Sensitization of human colon cancer cells to TRAIL-mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor (TNF) family, is thought to induce apoptosis preferentially in cancer cells; however, increasing evidence suggests that a number of cancers are resistant to TRAIL treatment. FLICE-like inhibitory protein (FLIP), which structurally resembles caspase-8, can act as an inhibitor of apoptosis when expressed at high levels in certain cancer cells. The purpose of our present study was to determine whether human colon cancer cells are sensitive to TRAIL treatment and, if not, to identify potential mechanisms of resistance. Colon cancer cells of different metastatic potential (KM12C, KML4A, and KM20) were found to be resistant to the effects of TRAIL when used as a single agent. FLIP expression levels were increased in all three KM cell lines. Treatment with either actinomycin D (Act D;10 :g/ml) or cycloheximide (CHX; 10 :g/ml) decreased FLIP expression levels in all three cell lines. The decrease in cellular levels of FLIP was associated with sensitization to TRAIL-mediated apoptosis, as demonstrated by enhanced cell death and caspase-3 activity compared with either Act D or CHX alone. Our findings suggest that reduction of FLIP levels by Act D or CHX renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL along with agents such as Act D or CHX, which target proteins that prevent cell death, may provide a more effective and less toxic regimen for treatment of resistant colon cancers.

Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation.

BACKGROUND & AIMS: Phosphatidylinositol 3-kinase (PI3K), an important mediator of intracellular signal transduction, has been shown to affect proliferation, differentiation, and apoptosis in a number of cells; the role of PI3K in intestinal cell differentiation is not known. METHODS: The effect of PI3K inhibition on enterocyte-like differentiation of the human colon cancer cells, HT29 and Caco-2, was assessed using complementary approaches (i.e., chemical inhibition with wortmannin, transfection with a dominant negative p85 mutant, or overexpression of the tumor suppressor gene phosphatase and tensin homologue deleted on chromosome 10 [PTEN]). Brush-border enzyme (intestinal alkaline phosphatase [IAP] and sucrase) activities, IAP messenger RNA levels, and IAP promoter induction were measured. RESULTS: The PI3K inhibitor, wortmannin, in combination with sodium butyrate, synergistically induced IAP and sucrase enzyme activities and IAP messenger RNA levels in a time- and dose-dependent fashion. Consistent with these results, cotransfection using the dominant negative mutant of p85 (Deltap85) induced IAP promoter activity. Moreover, overexpression of PTEN, which antagonizes PI3K, significantly augmented the induction of IAP enzyme activity in HT29 and Caco-2 cells treated with sodium butyrate and in spontaneously differentiated Caco-2 cells. CONCLUSIONS: Our results show that inhibition of PI3K significantly enhances enterocyte-like differentiation of HT29 and Caco-2 cells. Taken together, our findings suggest a contributory role for the PI3K/PTEN pathway in intestinal cell differentiation.

Inhibition of proliferation, invasion and adhesion of liver cancer cells by 5-azacytidine and butyrate.

BACKGROUND: The prognosis for liver cancer is poor with current chemotherapeutic agents, for the most part, ineffective. We have recently shown that 5-azacytidine (5-azaC) and butyrate stimulate apoptosis in two human liver cancer cell lines (HepG2 and Hep3B). The purpose of our present study was to determine the effects of these agents on proliferation, invasion and adhesion of liver cancer cells, and to assess potential cellular mechanisms for these effects. MATERIALS AND METHODS: HepG2 and Hep3B cells were treated with either 5-azaC (8 microM), sodium butyrate (35 mM), 5-azaC + butyrate or vehicle (control); proliferation, cellular invasion and adherence were determined. Western blots were performed to assess expression levels of p21waf1, p27kip1 and p53. RESULTS: Treatment with 5-azaC alone inhibited invasion of Hep3B cells whereas butyrate alone inhibited invasion of HepG2 cells; the combination of 5-azaC + butyrate completely suppressed the invasion of both cell lines. Moreover, cellular adhesion and proliferation were inhibited in both cell lines by combination treatment. Levels of the Cdk inhibitor p21waf1 were increased in HepG2 cells after 5-azaC and in both cell lines after butyrate treatment; levels of p27kip1 were increased in both cell lines after either 5-azaC or butyrate treatment. CONCLUSIONS: Our results demonstrate that the combination of 5-azaC and butyrate effectively blocks proliferation, invasion and cellular adhesion of both HepG2 and Hep3B cells. Increases in the expression of the cell cycle inhibitory proteins, p21waf1 and p27kip1 suggest that these effects may be mediated through the induction of these inhibitory proteins. Agents such as 5-azaC and butyrate that target the cell cycle pathway may prove clinically useful in the adjuvant treatment of liver cancers.

Inhibition of heat-shock protein 70 induction in intestinal cells overexpressing cyclooxygenase 2.

BACKGROUND & AIMS: The cyclooxygenase (COX) enzymes catalyze the initial step of prostaglandin formation; the inducible form, COX-2, plays a role in inflammation. Heat-shock protein 70 (hsp70) is a stress-responsive gene important for cell survival; induction of hsp70 appears to be mediated, in part, by the prostaglandin pathway. We determined the effect of COX-2 overexpression on hsp70 induction in rat intestinal epithelial (RIE) cells. METHODS: RIE cells transfected with COX-2 complementary DNA oriented in the sense (RIE-S) or antisense (RIE-AS) direction were subjected to a heat shock; RNA and protein were harvested and analyzed by Northern and Western blots, respectively. Gel shift assays were performed to assess DNA binding. RESULTS: Both hsp70 messenger RNA and HSP70 protein levels were increased in the RIE-AS cells, whereas induction was markedly inhibited in the RIE-S cells after heat shock. Inhibition of heat-shock factor binding was noted in RIE-S cells, suggesting that heat-shock transcription factor regulation may explain the inhibition of hsp70. The COX-2 selective inhibitor, NS-398, reversed the effects of COX-2 overexpression. CONCLUSIONS: The results support a functional role for the prostaglandin/COX pathway in the induction of hsp70. The findings underscore a potential regulatory mechanism involving an inverse relationship between COX-2 expression and hsp70 induction.

Glutamine-mediated regulation of heat shock protein expression in intestinal cells.

BACKGROUND: Glutamine is crucial in the growth and protection of intestinal mucosa; the molecular mechanisms for these effects are not known. Heat shock proteins (hsp) are stress-responsive genes that protect cells from various injuries. The purpose of this study was to determine whether glutamine alters hsp expression in intestinal cells after injury. METHODS: IEC-6 (rat intestinal crypt) cells were grown to 80% confluence, rinsed free of medium and serum, and then grown in medium containing various concentrations of L-glutamine (0.5 to 8.0 mmol/L) for 24 hours. Cells were then exposed to heat shock (43 degrees C for 1 hour) and RNA and protein were extracted at 0, 2, 8, and 24 hours after heat shock. Northern hybridizations were performed using an hsp 70 complementary DNA. Western blots were performed by using antibodies specific to the inducible and constitutive forms of hsp70. RESULTS: Northern blot analysis demonstrated a fourfold stimulation of hsp70 messenger RNA 2 hours after heat shock in IEC-6 cells supplemented with 8.0 mmol/L glutamine compared with 0.5 and 1.0 mmol/L glutamine. In addition, inducible Hsp70 protein levels were increased threefold in IEC-6 cells given glutamine (8.0 mmol/L) at 24 hours after heat shock. CONCLUSIONS: Glutamine augments hsp70 messenger RNA and protein expression in gut-derived IEC-6 cells after injury. Our findings demonstrate a novel mechanism for the enhanced cytoprotection of gut mucosa conferred by administration of glutamine and suggest that supplemental glutamine may be required after injury to enhance expression of stress-responsive genes in the gut.

Neurotensin expression and release in human colon cancers.

Neurotensin (NT), a distal gut peptide released by intraluminal fats, is trophic for normal small bowel and colonic mucosa. In addition, NT stimulates growth of certain colon cancers; the mechanism for this effect is not known. The purpose of this study was to determine whether human colon cancers (HCC) (1) express the mRNA for NT/neuromedin N (N), (2) produce NT peptide, and (3) express the mRNA for a functional NT receptor (NTR). RNA was extracted from four HCC cell lines in culture, nine HCC lines established in athymic nude mice, and from six HCC and adjacent normal mucosa from freshly resected operative specimens; the RNA was analyzed for NT/N mRNA by Northern hybridization with a complementary DNA probe. Neurotensin peptide content, NTR expression, and intracellular Ca++ ([Ca++]i) mobilization in response to NT were evaluated in three HCC cell lines (LoVo, HT29, HCT116). Neurotensin/N mRNA transcripts were identified in all four of the HCC cell lines and in one of nine HCC in nude mice. Neurotensin expression was found in two of six freshly resected HCC and in none of the six corresponding samples of normal mucosa. Neurotensin peptide was identified by RIA in LoVo, HT29, and HCT116. In addition, NTR mRNA was found in HT29 and HCT116. Neurotensin stimulated [Ca++]i mobilization in HCT116 (without serum) and in LoVo (with 0.25% serum). These findings demonstrate the presence of NT/N mRNA and NT peptide and the presence of a functional NTR in certain HCC. Neurotensin, a potent trophic factor for normal gut mucosa, may function as an autocrine growth factor in certain human colon cancers.

Effect of total jejunoileal denervation on fat-stimulated release of peptide YY and cholecystokinin.

We have investigated the effect of total denervation of the jejunoileum (JDNv) on stimulated release of peptide YY (PYY) and cholecystokinin-33/39 in five dogs prepared with chronic gastric and duodenal cannulas. JDNv was performed by stripping the adventitia from the superior mesenteric artery and vein, transecting the small bowel mesentery, and division (with reanastomosis) of the small bowel at the ligament of Treitz and ileocecal junctio. Introduodenal corn oil (3 ml/kg/hr) was given before JDNv and 1 and 2 months after JDNv. Intravenous bombesin (400 pmol/kg/hr) was given (on nonconsecutive days) before JDNv and 1 month after JDNv. Plasma PYY and cholecystokinin levels were measured by specific radioimmunoassay. Release of PYY was enhanced after JDNv. The integrated release of PYY (ng.[0 to 60 min]/ml) after intraduodenal corn oil was as follows: before JDNv, 4.1 +/- 1.2; 1 month after JDNv, 16.0 +/- 2.7; and 2 months after JDNv, 10.3 +/- 2.2. Similar results were noted with intravenous bombesin (3.7 +/- 0.9 [before JDNv] vs 12.0 +/- 0.7 [1 month after JDNv]). Corn oil-stimulated release of cholecystokinin was abolished after JDNv (before JDNv 2.2 +/- 1.1; 1 month after JDNv, 0.6 +/- 0.3; and 2 months after JDNv, 0.4 +/- 0.6). Basal plasma levels of PYY and cholecystokinin were not affected by JDNv. We conclude that JDNv enhances PYY and abolished cholecystokinin release, which provides evidence for different mechanisms of neural control.

Multiple adenocarcinomas of the colon and rectum. An analysis of incidences and current trends.

Three hundred forty-five colorectal cancers were identified in 320 patients over a nine-year period. Twenty-one patients (7 percent) had synchronous cancers. Metachronous cancers were identified in five patients (2 percent). Thirteen of the synchronous cancers were foci of invasive adenocarcinoma in polyps with elements of benign neoplastic tissue. There was a trend for younger patients to have multiple colon cancers. Fifteen percent of the synchronous colon cancer patients were less than 50 years of age. The mean age of patients who presented with metachronous cancer was 54, and 11 years was the average time interval between the diagnosis of the initial and the metachronous tumor. Colonoscopy proved to be more reliable than barium-enema examinations in identifying synchronous cancers. It is concluded from this review that before elective resections, colonoscopy should be used to effectively screen patients for synchronous cancers, and following curative resection, the residual colon should be periodically examined for the remainder of the patient's life.