Oncogenic Ras, but not (V600E)B-RAF, protects from cholesterol depletion-induced apoptosis through the PI3K/AKT pathway in colorectal cancer cells.

Cholesterol is necessary for proliferation and survival of transformed cells. Here we analyse the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in colorectal cancer cells carrying oncogenic Ras or (V600E)B-RAF mutations. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment results in a significant increase in apoptosis in HT-29 and Colo-205 cells containing the (V600E)B-RAF mutation, but not in HCT-116 and LoVo cells harbouring the (G13D)Ras mutation, or BE cells, which possess two mutations, (G13D)Ras and (G463V)B-RAF. We also demonstrate that oncogenic Ras protects from apoptosis induced by cholesterol depletion through constitutive activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. The specific activation of the PI3K/AKT pathway by overexpression of the (V12)RasC40 mutant or a constitutively active AKT decreases the LPDS plus 25-HC-induced apoptosis in HT-29 cells, whereas PI3K inhibition or abrogation of AKT expression renders HCT-116 sensitive to cholesterol depletion-induced apoptosis. Moreover, our data show that LPDS plus 25-HC increases the activity of c-Jun N-terminal kinase proteins only in HT-29 cells and that the inhibition of this kinase blocks the apoptosis induced by LPDS plus 25-HC. Finally, we demonstrate that AKT hyperactivation by oncogenic Ras protects from apoptosis, preventing the activation of c-Jun N-terminal kinase by cholesterol depletion. Thus, our data demonstrate that low levels of cholesterol induce apoptosis in colorectal cancer cells without oncogenic Ras mutations. These results reveal a novel molecular characteristic of colon tumours containing Ras or B-RAF mutations and should help in defining new targets for cancer therapy.

A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells.

Cancer develops when molecular pathways that control the fine balance between proliferation, differentiation, autophagy and cell death undergo genetic deregulation. The prospects for further substantial advances in the management of colorectal cancer reside in a systematic genetic and functional dissection of these pathways in tumor cells. In an effort to evaluate the impact of p38 signaling on colorectal cancer cell fate, we treated HT29, Caco2, Hct116, LS174T and SW480 cell lines with the inhibitor SB202190 specific for p38alpha/beta kinases. We report that p38alpha is required for colorectal cancer cell homeostasis as the inhibition of its kinase function by pharmacological blockade or genetic inactivation causes cell cycle arrest, autophagy and cell death in a cell type-specific manner. Deficiency of p38alpha activity induces a tissue-restricted upregulation of the GABARAP gene, an essential component of autophagic vacuoles and autophagosomes, whereas simultaneous inhibition of autophagy significantly increases cell death by triggering apoptosis. These data identify p38alpha as a central mediator of colorectal cancer cell homeostasis and establish a rationale for the evaluation of the pharmacological manipulation of the p38alpha pathway in the treatment of colorectal cancer.

Chemopreventive effect of chitosan oligosaccharide against colon carcinogenesis.

Chitosan oligosaccharide (COS, 3 kDa

Therapeutic potential of antisense oligodeoxynucleotides to down-regulate thymidylate synthase in mesothelioma.

Malignant mesothelioma is an aggressive tumor of the serosal surfaces of the lungs, heart, and abdomen. Survival rates are poor and effective treatments are not available. However, recent therapeutic regimens targeting thymidylate synthase (TS) in malignant mesothelioma patients have shown promise. We have reported the use of an antisense oligodeoxynucleotide targeting TS mRNA (antisense TS ODN 83) to inhibit growth of human tumor cells. To test the potential for antisense targeting of TS mRNA in treatment of malignant mesothelioma, we assessed and compared the effects of antisense TS ODN 83 on three human malignant mesothelioma cell lines (211H, H2052, and H28) and human nonmalignant mesothelioma cells (HT29 colorectal adenocarcinoma, HeLa cervical carcinoma, and MCF7 breast tumor cell lines). We report that ODN 83 applied as a single agent effectively reduced TS mRNA and protein in malignant mesothelioma cell lines. Furthermore, it inhibited malignant mesothelioma growth significantly more effectively than it inhibited growth of nonmalignant mesothelioma human tumor cell lines: a difference in susceptibility was not observed in response to treatment with TS protein-targeting drugs. In malignant mesothelioma cells, antisense TS both induced apoptotic cell death and reduced proliferation. In nonmalignant mesothelioma cells, only reduced proliferation was observed. Thus, antisense TS-mediated induction of apoptosis may be the basis for the high malignant mesothelioma sensitivity to antisense targeting of TS. Further preclinical and clinical study of TS antisense oligodeoxynucleotides, alone and in combination with TS-targeting chemotherapy drugs, in mesothelioma is warranted.

Chemopreventive effect of protein extract of Asterina pectinifera in HT-29 human colon adenocarcinoma cells.

We investigated the effect of protein extract of Asterina pectinifera on the activity of 4 enzymes that may play a role in adenocarcinoma of the colon: quinone reductase (QR), glutathione S-transferase (GST), ornithine decarboxylase (ODC), and cyclooxygenase (COX)-2. QR and GST activity increased in HT-29 human colon adenocarcinoma cells increased that had been exposed to 4 concentrations of the protein extract (80, 160, 200, and 240 microg/mL). Additionally, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC activity decreased significantly in cells exposed to the extract in concentrations of 160 microg/mL (p<0.05), 200 microg/mL (p<0.005), and 240 microg/mL (p<0.005). TPA-induced COX-2 activity also decreased in cells exposed to extract concentrations of 10, 20, 40, and 60 microg/mL. COX-2 expression was also inhibited in cells exposed to this extract. These results suggest that this protein extract of A pectinifera has chemopreventive activity in HT-29 human colon adenocarcinoma cells, and therefore, may have the potential to function as a chemopreventive agent in human colorectal cancer.

Regulation of expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor in human colon carcinoma cells.

The enzyme/cytokine thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) has diverse functions within cells, including the regulation of steady-state thymidine levels, the conversion of cancer chemotherapeutic agent 5-fluorouracil to an active metabolite, and the mediation of angiogenesis in normal and malignant cells. Although the level of TP/PD-ECGF expression varies substantially among different individuals, is usually elevated in colorectal tumors compared to nonmalignant tissue, and has been shown to be directly associated with poor clinical prognosis, little is known about the mechanisms for control of TP/PD-ECGF expression. TP/PD-ECGF mRNA levels are extremely low in most cell lines in vitro, including HT29 human colon carcinoma cells. IFN-alpha and IFN-beta induced an increase in TP/PD-ECGF enzyme activity and mRNA levels. The induction of TP/PD-ECGF expression by IFN was not as strong as that of another IFN-inducible gene, 2'-5' oligoadenylate synthetase, but in contrast to 2'-5' oligoadenylate synthetase, TP/PD-ECGF mRNA levels remained elevated for up to 72 h. Experiments suggested that this was due to the combination of a rapid but transient increase in the rate of TP/PD-ECGF transcription that was accompanied by a more prolonged stabilization of TP/PD-ECGF mRNA. Using an electrophoretic mobility shift assay, IFN was found to rapidly and transiently induce nuclear factors that bound to a putative IFN response element in the TP/PD-ECGF promoter. The complex observed was similar but not identical to that seen using the consensus IFN-stimulated response element sequence as a target. TP/PD-ECGF mRNA also has a pyrimidine-rich sequence at its 3' end that was similar to a motif that has been reported to mediate increased mRNA stability in other genes. These studies indicate that TP/PD-ECGF gene expression was subject to regulation by both transcriptional and posttranscriptional mechanisms.

Yeast cytosine deaminase improves radiosensitization and bystander effect by 5-fluorocytosine of human colorectal cancer xenografts.

The efficacy of cancer gene therapy using bacterial cytosine deaminase (bCD)/5-fluorocytosine (5-FC) enzyme/prodrug strategy is limited by the inefficiency of cytosine deaminase (CD)-catalyzed conversion of 5-FC into 5-fluorouracil (5-FU). We have shown previously that yeast CD (yCD) is more efficient at the conversion of 5-FC than bCD. In the current study, we hypothesized that the increased production of 5-FU by yCD would enhance the efficacy of the CD/5-FC treatment strategy by increasing the bystander effect as well as the efficacy of radiotherapy because of the radiosensitizing capacity of 5-FU. To test this hypothesis, we generated stable HT29 human colon cancer cell lines expressing either bCD (HT29/bCD) or yCD (HT29/yCD). The amount of 5-FU produced in HT29/yCD tumors after a single injection of 5-FC (1000 mg/kg, i.p.) was 15-fold higher than that produced in HT29/bCD tumors. In tumor-bearing nude mice, the average minimum relative tumor size (compared with pretreatment values) of HT29/bCD tumors treated with 5-FC and radiation (500 mg/kg i.p. and 3 Gy, 5 days a week for 2 weeks) was 0.55+/-0.1, compared with 0.01+/-0.01 in HT29/yCD tumors (P = 0.002). Moreover, an increased cytotoxic and radiosensitizing effect of 5-FC on bystander cells was observed in vitro and in vivo when yCD was expressed in HT29 cells instead of bCD. In mice bearing HT29 tumors containing 10% HT29/yCD cells, the combined treatment resulted in a minimum tumor size of 0.20+/-0.07 compared with 0.60+/-0.1 in 10% HT29/bCD cells (P < 0.001). These results demonstrate that the use of yCD in the CD/5-FC strategy has a high potential to improve the therapeutic outcome of combined gene therapy and radiotherapy in cancer patients.

Atypical protein kinase C zeta as a target for chemosensitization of tumor cells.

Exposure of tumor cells to cytotoxic agents simultaneously activates a variety of intracellular signaling pathways. Some of these pathways involve enzymes from the protein kinase C (PKC) family of serine/threonine kinases. This family includes isoenzymes that negatively influence cell death, whereas other demonstrate an opposite effect. The present study analyzes the role of the zeta atypical PKC isoform in tumor cell response to cytotoxic agents. Using a histone H1 phosphorylation assay, we showed that both tumor necrosis factor alpha and etoposide activate PKCzeta in U937 human leukemic cells. Stable transfection of a kinase-dead, dominant-negative PKCzeta mutant in U937 cells decreases Bcl-2 expression while increasing the expression of Bax and several procaspases. This transfection also prevents etoposide-induced nuclear factor-kappaB nuclear translocation and accumulation of X-linked inhibitor of apoptosis protein. PKCzeta inhibition accelerates the occurrence of apoptosis in leukemic cells exposed to etoposide and tumor necrosis factor alpha. This sensitization was confirmed in vitro by use of a clonogenic assay. In addition, PKCzeta inhibition sensitized tumor cells grown in nude mice to etoposide. These results indicate that PKCzeta isoform is a protective signals that is activated in tumor cells exposed to a cytotoxic agent. This inducible resistance factor thus appears an attractive target for chemosensitization of tumor cells.

Isoform-specific induction of a human aldo-keto reductase by polycyclic aromatic hydrocarbons (PAHs), electrophiles, and oxidative stress: implications for the alternative pathway of PAH activation catalyzed by human dihydrodiol dehydrogenase.

Human dihydrodiol dehydrogenase (DD) isoforms are aldo-keto reductases (AKRs) that activate polycyclic aromatic hydrocarbons (PAHs) by oxidizing trans-dihydrodiol proximate carcinogens to reactive and redox-active ortho-quinones. Of these, human AKR1C1 (DD1) and AKR1C2 (DD2) oxidize trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the cytotoxic and genotoxic metabolite benzo[a]pyrene-7,8-dione (BPQ) with the highest catalytic efficiency. Exposure of HepG2 cells to a panel of inducers revealed that mRNA encoding one or more human AKR1C member(s) was induced (3- to 10-fold) by benzo[a]pyrene and other polycyclic aromatic compounds (bi-functional inducers), electrophilic Michael acceptors and phenolic antioxidants (monofunctional inducers), and reactive oxygen species (ROS). The induction of AKR1C mRNA by bifunctional inducers was delayed with respect to the induction of CYP1A1 mRNA, and AKR1C mRNA was not induced by the nonmetabolizable aryl hydrocarbon receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These data suggest that, in contrast to the CYPs, induction of AKR1C member(s) by PAHs and other bifunctional inducers is mediated indirectly via an antioxidant response element rather than a xenobiotic response element. Immunoblot and enzymatic assays confirmed that the increases in AKR1C mRNA were faithfully translated into functional AKR1C protein(s). The increased DD activity in HepG2 lysates was inhibited only by high concentrations of ursodeoxycholate, which suggested that AKR1C2 (DD2, bile-acid-binding protein) was not the isoform induced. RNase protection assays identified AKR1C1 (DD1) mRNA as the transcript which was up-regulated by mono- and bi-functional inducers and ROS in both human hepatoma (HepG2) and colon carcinoma (HT29) cells. BPQ, the electrophilic and redox-cycling product of the AKR1C1 reaction, also induced AKR1C1 expression. Thus, BPQ formation by AKR1C1 results in both a chemical (redox-cycling) and a genetic (AKR1C1 induction) amplification of ROS in PAH-exposed cells. Because ROS have been implicated in both tumor initiation and tumor promotion, the amplification of ROS by this pathway may play a significant role in PAH carcinogenesis.

The farnesyltransferase inhibitor L744,832 reduces hypoxia in tumors expressing activated H-ras.

Many tumors contain extensive regions of hypoxia. Because hypoxic cells are markedly more resistant to killing by radiation, repeated attempts have been made to improve the oxygenation of tumors to enhance radiotherapy. We have studied the oxygenation of tumor xenografts in nude mice after treatment with the farnesyltransferase inhibitor L744,832. Hypoxia was assessed by measuring the binding of the hypoxic cell marker pentafluorinated 2-nitroimidazole. We show that xenografts from two tumor cell lines with mutations in H-ras had markedly improved oxygenation after farnesyltransferase treatment. In contrast, xenografts from two tumors without ras mutations had equivalent hypoxia regardless of treatment. The effect on tumor oxygenation could be detected at 3 days and remained after 7 days of treatment. These results indicate that treatment with farnesyltransferase inhibitors can alter the oxygenation of certain tumors and suggest that such treatment might be useful in the radiosensitization of these tumors.

Regulation of the Src tyrosine kinase and Syp tyrosine phosphatase by their cellular association.

The specific activity of the Src tyrosine kinase is elevated in human colon carcinoma cells. To identify Src-binding proteins that might upregulate Src activity in these cells, a human colon carcinoma lambda gt11 expression library was screened with purified, 32P-labeled Src. The SH-PTP2 (Syp) tyrosine phosphatase was isolated and shown to associate with Src. In vitro studies demonstrated that: (1) transforming F527 Src phosphorylates Syp, and (2) Syp dephosphorylates Src at Tyr 527. Both events are known to upregulate enzyme activity. Others have shown that overexpression of the receptor tyrosine phosphatase alpha in rat embryo fibroblasts results in Src activation by dephosphorylation of Tyr 527, cell transformation and tumorigenesis. Thus, transmembrane tyrosine phosphatases may be involved in cell transformation exerting at least some of their effects through activation of Src. To the best of our knowledge, this is the first identification of an intracellular tyrosine, phosphatase which may activate Src by a similar mechanism.

Change of calretinin expression in the human colon adenocarcinoma cell line HT29 after differentiation.

Calretinin is a Ca(2+)-binding protein of the EF-hand family which is expressed in colon adenocarcinomas and colon-derived tumor cell lines (e.g. WiDr), but is absent from normal human enterocytes. Its function has not as yet been elucidated, but some lines of evidence lead us to postulate its involvement in cell proliferation in these cells. In order to test whether calretinin is correlated with an undifferentiated, proliferating, or with a differentiated, state of cells, its expression was studied in the human colon adenocarcinoma clonal cell line HT29-18, which can be caused to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (glucose starvation differentiation). Treatment of HT29-18 cells with galactose led to a drop in the calretinin mRNA level and in protein expression as evidenced by immunocytochemical staining and Western blot analysis of cytosolic cell extracts. These results suggest that calretinin is present in HT29-18 cancer cells, mostly in those which are in the undifferentiated state. The possibility that calretinin is involved in maintaining the cells in an undifferentiated (cancerous) state is discussed.

Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA.

Previous studies have shown that the cytotoxicity of fluoropyrimidines is mediated, in large part, by inhibition of the enzyme thymidylate synthase (TS). The aim of this study was to determine whether the chemosensitivity of human cancer cells to fluoropyrimidines could be increased by decreasing TS expression with antisense oligodeoxyribonucleotides (ODNs). ODNs (18-mers) targeted at the AUG translational initiation site of TS mRNA inhibited translation in a sequence- and dose-dependent manner in a rabbit reticulocyte lysate in vitro translation system. Treatment of human colon cancer HT-29 cells with antisense ODNs decreased TS catalytic activity in the cells in a dose-dependent manner over a short period, but the longer-term effect of the TS antisense ODN treatment was actually to increase the amount of TS in the cells and to decrease their sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd). However, when human nasopharyngeal cancer KB31 cells were transfected with a plasmid (pHaMAGRP) construct containing the TS antisense fragment (+ 1 to + 422) under the control of a glucose-regulated promoter, the expression of both TS protein and TS catalytic activity was decreased by nearly 30% (P = 0.014), and sensitivity of these cells to FdUrd was enhanced by approximately 8-fold (P = 0.021). No changes in the levels of expression of TS protein or FdUrd-associated cytotoxicity were observed in control, vector-transfected cells. No change was observed in the sensitivity of transfected cells toward either cisplatin or Adriamycin. These results show that the level of expression of TS in human malignant cells can be down-regulated with antisense TS RNA, and their sensitivity to fluoropyrimidines can, thereby, be increased.

[Establishment and biological characterization of 5-fluorouracil-resistant human colon cancer HT-29/5-FU cell line].

OBJECTIVE: To establish 5-fluorouracil (5-FU)-resistant human colon cancer HT-29 cell line (HT-29/5-FU) in vitro and observe its biological properties. METHODS: The HT-29/5-FU cell line was established by continuously exposing the HT-29 cells to ascending doses of 5-FU. The morphology and colony formation rate were detected. The growth curve and the chemosensitivity of HT-29/5-FU cell line were determined by MTT assay. Western blotting was used to analyze the expressions of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). Cell cycle and apoptosis were measured by flow cytometry. RESULTS: The resistance of HT-29/5-FU cells to 5-FU was 3.59-fold greater than that of HT-29 cells. The morphology of HT-29/5-FU cells differed from that of HT-29 cells. Compared with HT-29 cells, HT-29/5-FU cells showed remarkable reduction of cell proliferation and colony formation, higher expressions of TS and DPD, higher percentage of cells in the S phase, and stronger ability of resistance to apoptosis induced by 5-FU. CONCLUSION: The biological characters of HT-29/5-FU cell line may play an important role in 5-FU-resistant mechanisms, which may also contribute to elucidate the potential mechanisms of tumor drug resistance as well as resistance reversal.

In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine.

To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU.

Effects of sulindac, sulindac metabolites, and aspirin on the activity of detoxification enzymes in HT-29 human colon adenocarcinoma cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been found to reduce cancer rates in various segments of the gastro-intestinal tract in both animals and humans. In this study we examined the effect of sulindac, sulindac sulfide, sulindac sulfone and aspirin on QR and GST activity. We found that sulindac itself increased QR activity as much as 2-fold over controls but had no effect on GST activity. Sulindac sulfone, a metabolite of sulindac which lacks the ability to inhibit prostaglandin (PG) synthesis, increased QR and GST to 1.5-fold over controls in both cases. Aspirin increased QR and GST to 1.5-fold and 3.5-fold over controls respectively. These data indicate that NSAIDs increase phase II enzyme detoxification enzyme activity. Consequently, this effect may contribute to the protective effect of NSAIDs against colon cancer and may be an anticarcinogenic effect of these drugs that is distinct from their ability to inhibit PG synthesis.

Geldanamycin-induced cytotoxicity in human colon-cancer cell lines: evidence against the involvement of c-Src or DT-diaphorase.

We investigated two of the major proposed modes of action of the benzoquinoid ansamycin geldanamycin using a pair of human colon-carcinoma cell lines, BE and HT29. One potential mechanism of action in colorectal cancer is the inhibition of c-Src kinase activity, since this proto-oncogene is hyperexpressed in human large-bowel tumours. Our results show that despite the 9-fold higher level of c-Src kinase activity found in HT29 cells, there was only a 1.4-fold difference in cytotoxicity as compared with BE cells, the latter being the most sensitive. Moreover, even at concentrations of geldanamycin that resulted in cell kill of 80% or more after a 24-h period of exposure, there was no effect on c-Src kinase activity in HT29 cells, although c-Src protein was depleted at supralethal levels of exposure. We also investigated the metabolism of the quinone moiety of geldanamycin by DT-diaphorase, an enzyme that activates certain quinone antibiotics such as mitomycin C and is hyperexpressed in colorectal cancer cells. Geldanamycin was shown to be a substrate for DT-diaphorase present in HT29 cells. However, the lack of a major differential in cytotoxicity between HT29 and BE cells indicates that this is unlikely to be pharmacologically significant, since the former contains high levels of enzyme activity, whereas BE cells have no significant activity due to a point mutation in the DT-diaphorase (NQO1) gene. Although reduction of geldanamycin was also catalysed by non-DT-diaphorase reductases in HT29 and BE cells, providing the potential for free radical induction, this is unlikely to be significant since studies previously reported by us elsewhere showed that cells exposed to geldanamycin exhibited no evidence of DNA damage. Thus, as far as the mode of action of geldanamycin in human colon-carcinoma cells is concerned, the present results rule out two major possibilities, namely, the involvement of c-Src tyrosine kinase inhibition and DT-diaphorase metabolism.

Butyrate and propionate downregulate ERK phosphorylation in HT-29 colon carcinoma cells prior to differentiation.

We have characterized the effects of different short-chain fatty acids (SCFAs) on cell growth and differentiation as well as the phosphorylation state of ERK1 and 2 in the human colon adenocarcinoma cell line HT-29. Of the five SCFAs tested, only butyrate and propionate impaired cellular proliferation. Moreover, butyrate and propionate specifically resulted in a decrease in ERK1 and 2 phosphorylation at 3 and 6 hours post-treatment, suggesting a correlation between the ability of these SCFAs to inhibit cellular proliferation and decrease ERK phosphorylation. Notably, the decrease in ERK phosphorylation was observed prior to the induction of the differentiation markers alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) by butyrate and propionate from days 6 to 18 post-treatment. In the case of butyrate- and propionate-induced differentiation, ERK phosphorylation is a marker and may play a role in the proliferation and/or differentiation states of this cell line.

Tumor necrosis factor-alpha induces apoptosis associated with poly(ADP-ribose) polymerase cleavage in HT-29 colon cancer cells.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is known for its selective cytotoxic activity on tumour cells. We analysed the response of HT-29 human colon carcinoma cells to this cytokine. MATERIALS AND METHODS: After TNF-alpha treatment, cell proliferation, cell cycle, reactive oxygen species (ROS) production (flow cytometry), the amount of apoptotic cells (flow cytometry, fluorescence microscopy), cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 activity (Western blotting) were detected. RESULTS: TNF-alpha induced a decrease of cell growth and viability, an accumulation of cells in the S-phase of the cell cycle, an increase of subdiploid cell population and nuclear chromatin condensation and fragmentation, but not sooner than 96-120 hours. However, earlier events characteristic of apoptosis occurred, such as caspase-3 activation, PARP cleavage to 89 kDa fragment and changes in ROS production. CONCLUSION: We demonstrated that, in addition to being an early marker of apoptosis, activation of caspase-3 and degradation of PARP may play a causative role in HT-29 cell death induced by TNF-alpha.

Antiangiogenic and antitumor effects of a protein kinase Cbeta inhibitor in human HT-29 colon carcinoma and human CaKi1 renal cell carcinoma xenografts.

The compound 317615 x 2HCl, a selective protein kinase Cbeta inhibitor, was not very cytotoxic toward human CaKi1 renal cell carcinoma cells or human HT-29 colon carcinoma cells in monolayer culture. Isobologram analysis was used to determine additivity or synergy of the combination regimens. Exposure of CaKi1 cells to 317615 x 2HCl (10 or 100 mM) along with gemcitabine or 5-fluorouracil for 24 hours resulted in cytotoxicity that appeared to be less-than-additive to additive for the two agents. Exposure of HT-29 cells to gemcitabine along with 317615 x 2HCl (10 mM or 100 mM) resulted in a synergistic cytotoxicity while combinations with 5-fluorouracil resulted in additive to greater-than-additive cytotoxicity for the agents. After treatment of CaKi1 or HT-29 xenograft-bearing mice with 317615 x 2HCl, immunohistochemical staining for expression of endothelial specific markers, either CD31 or CD105, was used to quantify the number of intratumoral vessels in the samples. CaKi1 tumor angiogenesis was very responsive to treatment with 317615 x 2HCl such that the number of intratumoral vessels stained by CD31 or CD105 was decreased to 20% of the control. The HT-29 colon carcinoma angiogenesis was also responsive to 317615 x 2HCl, such that the number of intratumoral vessels stained by CD31 or CD105 was decreased to 40% to 50% of the controL 5-fluorouracil, cisplatin or fractionated radiation therapy was combined with treatment with 317615 x 2HCl in the simultaneous combination treatment regimen in animals bearing HT-29 colon carcinoma xenografts. The resulting tumor growth delays indicated that administration of 317615 x 2HCl increased the effects of the cytotoxic therapy. Both a simultaneous or an overlapping treatment regimen and a sequential treatment regimen were used to assess 317615 x 2HCl alone and along with fractionated radiation therapy or gemcitabine against the human CaKi1 renal cell carcinoma xenograft. The CaKi1 tumor was quite sensitive to fractionated radiation therapy and to gemcitabine and, although 317615 x 2HCl was an effective single agent in this tumor, the combination regimens did not reach additivity for the combination regimens in vivo. 317615 x 2HCl is in early clinical testing.