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Transforming growth factor (TGF)-beta-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-beta inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-beta-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-beta-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-beta/Smad3. Total RNA samples were analyzed using Affymetrix oligonucleotide microarrays. We found that TGF-beta regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-beta activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-beta induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-beta-induced apoptosis in RIE-1/Smad3 cells.

Glutamine starvation induces apoptosis in enterocytes; therefore glutamine is important in the maintenance of gut mucosal homeostasis. However, the molecular mechanisms are unknown. The caspase family of proteases constitutes the molecular machinery that drives apoptosis. Caspases are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were to (1) identify specific caspases activated by glutamine starvation and (2) determine whether a general caspase inhibitor blocks glutamine starvation-induced apoptosis in intestinal epithelial cells. Rat intestinal epithelial (RIE-1) cells were deprived of glutamine. Specific caspase activation was measured using fluorogenic substrate assay. Apoptosis was quantified by DNA fragmentation and Hoechst nuclear staining. Glutamine starvation of RIE-1 cells resulted in the time-dependent activation of caspases 3 (10 hours) and 2 (18 hours), and the induction of DNA fragmentation (12 hours). Caspases 1 and 8 remained inactive ZVAD-fluoromethyl ketone, a general caspase inhibitor, completely blocked glutamine starvation-induced caspase activation, DNA fragmentation, and nuclear condensation. These results indicate that glutamine starvation selectively activates specific caspases, which leads to the induction of apoptosis in RIE-1 cells. Furthermore, inhibition of caspase activity blocked the induction of apoptosis, suggesting that caspases are potential molecular targets to attenuate apoptotic responses in the gut.

Background: The prognosis for gastric cancer remains dismal; novel agents that target specific molecular pathways are needed as adjuvant therapy. Camptothecin (CPT), on inhibitor of topoisomerase I, is effective in the treatment of certain solid tumors; its effects on gastric cancer are largely undefined. The purpose of this study was to (1) characterize the effects of CPT on the growth of a human gastric cancer and (2) assess potential cellular mechanisms responsible for CPT-mediated growth inhibition., Methods: The human gastric cancer SIIA was transplanted subcutaneously into athymic nude mice. After tumors reached approximately 100 mm2, mice were randomized into 3 groups to receive either CPT (5 or 10 mg/kg) or vehicle (control) intraperitoneally 3 days per week for 3 weeks; tumor size was measured biweekly. To assess potential mechanisms of CPT-mediated inhibition, SIIA cells were treated with CPT (20 mumol/L) and cells were counted over a time course; apoptosis was assessed by Hoechst stain and DNA laddering. Expression of p53 (a tumor suppressor), p21Waf1 and p27Kip1 (cell cycle inhibitors), and Bcl-2 and Bcl-XL (antiapoptotic proteins) was determined., Results: CPT (5 and 10 mg/kg) significantly inhibited tumor growth of SIIA gastric cancers compared with controls. CPT-mediated inhibition of SIIA cell proliferation was associated with an increase in apoptosis. Moreover, CPT treatment resulted in induction of p53, p21Waf1, and p27Kip1 and a decrease in Bcl-2 and Bcl-XL RNA and protein levels., Conclusions: Treatment with CPT effectively inhibited the growth of the human gastric cancer SIIA; the mechanism involved was induction of apoptosis mediated by up-regulation of p53, p21Waf1/Cip1, and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL. Novel agents such as CPT, which target specific molecular pathways, may prove clinically useful in the adjuvant treatment of gastric cancers.

Background: Glutamine is the most abundant amino acid in the blood, and its deprivation leads to gut mucosal atrophy. The small intestinal mucosa is maintained by a balance between cell proliferation and cell death by apoptosis. We reported that glutamine is required for nitrogen-stimulated proliferation in intestinal epithelial cells. We do not know whether glutamine regulates apoptosis in the gut. The purpose of this study is to determine whether glutamine deprivation induces apoptosis in rat intestinal epithelial (RIE-1) cells and to compare the effect of glutamine starvation with that of methionine and cysteine (Met/Cys) starvation., Methods: RIE-1 cells were deprived of either glutamine or Met/Cys for 24 hours. Cell numbers were determined by cell counting and tetrazolium enzymatic assay. Apoptosis was quantified by Annexin V assay and confirmed by DNA gel electrophoresis and Hoecsht nuclear staining., Results: Deprivation of glutamine or Met/Cys resulted in decreased cell numbers. However, only the glutamine-deprived group showed significant induction of apoptosis with increased Annexin V staining, DNA laddering, and nuclear condensation., Conclusions: This study provides biochemical and morphologic evidence that glutamine deprivation induces apoptosis in rat intestinal epithelial cells. In contrast, Met/Cys starvation suppresses cell number without induction of apoptosis. These results suggest that glutamine serves as a specific survival factor in enterocytes.