Lack of cyclooxygenase-2 activity in HT-29 human colorectal carcinoma cells

Induction of cyclooxygenase-2 (COX-2) is an early event in the sequence of polyp formation to colon carcinogenesis. COX-2 is at elevated levels in human colorectal cancers and in tumors and polyps of mouse models of colorectal cancer. Mutation of the adenomatous polyposis coli (APC) gene is the initial event leading to colorectal cancer. Colorectal cells in culture which express mutant APC are often used to examine the association of COX-2 expression and apoptosis. The expression of full-length APC in HT-29 cells, a human colorectal carcinoma cell line which normally expresses truncated APC and highly expresses COX-2, inhibits cell growth through increased apoptosis and results in a down-regulation of COX-2 protein. In this report, we examine whether down-regulation of COX-2 is directly linked to the increase in apoptosis observed in these HT-29-APC cells. We present evidence that COX-2 and apoptosis are not linked since COX-2, although expressed, is catalytically inactive. Interestingly, the COX-2 cloned from HT-29 cells is catalytically active when transfected into HCT-116 cells, a colorectal cell line which normally does not express COX-2, but is not active in the HT-29 cell line itself.     

Role of leukotriene B4 in celecoxib-mediated anticancer effect

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has anticancer effect on many cancers associated with chronic inflammation by both COX-2-dependent and COX-2-independent mechanisms. The non-COX-2 targets of celecoxib, however, are still a matter of research. Leukotriene B₄ (LTB₄) has been implicated in prostate and colon carcinogenesis, but little is known about the potential role of LTB₄ in celecoxib-mediated anticancer effect. In this study, we evaluated whether LTB₄ was involved in celecoxib-mediated inhibitory effect on human colon cancer HT-29 cells and human prostate cancer PC-3 cells. Our data showed that survival of both cell lines was obviously suppressed after celecoxib treatment for 72 h in a concentration-dependent manner. However, only in HT-29 cells, this inhibitory effect could be reversed by LTB₄, which promoted survival of HT-29 cells rather than PC-3 cells. Consistent with these results, lioxygenase (LOX) potent inhibitor nordihydroguaiaretic acid (NDGA) had a higher inhibitory effect on HT-29 cells than PC-3 cells. Additionally, ELISA results showed that celecoxib could suppress expression of LTB₄ in both cell lines, whereas, inhibition of PGE₂ was only detected in HT-29 cells. These results indicate that the anticancer effect of celecoxib is COX-2-independent in HT-29 and PC-3 cells and in HT-29 cells primarily via down-regulating LTB₄ production.     

Chemopreventive effect of chitosan oligosaccharide against colon carcinogenesis

Chitosan oligosaccharide (COS, 3 kDa相似文献   

Oroxylin A improves the sensitivity of HT-29 human colon cancer cells to 5-FU through modulation of the COX-2 signaling pathway

5-Fluorouracil (5-FU) is a principal drug for the treatment of colorectal cancer. Due to its low response and high toxicity, synergistic effects of 5-FU in combination with other drugs have been widely researched. This study investigated whether oroxylin A improved the sensitivity of HT-29 human colon cancer cells to 5-FU. A correlation between COX-2 inhibition by oroxylin A and a synergistic effect of 5-FU on the growth of HT-29 cells was observed, and a COX-2 pathway for this effect was recognized; oroxylin A evidently elevated the level of reactive oxygen species in HT-29 cells, which subsequently inhibited COX-2 expression and enhanced the susceptibility of HT-29 cells to 5-FU. Likely also related to COX-2 inhibition, oroxylin A decreased PGE(2) levels in HT-29 cells. The synergistic effect of 5-FU induced by oroxylin A was also found in the suppression of Bcl-2 and in the activation of P53, Bax, PARP, and procaspase-3 proteins in HT-29 cells. Ultimately, a combination of 5-FU with oroxylin A significantly reduced the growth of HT-29 tumors in nude mice compared with treatment with 5-FU or oroxylin A alone. In conclusion, a combination of 5-FU and oroxylin A has a significant synergistic effect in the inhibition of HT-29 cell proliferation in vitro and controls HT-29 tumor growth in vivo. This synergistic effect may be mainly related to COX-2 inhibition by oroxylin A in HT-29 cells.     

A novel nitro-oxy substituted analogue of rofecoxib reduces human colon cancer cell growth

Rofecoxib is a specific COX-2 inhibitor able to exert antiproliferative activity against colorectal cancer cells. It was withdrawn from the market after the demonstration of an increased risk of cardiovascular complications after prolonged use. Nevertheless, it remains an interesting compound for laboratory research as an experimental COX-2 inhibitor. In this study, the antiproliferative activity of a novel dinitro-oxy-substituted analogue of rofecoxib (NO-rofe), potentially less cardiotoxic, has been investigated in vitro on human colon cancer cells and compared with the action of the parent drug. Due to the fact that COX-2 inhibition is the main characteristic of coxibs, we performed all experiments in COX-2-overexpressing (HT-29) and COX-2-negative (SW-480) human colon cancer cells, to elucidate whether the observed effects were dependent on COX-2 inhibition. Moreover, experiments were performed in order to evaluate whether COX-2 pharmacological inhibition may affect beta-catenin/E-cadherin signaling pathway. NO-rofe exerted a significant antiproliferative activity on COX-2 positive HT-29 human colon cancer cells, being less effective on the COX-2 negative SW-480 human colon cancer cell line. In particular, the rofecoxib analogue retained similar potencies with respect to COX-2 inhibition but was much more active than rofecoxib in inhibiting the growth of human colon cancer cells in vitro. In addition, this novel compound resulted in the induction of membrane β-catenin/E-cadherin expression, a feature that may significantly contribute to its antiproliferative activity.     

Two opposing effects of non-steroidal anti-inflammatory drugs on the expression of the inducible cyclooxygenase. Mediation through different signaling pathways

The efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is considered to be a result of their inhibitory effect on cyclooxygenase (COX) activity. Here, we report that flufenamic acid shows two opposing effects on COX-2 expression; it induces COX-2 expression in the colon cancer cell line (HT-29) and macrophage cell line (RAW 264.7); conversely, it inhibits tumor necrosis factor alpha (TNFalpha)- or lipopolysaccharide (LPS)-induced COX-2 expression. This inhibition correlates with the suppression of TNFalpha- or LPS-induced NFkappaB activation by flufenamic acid. The inhibitor of extracellular signal-regulated protein kinase, p38, or NFkappaB does not affect the NSAID-induced COX-2 expression. These results suggest that the NSAID-induced COX-2 expression is not mediated through activation of NFkappaB and mitogen-activated protein kinases. An activator of peroxisome proliferator-activated receptor gamma, 15-deoxy-Delta(12,14)-prostaglandin J(2), also induces COX-2 expression and inhibits TNFalpha-induced NFkappaB activation and COX-2 expression. Flufenamic acid and 15-deoxy-Delta(12,14)-prostaglandin J(2) also inhibit LPS-induced expression of inducible form of nitric-oxide synthase and interleukin-1alpha in RAW 264.7 cells. Together, these results indicate that the NSAIDs inhibit mitogen-induced COX-2 expression while they induce COX-2 expression. Furthermore, the results suggest that the anti-inflammatory effects of flufenamic acid and some other NSAIDs are due to their inhibitory action on the mitogen-induced expression of COX-2 and downstream markers of inflammation in addition to their inhibitory effect on COX enzyme activity.     

Cyclooxygenase-independent effects of aspirin on HT-29 human colon cancer cells, revealed by oligonucleotide microarrays

Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation of human colon cancer cells in vitro. Transmission electron microscope detected morphological features of apoptosis in the aspirin-treated (5 mM, 72 h) HT-29 cells in which cyclooxygenoase-2 is catalytically inactive. We investigated aspirin-induced genome-wide expression changes in HT-29 cells and further studied the time- and concentration-dependent expression changes in 374 apoptosis-related genes, which is the first to show stimulation of genome-wide expression of HT-29 cells by aspirin. The most marked effects of aspirin are on ribosome assembly and rRNA metabolism, which could explain why the quasi-apoptotic morphological changes are not accompanied by a classical DNA ladder. These findings demonstrate that aspirin induces apoptosis in HT-29 cells, bolstering the hypothesis that apoptosis may be a mechanism by which NSAIDs inhibit colon carcinogenesis.     

Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy in experimental systems

The elevated cyclooxygenase-2 (COX-2) expression has been shown to affect the carcinogenesis and tumor progression processes, including cell proliferation, motility and angiogenesis. COX-2 is overexpressed in approximately 80% of sporadic colorectal carcinomas and COX-2 enzyme is the best defined target of non-steroidal anti-inflammatory drugs (NSAIDs). In the chemotherapy of colorectal carcinomas 5-fluorouracil (5-FU) has been the most important of the basic drugs for more than 40 years. In order to improve the effectiveness of 5-FU therapy different biological modifiers i.e. inhibitors of its catabolism or activators of anabolism have been studied recently. The rate-limiting enzyme of 5-FU catabolism is dihydropyrimidine dehydrogenase (DPD) since more than 80% of the administered 5-FU is catabolized by DPD. Tumoral DPD has become of clinical interest because elevated intratumoral DPD can decrease the tumor response to 5-FU therapy. The main purpose of our experiments was to investigate the effect of COX inhibitors on the efficacy of 5-FU on high and low COX-2 expressing HCA-7 and HT-29 human colon adenocarcinoma cell lines, respectively, and also on xenografts derived from HT-29 cells. The cytotoxic and antitumor effects of 5-FU in the presence of low doses of indomethacin (non-selective COX-2 inhibitor) and that of NS-398 (highly selective COX-2 inhibitor) on HT-29 and HCA-7 cells and also on the HT-29 xenograft were investigated. In addition, our intention was to understand the mechanism(s) by which NSAIDs could enhance the cytotoxic effect of 5-FU. Our data indicated that the elevated COX-2 expression of HCA-7, the collagen-induced HT-29-C cells and of the HT-29 xenograft were associated with reduced 5-FU sensitivity. Based on the fact that at the same time DPD activity was also increased it might be conceivable that a possible explanation for the decrease of 5-FU sensitivity is the co-existence of high COX-2 and DPD activity. Indomethacin or NS-398 enhanced in a simultaneous and significant manner the sensitivity and cytotoxic effect of 5-FU on high COX-2 expressing cells and xenografts through the modulation of DPD - decrease of its mRNA expression and/or enzyme activity. Based on our results it could be presumable that 5-FU efficacy is limited by the COX-2 associated high DPD expression and activity in patients with colorectal cancer as well, therefore further clinical studies are warranted to decide if NSAIDs in the therapeutic protocol might improve the antitumor potency of 5-FU. Réti A. Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy in experimental systems.     

Glycine-extended gastrin stimulates proliferation and inhibits apoptosis in colon cancer cells via cyclo-oxygenase-independent pathways

Glycine-extended gastrin (G-Gly) is an end product of processing of the progastrin precursor peptide that has a different spectrum of activity to amidated gastrin. G-Gly promotes cell proliferation in normal and malignant colonic epithelium but the mechanisms responsible are poorly understood. Prostaglandins produced by the cyclo-oxygenase (COX) enzymes have been implicated as downstream mediators of several growth factors, and COX inhibitors such as non-steroidal anti-inflammatory drugs inhibit the proliferation and invasiveness of colonic cancer and reduce the incidence of colon cancer. We have examined the mechanisms of the actions of G-Gly in HT-29 colon cancer cells. G-Gly induced a dose-dependent increase in cell proliferation that was insensitive to inhibition of either COX-1 or COX-2, but was abolished by inhibition of the p38 MAP kinase, ERK and NF-kappaB pathways. G-Gly did not increase prostaglandin E2 production. Celecoxib induced apoptosis and reduced viable cell numbers in a COX-independent manner. G-Gly significantly reduced serum-starvation and celecoxib-induced apoptosis and this effect was also blocked by inhibition of the p38 MAP kinase, ERK and NF-kappaB pathways. Stimulation of HT-29 cells with G-Gly led to a rapid increase in ERK and p38 MAP kinase phosphorylation and increased nuclear translocation of active NF-kappaB. Activation of NF-kappaB was independent of ERK and p38 MAP kinase. G-Gly stimulates proliferation and inhibits apoptosis in colon cancer cells via COX-independent and ERK-, p38 MAP kinase-, and NF-kappaB-dependant pathways. Locally and systemically produced G-Gly may be important in reducing the beneficial effects of chemopreventative agents in colon cancer.     

Effects of adrenaline in human colon adenocarcinoma HT-29 cells

AimsStress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved.Main methodsThe effect of adrenaline on HT-29 cell proliferation was determined by [³H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E₂ (PGE₂) release were determined by zymography and enzyme immunoassay, respectively.Key findingsAdrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE₂ release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β₁- and β₂-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE₂ release in HT-29 cells.SignificanceThese results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β₁- and β₂-adrenoceptors by a COX-2 dependent pathway.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10-40 microM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6-24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E(2) by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.     

Celecoxib and tauro-ursodeoxycholic acid co-treatment inhibits cell growth in familial adenomatous polyposis derived LT97 colon adenoma cells

Chemoprevention would be a desirable strategy to avoid duodenectomy in patients with familial adenomatous polyposis (FAP) suffering from duodenal adenomatosis. We investigated the in vitro effects on cell proliferation, apoptosis, and COX-2 expression of the potential chemopreventives celecoxib and tauro-ursodeoxycholic acid (UDCA). HT-29 colon cancer cells and LT97 colorectal micro-adenoma cells derived from a patient with FAP, were exposed to low dose celecoxib and UDCA alone or in combination with tauro-cholic acid (CA) and tauro-chenodeoxycholic acid (CDCA), mimicking bile of FAP patients treated with UDCA. In HT-29 cells, co-treatment with low dose celecoxib and UDCA resulted in a decreased cell growth (14-17%, p<0.01). A more pronounced decrease (23-27%, p<0.01) was observed in LT97 cells. Cell growth of HT-29 cells exposed to 'artificial bile' enriched with UDCA, was decreased (p<0.001), either in the absence or presence of celecoxib. In LT97 cells incubated with 'artificial bile' enriched with UDCA, cell growth was decreased only in the presence of celecoxib (p<0.05). No clear evidence was found for involvement of proliferating cell nuclear antigen, caspase-3, or COX-2 in the cellular processes leading to the observed changes in cell growth. In conclusion, co-treatment with low dose celecoxib and UDCA has growth inhibitory effects on colorectal adenoma cells derived from a patient with FAP, and further research on this combination as promising chemopreventive strategy is desired.     

UDN glycoprotein inhibits activator protein-1 and matrix metalloproteinase-9 via blocking of oxygen radicals in HT-29 cells

The present study was performed to investigate the anti-inflammatory potential of a 116-kDa glycoprotein isolated from Ulmus davidiana Nakai (UDN glycoprotein) in lipopolysaccaride (LPS)-treated cancerous human colon epithelial cells (HT-29 cells). UDN glycoprotein inhibited the production of intracellular superoxide anion (O₂^·−), hydrogen peroxides (H₂O₂), and nitric oxide (NO), whereas normalized the activity of anti-oxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX)], accompanying the inhibition of manganese-superoxide dismutases (Mn-SOD) activity in LPS-treated HT-29 cells. In addition, UDN glycoprotein blocked the DNA binding activity of activator protein-1 (AP-1) through suppression of c-Jun and c-Fos activities, respectively. We also evaluated the anti-inflammatory potential of UDN glycoprotein based on the activity of the pro-inflammatory signal mediators [inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinases-9 (MMP-9)]. The results showed that UDN glycoprotein (200 μg/ml) has an inhibitory effect on the activation of iNOS, COX-2, and MMP-9 proteins in the LPS-treated HT-29 cells. From these results, we suggest that UDN glycoprotein is one of the potential anti-inflammatory agents that blocks LPS-mediated inflammatory signal pathway in HT-29 cells. Here, we speculate that UDN glycoprotein could be used as an antioxidative agent for inflammatory gastrointestinal cancers.     

Selective inhibition of cyclooxygenase-2 (COX-2) by 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3, a less calcemic vitamin D analog

Inducible cyclooxygenase-2 (COX-2) has been implicated to play a role in inflammation and carcinogenesis and selective COX-2 inhibitors have been considered as anti-inflammatory and cancer chemopreventive agents. 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1alpha,25(OH)2D3 and one of its less calcemic synthetic analogs, 1alpha,25(OH)2-16-ene-23-yne-D3 on the activity of both COX-1 and COX-2 in an in vitro enzyme assay. Preliminary data indicated that both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited selectively the activity of COX-2 with no effect on the activity of COX-1. Out of the two compounds, 1alpha,25(OH)2-16-ene-23-yne-D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1alpha,25(OH)2-16-ene-23-yne-D3 only. 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX-2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin-2 (IL-2). Furthermore, 1alpha,25(OH)2-16-ene-23-yne-D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX-2, iNOS, and IL-2 in the tissues of the same air pouch. In both cases, 1alpha,25(OH)2-16-ene-23-yne-D3 did not show any effect on the expression of COX-1. In summary, our results indicate that 1alpha,25(OH)2-16-ene-23-yne-D3, a less calcemic vitamin D analog, exhibits potent anti-inflammatory effects and is a selective COX-2 inhibitor.     

Prostaglandin-independent effects of aspirin on cell cycle and putrescine synthesis in human colon carcinoma cells

Aspirin consumption has been reported to be able to reduce colorectal cancer risk in humans and in animal models of colon carcinogenesis. Although the mechanism involved in such an effect is not yet clear, both prostaglandin-dependent and -independent effects have been proposed. Using HT-29 Glc(-/+)cells, which originate from a human colon adenocarcinoma, we demonstrated in this study a dose-dependent effect of millimolar concentration of aspirin on cell growth that was concomitant with a rapid accumulation of the cells in the G0/G1 phase, followed by an accumulation in the G2/M phase and by a minor increase in the proportion of cells undergoing nuclear condensation. Cell membrane integrity and cell release into the culture medium were not affected by this treatment. The aspirin effects were apparently unrelated to prostaglandin biosynthesis inhibition, since although these cells were found to express high levels of cyclooxygenase 1 (COX-1) and low levels of COX-2 proteins, they did not produce any measurable net amounts of prostaglandins, based on both utilization of radiolabelled arachidonic acid and the radioimmunoassay of prostaglandins E2 and F2 alpha. In contrast, we identified polyamine biosynthesis as a cellular target of aspirin, since the treatment of HT-29 Glc(-/+) cells with aspirin reduced the flux of L-ornithine through ornithine decarboxylase, an effect that could not be explained by an acute action of the drug on the ornithine decarboxylase catalytic activity. Since polyamine biosynthesis is strictly necessary for HT-29 cell growth, our data suggest that reduced flux through ornithine decarboxylase may participate in the antiproliferative activity of aspirin towards colonic tumoral cells. It is concluded that in HT-29 Glc(-/+) cells that are not functional for prostaglandin production, aspirin can affect cell growth, cell cycle, and polyamine biosynthesis without affecting cell membrane integrity.     

Survivin and Cyclooxygenase-2 are co-expressed in human and mouse colon carcinoma and in terminally differentiated colonocytes

In the evolution of colon rectal cancer (CRC) the imbalance between cell proliferation and apoptosis is considered one of the prominent causes of tumor induction and/or progression. In order to establish the role of anti apoptotic proteins in colon cancer development, we studied with immunohistochemical techniques the expression of Survivin in a mouse model of colon carcinogenesis induced by 1,2-dimethyl-hydrazine treatment. In this mouse model Survivin was over-expressed during tumor development, showing a distribution mimicking that described in the correspondent human malignancies. We also correlated Survivin distribution with COX-2 and beta-Catenin expression patterns. The co-localization of COX-2/beta-Catenin/Survivin in the same epithelial cells in tumor samples lends credence to possible in vivo regulatory effects of COX-2 and beta-Catenin on the intracellular Survivin levels in mouse and human colon cancer.     

Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT-29 cell line

Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity. This effect was correlated with inhibition of the phosphorylation of the PDK1 downstream substrate Akt/protein kinase B (PKB) on two regulatory sites, Thr(308) and Ser(473). However, expression of a constitutive active form of Akt/PKB (myristoylated PKB) has a low protective effect toward celecoxib-induced cell death. In contrast, overexpression of constitutive active mutant of PDK1 (PDK1(A280V)) was as potent as the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, to impair celecoxib-induced apoptosis. By contrast, cells expressing a kinase-defective mutant of PDK1 (PDK1(K114G)) remained sensitive to celecoxib. Furthermore, in vitro measurement reveals that celecoxib was a potential inhibitor of PDK1 activity with an IC(50) = 3.5 microm. These data indicate that inhibition of PDK1 signaling is involved in the proapoptotic effect of celecoxib in HT-29 cells.     

Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29

Autophagy is a major catabolic process allowing the renewal of intracellular organelles by which cells maintain their homeostasis. We have previously shown that autophagy is controlled by two transduction pathways mediated by a heterotrimeric Gi3 protein and phosphatidylinositol 3-kinase activities in the human colon cancer cell line HT-29. Here, we show that 3-methyladenine, an inhibitor of autophagy, increases the sensitivity of HT-29 cells to apoptosis induced by sulindac sulfide, a nonsteroidal anti-inflammatory drug which inhibits the cyclooxygenases. Similarly, HT-29 cells overexpressing a GTPase-deficient mutant of the G(alpha i3) protein (Q204L), which have a low rate of autophagy, were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells. In both cell populations we did not observe differences in the expression patterns of COX-2, Bcl-2, Bcl(XL), Bax, and Akt/PKB activity. However, the rate of cytochrome c release was higher in Q204L-overexpressing cells than in HT-29 cells. These results suggest that autophagy could retard apoptosis in colon cancer cells by sequestering mitochondrial death-promoting factors such as cytochrome c.     

The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.