result was then applied in the chitosan encapsulation of QDs. The biocompatibility of both types of nanoparticles was assessed in cell culture studies using HT29 human colon carcinoma and NIH 3T3 mouse fibroblast cells. MTT and trypan blue exclusion assays revealed that both chitosan-encapsulated Au nanoparticles and QDs exhibited improved biocompatibility over their bare, nonencapsulated counterparts. Therefore, this study showed that chitosan could be used to encapsulate both Au nanoparticles and QDs in order to enhance their biocompatibility. The approaches developed in this study can also be extended to other nanoparticles for bioapplications as well.

The importance of colonic butyrate transport to the regulation of genes associated with colonic tissue homoeostasis

The transition from normality to malignancy in colorectal cancer is characterized by alterations in the expression of genes associated with the maintenance of tissue homoeostasis. Butyrate, a product of microbial fermentation of dietary fibre in the colon, is known to regulate a number of genes associated with the processes of proliferation, differentiation and apoptosis of colonic epithelial cells, and, hence, homoeostasis of colonic tissue. We have shown previously that the transport of butyrate into colonocytes is of fundamental importance to butyrate's regulatory ability, and therefore sought to assess the expression profile of butyrate-responsive genes in colon cancer tissue, where the expression of the colonic luminal-membrane butyrate transporter, MCT1 (monocarboxylate transporter 1), is significantly down-regulated. In the present paper, we first employed microarray analysis to assess global changes in butyrate-responsive genes using HT29 human colon carcinoma cells treated with butyrate. There was consistency in the butyrate response of selected genes in two other human colonic cell lines (HCT116 and AA/C1) using quantitative real-time PCR. Furthermore, we report that expression levels of selected butyrate-responsive genes involved in the processes of proliferation, differentiation and apoptosis, are deregulated in colon cancer tissue, correlating with decreased expression of MCT1. These findings support our hypothesis that a reduction in MCT1 expression, and hence butyrate transport, can lead to a reduction in the intracellular butyrate levels required to regulate gene expression. Collectively, our results highlight the important contribution of butyrate transport to the maintenance of tissue homoeostasis and disease prevention.

Long-Term Exposure of the HT-29 Human Intestinal Epithelial Cell Line to TNF Causes Sustained Up-Regulation of the Polymeric Ig Receptor and Proinflammatory Genes through Transcriptional and Posttranscriptional Mechanisms

Transport of IgA Abs across intestinal epithelial cells into gut secretions is mediated by the polymeric Ig receptor (pIgR). The cytokine TNF plays a central role in initiating and amplifying inflammatory reactions, and is implicated in the pathogenesis of inflammatory bowel diseases. Acute exposure of intestinal epithelial cell lines to TNF has been shown to up-regulate transcription of genes encoding pIgR and a number of proinflammatory factors, but the effects of chronic exposure to TNF have not been studied. We found that exposure of HT-29 human colon carcinoma cells to TNF for up to 20 days reduced the rate of cell proliferation, but did not cause gross morphological changes. Expression of mRNA encoding pIgR and several proinflammatory genes increased acutely, and then diminished but remained elevated above control levels throughout the experiment. Changes in gene expression were paralleled by increased expression of the transcription factors IFN regulatory factor-1 and the RelB subunit of NF-kappaB. HT-29 cells activated the endogenous TNF gene in response to TNF treatment, but the level of TNF production was insufficient to maintain pIgR and proinflammatory gene expression after withdrawal of exogenous TNF. Chronic exposure to TNF caused a marked increase in pIgR mRNA stability and a small but significant decrease in TNF mRNA stability, but no change in the half-lives of IL-8, c-Myc, and GAPDH. In summary, we observed different effects of acute vs chronic exposure to TNF on gene expression, and found evidence for transcriptional and posttranscriptional regulation of expression of the pIgR.

Inhibition of colon cancer cell proliferation by the dietary compound conjugated linoleic acid is mediated by the CDK inhibitor p21CIP1/WAF1

Our previous studies indicated that dietary conjugated linoleic acid (CLA) inhibits colon tumor cell proliferation in vitro and in vivo. To identify mechanisms by which CLA regulates growth arrest, the HT-29 human colon carcinoma cell line was treated with various physiological concentrations of CLA and analyzed by flow cytometry. We detected a dose-dependent increase in the percentage of cells arrested in G1 after CLA treatment that was accompanied by induction of the cyclin dependent kinase (CDK) inhibitor p21CIP1/WAF. CLA addition also led to increased p21 expression in HCT116 and SW480 cells, indicating that p21 induction is a general consequence of CLA treatment in colon cancer cells. Since both HT-29 and SW480 cells have mutant p53, our data indicate that p53 is not essential for induction of p21. In addition to an increase in p21 levels, HT-29 cell growth arrest was also accompanied by moderate decreases in Cyclin A, D1, E, and proliferating cell nuclear antigen (PCNA) levels. Following CLA treatment, p21 associated with and inhibited CDK4 and CDK2, and this correlated with reduced phosphorylation of retinoblastoma proteins. Increased association of p21 with PCNA was also detected. Dietary CLA inhibits cell cycle progression by inducing p21, which negatively regulates the growth promoting activities of CDK/cyclins and PCNA. These studies indicate that physiological concentrations of CLA inhibit growth of colon cancer cells with either wild-type or mutant p53, and may have therapeutic benefits in vivo.

Role of Intracellular Glutathione in Cell Sensitivity to the Apoptosis Induced by Tumor Necrosis Factor α–Related Apoptosis-Inducing Ligand/Anticancer Drug Combinations

We have recently shown that combination of tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) with anticancer drugs induced an apoptotic cell death pathway involving both caspases and mitochondria. The present work further explores the role of intracellular reduced glutathione (GSH) level in cell sensitivity to this cell death pathway. Intracellular GSH level was measured by high-performance liquid chromatography. Cell death was detected by immunofluorescence after Hoechst 33342/propidium iodide staining. Reactive oxygen species production was evaluated by flow cytometry after dihydroethidium probe labeling. Western blot analysis was done to study stress-activated protein kinase/c-jun NH(2)-terminal kinase (SAPK/JNK) phosphorylation. The Student's t test was used to determine significance of the results. Three to six experiments were done. GSH depletion enhanced apoptosis induced by TRAIL/cisplatin (CDDP) or TRAIL/5-fluorouracil (5-FU) combinations in both human HT29 colon carcinoma and HepG2 hepatocarcinoma cells, whereas it enhanced cytotoxicity induced only by TRAIL/CDDP in human primary hepatocytes. Our results further suggested that GSH depletion enhanced SAPK/JNK phosphorylation upon TRAIL/5-FU exposure and likely reduced the detoxification mechanisms of CDDP in HT29 cells. Resistance of Bcl-2-expressing HT29 and HepG2 cells to combined treatment was not overcome by GSH depletion, thus indicating that Bcl-2-mediated antiapoptotic effect occurs independently of intracellular GSH level. GSH depletion could be useful to increase the therapeutic efficacy of cancer treatment by TRAIL/anticancer drug combinations. Furthermore, TRAIL/5-FU combination might be a potential anticancer treatment of human tumors, being ineffective on human primary hepatocytes and thus could be of interest in clinical cancer treatment. Nevertheless, Bcl-2 expression remains an important resistance factor.

Increased BNip3 and decreased mutant p53 in cisplatin-sensitive PDT-resistant HT29 cells

We have reported previously the isolation of three photodynamic therapy (PDT)-resistant human colon carcinoma HT29 cell lines that show increased expression of the Hsp27 and BNip3 protein and a decreased expression of the mutant p53 protein compared to parental HT29 cells. Since mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. We report here that the PDT-resistant HT29 cell lines showed a significant increase in cisplatin sensitivity and an increase in both spontaneous and cisplatin-induced apoptosis compared to parental HT29 cells. In addition, the cisplatin sensitivity of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells correlated with increased BNip3 and decreased mutant p53 protein levels, but not Hsp27 protein levels.