Taurocholic acid does not induce apoptosis in HCT116 cells regardless of its intracellular concentration

Hydrophobic bile acids but not hydrophilic bile acids induce apoptosis in HCT116 cells. We expressed sodium-dependent bile acid transporters in HCT116 cells, and the intracellular concentration of hydrophilic bile acids increased to that of the hydrophobic bile acids. But no sign of apoptosis was observed, which suggests a hydrophobic-bile acid-specific mechanism for the induction of apoptosis in HCT116 cells.     

Characteristics of apoptosis in HCT116 colon cancer cells induced by deoxycholic acid

Hydrophobic bile acids induce apoptosis in both colon cancer cells and hepatocytes. The mechanism by which colon cancer cells respond to bile acids is thought to be different from that of hepatocytes. Therefore, we investigated the characteristics of apoptosis in colon cancer cell line HCT116. Hydrophobic bile acids, i.e., deoxycholic acid (DCA), and chenodeoxycholic acid, induced apoptosis in HCT116 cells. Apoptotic indications were detectable at as early as 30 min and the extent increased in time- and concentration-dependent manners. SDS and a hydrophilic bile acid, cholic acid, did not induce apoptosis even at cytotoxic concentrations. Pretreatment with cycloheximide failed to inhibit apoptosis, suggesting that protein synthesis is not involved in the apoptotic response. Release of cytochrome c from mitochondria and activation of caspase-9 were detectable after 5 and 10 min, respectively, whereas remarkable activation of Bid was not detected. Ursodeoxycholic acid (UDCA) protected HCT116 cells from DCA-induced apoptosis but a preincubation period of > or =5 h was required. Nevertheless, UDCA did not inhibit cytochrome c release from mitochondria. Our results indicate that hydrophobic bile acids induce apoptosis in HCT116 cells by releasing cytochrome c from mitochondria via an undefined but specific mechanism, and that UDCA protects HCT116 cells by acting downstream of cytochrome c release.     

The antiapoptotic role of pregnane X receptor in human colon cancer cells

The orphan nuclear receptor pregnane X receptor (PXR) plays an important role in the detoxification of foreign and endogenous chemicals, including bile acids. PXR promotes bile acid elimination by activating bile acid-detoxifying enzymes and transporters. Certain bile acids are known to promote colonic carcinogenesis by inducing colon cancer cell apoptosis. However, whether and how PXR plays a role in colon cancer apoptosis has not been reported. In this study, we showed that activation of PXR by genetic (using a constitutively activated PXR) or pharmacological (using PXR agonist rifampicin) means protected the PXR-overexpressing colon cancer HCT116 cells from deoxycholic acid-induced apoptosis. Interestingly, activation of PXR also protected HCT116 cells from adriamycin-induced cell death, suggesting that the antiapoptotic effect of PXR was not bile acid specific. Moreover, the antiapoptotic effect of PXR in HCT116 cells appeared to be independent of xenobiotic enzyme regulation, because these cells had little basal and inducible expression of bile acid-detoxifying enzymes. Instead, SuperArray analysis showed that PXR-mediated deoxycholic acid resistance was associated with up-regulation of multiple antiapoptotic genes, including BAG3, BIRC2, and MCL-1, and down-regulation of proapoptotic genes, such as BAK1 and TP53/p53. Treatment with rifampicin in colon cancer LS180 cells, a cell line known to express endogenous PXR, also inhibited apoptosis. Activation of PXR in transgenic mice inhibited bile acid-induced colonic epithelial apoptosis and sensitized mice to dimethylhydrazine-induced colonic carcinogenesis, suggesting that the antiapoptotic effect of PXR is conserved in normal colon epithelium. In summary, our results have established the antiapoptotic role of PXR in both human colon cancer cells and normal mouse colon epithelium.     

Deoxycholate induces DNA damage and apoptosis in human colon epithelial cells expressing either mutant or wild-type p53

Diets rich in fat result in higher concentrations of secondary bile acids or their salts in the colon, which may adversely affect cells of the colonic epithelium. Because secondary bile acids are thought to be genotoxic, exposing colon epithelial cells to secondary bile acids may induce DNA damage that might lead to apoptosis. The requirement for the p53 tumor suppressor gene in such events is unknown. In particular, the effects of secondary bile acids on colon epithelial cells having different p53 tumor suppressor gene status have not been examined. Therefore, HCT-116 and HCT-15 human colon adenocarcinoma cells, which express the wild-type and mutant p53 genes, respectively, were exposed to physiological concentrations of deoxycholate. The cells were then analyzed for evidence of DNA damage and apoptosis. After 2 h of incubation with 300 microM deoxycholate, both cell lines had greater levels of single-strand breaks in DNA as assessed by the comet assay. After 6 h of exposure to deoxycholate, HCT-116 and HCT-15 cells showed morphological signs of apoptosis, i.e., membrane blebbing and the presence of apoptotic bodies. Chromatin condensation and fragmentation were also seen after staining DNA with 4',6-diamidino-2-phenylindole. Other apoptotic assays revealed greater binding of annexin V-fluorescein isothiocyanate, as well as greater post-enzymatic labeling with dUTP-fluorescein isothiocyanate, by both cell lines exposed to deoxycholate. These data suggest that deoxycholate caused DNA damage in colon epithelial cells that was sufficient to trigger apoptosis in a p53-independent manner.     

Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.     

Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53(-/-) HCT116 and p21(-/-) HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53(-/-) HCT116 and p21(-/-) HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21(-/-) HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.     

Caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis: Protective role of the nitric oxide signaling module

Hydrophobic bile acids such as deoxycholate are known tumor promoters in the gastrointestinal tract. We have previously shown that deoxycholate induces apoptosis in colon epithelial cells and that these cells can be made resistant to deoxycholate-induced apoptosis. We now show that the nitric oxide synthase/nitric oxide/guanylate cyclase/cyclic guanosine monophosphate/cGMP-activated protein kinase (NOS/NO/GC/cGMP/PKG) signaling module contributes, in part, to the observed resistance of the cultured DOC-resistant colon epithelial cells (HCT-116R) using pharmacological inhibitors/antagonists (NS2028, Rp-8pCPT-cGMP, KT5823) of members of this signaling module. A novel finding from this study is the caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis of deoxycholate-sensitive HCT-116SA cells and the absence of guanylate cyclase alpha 1 cleavage in deoxycholate-treated HCT-116R resistant cells using Western blot analyses. This cleavage was specific to caspases as lysosomal, proteasomal, serine protease, cathepsin and calpain inhibitors failed to prevent the cleavage, whereas a general caspase inhibitor and a specific caspase-6 inhibitor did prevent guanylate cyclase alpha 1 cleavage.     

Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis

The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53-/- cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53-/- over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53-/- cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax-Bak-) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax-Bak+/HCT116Bax+Bak-) was only marginally effective after WithaD treatment. In HCT116p53-/- cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.     

Determination of molecular associations of some hydrophobic and hydrophilic bile acids by infrared and Raman spectroscopy

The aim of this study was to analyse the Raman and infrared spectra of eight common mammalian bile acids in order to identify intermolecular interactions between hydroxyl and carbonyl groups. The results are considered in the light of the new hydrophilic/hydrophobic classification of bile acids. The alcohol OH group of the hydrophobic bile acids forms different intermolecular bonds. The most hydrophobic bile acid, lithocholic acid forms polymers, and this may explain its very low water solubility. The hydrophilic bile acids have some of their alcohol OH groups free of any intermolecular interaction. The strongly hydrophilic murideoxycholic acid also forms dimers, again consistent with a very low water solubility. The proposed structural arrangements are in agreement with published crystallographic studies. Received: 7 November 1996 / Accepted: 8 December 1996     

Ionizing radiation enhances the expression of the nonsteroidal anti-inflammatory drug-activated gene (NAG1) by increasing the expression of TP53 in human colon cancer cells

The induction of apoptosis in cells of human colon cancer cell lines after gamma irradiation was investigated to determine whether apoptosis was mediated by TP53 and the subsequent expression of its downstream target, the NSAID-activated gene (NAG1). HCT116 (TP53(+/+)), HCT15 (TP53 mutant) and TP53 null HCT116 (TP53(-/-)) cells were irradiated with gamma rays, and apoptosis was measured at various times after irradiation. In HCT116 TP53(+/+) cells, apoptosis was increased after irradiation; the increase was dependent on the time after treatment and the dose of gamma rays. However, in HCT15 TP53 mutant cells and HCT116 TP53(-/-) cells, there were no remarkable changes in apoptosis. The expression of TP53 protein in HCT116 cells was increased after irradiation and was followed by an increase in the expression of NAG1 protein. In contrast, the expression of NAG1 protein in TP53 mutant cells and TP53(-/-) cells was not increased by the radiation treatment, suggesting that NAG1 was required for apoptosis. The expression of NAG1 increased apoptosis in HCT116 cells, but radiation treatment did not further increase apoptosis. The transfection of a NAG1 siRNA into HCT116 cells suppressed radiation-induced apoptosis and inhibited the induction of NAG1 protein without altering the expression of TP53. a NAG1 luciferase promoter construct that included both of the TP53 binding sites, was activated by radiation in dose-dependent manner, while the promoters lacking one or both of the TP53 binding sites in the NAG1 promoter activity either was less responsive or did not respond. The findings reported here indicate that gamma radiation activates the TP53 tumor suppressor, which then increases the expression of NAG1. NAG1 mediates the induction of apoptosis in human colorectal cells.     

Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells

The purine-derived analogs, roscovitine and purvalanol are selective synthetic inhibitors of cyclin-dependent kinases (CDKs) induced cell cycle arrest and lead to apoptotic cell death in various cancer cells. Although a number of studies investigated the molecular mechanism of each CDK inhibitor on apoptotic cell death mechanism with their therapeutic potential, their regulatory role on autophagy is not clarified yet. In this paper, our aim was to investigate molecular mechanism of CDK inhibitors on autophagy and apoptosis in wild type (wt) and Bax deficient HCT 116 cells. Exposure of HCT 116 wt and Bax^−/− cells to roscovitine or purvalanol for 24 h decreased cell viability in dose-dependent manner. However, Bax deficient HCT 116 cells were found more resistant against purvalanol treatment compared to wt cells. We also established that both CDK inhibitors induced apoptosis through activating mitochondria-mediated pathway in caspase-dependent manner regardless of Bax expression in HCT 116 colon cancer cells. Concomitantly, we determined that purvalanol was also effective on autophagy in HCT 116 colon cancer cells. Inhibition of autophagy by 3-MA treatment enhanced the purvalanol induced apoptotic cell death in HCT 116 Bax^−/− cells. Our results revealed that mechanistic action of each CDK inhibitor on cell death mechanism differs. While purvalanol treatment activated apoptosis and autophagy in HCT 116 cells, roscovitine was only effective on caspase-dependent apoptotic pathway. Another important difference between two CDK inhibitors, although roscovitine treatment overcame Bax-mediated drug resistance in HCT 116 cells, purvalanol did not exert same effect.     

siRNA抑制Survivin基因对大肠癌细胞HCT116增殖及凋亡的的影响

目的探讨si RNA沉默Survivin基因后对人大肠癌细胞系HCT116的Survivin蛋白表达、细胞增殖及凋亡的影响。方法以脂质体lip-2000为载体,用针对Survivin特异靶点的si RNA转染人大肠癌细胞系HCT116后,应用免疫细胞化学S-P法、Western Blot检测Survivin蛋白表达变化;MTT法检测细胞增殖;流式细胞技术检测细胞凋亡。结果免疫细胞化学结果显示:HCT116的正常对照组、脂质体对照组及阴性错配对照组细胞浆均呈强阳性表达,Survivin-si R-NA组细胞浆Survivin呈弱阳性表达;Western blot结果显示:HCT116细胞系Survivin-si RNA组细胞的蛋白条带亮度均明显低于正常对照组、脂质体对照组和阴性错配对照组;MTT检测结果:与阴性错配对照组相比,Survivin-si RNA组细胞生长出现明显的抑制(P0.05),不同时段(24h、48h、72h)肿瘤细胞增殖抑制率之间有显著差异(P0.05)。流式细胞术检测Survivin-si RNA组细胞凋亡比例为9.72%,明显高于空白对照组及阴性错配对照组(P0.01)。结论si RNA抑制Survivin基因可以抑制大肠癌细胞增殖,促进凋亡;Survivin有望成为大肠癌基因治疗的新靶点。     

Deoxycholic acid induces intracellular signaling through membrane perturbations

Secondary bile acids have long been postulated to be tumor promoters in the colon; however, their mechanism of action remains unclear. In this study, we examined the actions of bile acids at the cell membrane and found that they can perturb membrane structure by alteration of membrane microdomains. Depletion of membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed deoxycholic acid (DCA)-induced apoptosis, and staining for cholesterol with filipin showed that DCA caused a marked rearrangement of this lipid in the membrane. Likewise, DCA was found to affect membrane distribution of caveolin-1, a marker protein that is enriched in caveolae membrane microdomains. Additionally, fluorescence anisotropy revealed that DCA causes a decrease in membrane fluidity consistent with the increase in membrane cholesterol content observed after 4 h of DCA treatment of HCT116 cells. Significantly, by using radiolabeled bile acids, we found that bile acids are able to interact with and localize to microdomains differently depending on their physicochemical properties. DCA was also found to induce tyrosine phosphorylation and activate the receptor tyrosine kinase epidermal growth factor receptor in a ligand-independent manner. In contrast, ursodeoxycholic acid did not exhibit any of these effects even though it interacted significantly with the microdomains. Collectively, these data suggest that bile acid-induced signaling is initiated through alterations of the plasma membrane structure and the redistribution of cholesterol.     

Inhibition of proteasomal degradation of Mcl-1 by cobalt chloride suppresses cobalt chloride-induced apoptosis in HCT116 colorectal cancer cells

Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250–750 μM) which, at higher concentrations (500–750 μM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.     

The Ganglioside GM3 Is Associated with Cisplatin-Induced Apoptosis in Human Colon Cancer Cells

Cisplatin (cis-diamminedichloroplatinum, CDDP) is a well-known chemotherapeutic agent for the treatment of several cancers. However, the precise mechanism underlying apoptosis of cancer cells induced by CDDP remains unclear. In this study, we show mechanistically that CDDP induces GM3-mediated apoptosis of HCT116 cells by inhibiting cell proliferation, and increasing DNA fragmentation and mitochondria-dependent apoptosis signals. CDDP induced apoptosis within cells through the generation of reactive oxygen species (ROS), regulated the ROS-mediated expression of Bax, Bcl-2, and p53, and induced the degradation of the poly (ADP-ribosyl) polymerase (PARP). We also checked expression levels of different gangliosides in HCT116 cells in the presence or absence of CDDP. Interestingly, among the gangliosides, CDDP augmented the expression of only GM3 synthase and its product GM3. Reduction of the GM3 synthase level through ectopic expression of GM3 small interfering RNA (siRNA) rescued HCT116 cells from CDDP-induced apoptosis. This was evidenced by inhibition of apoptotic signals by reducing ROS production through the regulation of 12-lipoxigenase activity. Furthermore, the apoptotic sensitivity to CDDP was remarkably increased in GM3 synthase-transfected HCT116 cells compared to that in controls. In addition, GM3 synthase-transfected cells treated with CDDP exhibited an increased accumulation of intracellular ROS. These results suggest the CDDP-induced oxidative apoptosis of HCT116 cells is mediated by GM3.     

Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio

p21(WAF1) appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21(WAF1) response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21(-/-) cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21(-/-) cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21(+/+) cells, providing evidence that p21(WAF1) exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-alpha reduced the sensitivity of HCT116/p21(-/-) cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21(+/+) cells. Enhancement of p53 expression after disruption of p21(WAF1) resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14(ARF), an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14(ARF) in p53 stabilization, overexpression of p14(ARF) in HCT116/p21(+/+) cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the anti-apoptotic effect of p21(WAF1) consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14(ARF) expression.     

RNAi沉默Snail增加结肠癌对5氟尿嘧啶敏感性研究

目的:研究Snail的抑制是否能增加耐药结肠癌细胞对5-FU的敏感性,评估其可能的信号转导通路。方法:使用5-氟尿嘧啶耐药HCT116细胞(HCT116/5-FU),评估细胞形态及分子的变化。通过靶向人Snail基因小干扰RNA(si RNA)抑制Snail的表达。Annexin V/PI染色用于评估5-FU诱导的细胞凋亡。Western blot检测caspase以及可能的丝裂原活化蛋白激酶(MAPK)和线粒体途径。结果:HCT116细胞对5-Fu耐药性的获得诱导了与EMT一致的形态学变化。RNA干扰沉默Snail逆转HCT116/5-FU细胞EMT并增加了5-FU耐药HCT116细胞对5-FU的敏感性。可能的机制涉及JNK与线粒体途径的激活。结论:EMT样表型的改变与HCT116细胞对5-FU耐药相关;si RNA介导的Snail下调可能是一个潜在的克服5-FU化疗耐药的治疗方法。