Sulindac sulfide alters the expression of cyclin proteins in HT-29 colon adenocarcinoma cells

Sulindac sulfide (SS), the active metabolite of the colon cancer chemopreventive compound sulindac, inhibits the proliferation of HT-29 colon cancer cells mainly by inducing cell quiescence. We determined by bivariate flow-cytometric analysis both the DNA and cyclin protein content of individual cells. Thus, we assessed in detail the expression of several cyclins during the cell-cycle phases and demonstrated that SS (i) decreases the expression of cyclins B1 and E and (ii) increases the expression of cyclins D1, D2 and D3, particularly in the G₁ phase of the cell cycle. SS-induced apoptotic cells expressed both E- and D-type cyclins but not cyclin B1. The changes in cyclin expression combined with reduced catalytic activity of cyclin-dependent kinases could explain in molecular terms the anti-proliferative effect of SS on HT-29 colon cancer cells. These changes may contribute to the chemopreventive effect of sulindac. Int. J. Cancer 76:99–104, 1998.© 1998 Wiley-Liss, Inc.     

Growth-suppressive effect of non-steroidal anti-inflammatory drugs on 11 colon-cancer cell lines and fluorescence differential display of genes whose expression is influenced by sulindac

In addition to an anti-inflammatory effect, sulindac, one of the non-steroidal anti-inflammatory drugs (NSAIDs), has been shown to have a protective effect against the incidence and mortality of colorectal cancer. However, the molecular basis of its anti-proliferative function remains unclear. To investigate its molecular mechanism, we exposed 11 colon-cancer cell lines to NSAIDs such as aspirin, sulindac and the sulfide and sulfone metabolites of sulindac. Sensitivity to these drugs was dose- and time-dependent but varied from one cell line to another. Among the cell lines examined, sulindac showed a moderate anti-proliferative effect on HT-29 colon cancer cells and caused morphological changes, including an increase of cells with abnormal DNA content. We used the mRNA fluorescence differential display method with these cells to identify molecules that might contribute, through altered expression, to cellular changes in response to NSAIDs. Sixty-eight cDNA fragments were confirmed by RT-PCR to have significantly different expression levels following sulindac treatment. Thirty of these fragments proved to be novel cDNA sequences or identical to expressed sequence tags; the other 38 fragments were identical, or showed significant homology, to genes whose function was already known. Among the known genes differentially expressed in HT-29 cells after sulindac treatment were those encoding acetylglucosaminyltransferase, ferritin heavy chain, zinc finger protein 165, aldose reductase, carcinoembryonic antigen, aldoketoreductase, NF-kappaB-activating kinase, lysosome-associated protein, RhoE = 26 kDa GTPase homologue, NADH oxidoreductase, G/T mismatch bindingprotein, TM7SF3, ADP/ATP carrier-like protein and chromosome segregation protein. This variety among classes of proteins affected by sulindac in our experiments underscores the complexity of anti-proliferative mechanisms that may operate in colon-cancer cells treated with NSAIDs. Furthermore, identification of genes regulated by NSAIDs in colon-cancer cells should provide useful information to identify novel therapeutic targets for treatment and/or prevention of colon cancer.     

Antiproliferative effects of S-allylmercaptocysteine on colon cancer cells when tested alone or in combination with sulindac sulfide

Epidemiological studies link increased garlic (Allium sativum) consumption with a reduced incidence of colon cancer in various human populations. Experimental carcinogenesis studies in animal models and in cell culture systems indicate that several allium-derived compounds exhibit inhibitory effects and that the underlying mechanisms may involve both the initiation and promotion phases of carcinogenesis. To provide a better understanding of the effects of allium derivatives on the prevention of colon cancer, we examined two water-soluble derivatives of garlic, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), for their effects on proliferation and cell cycle progression in two human colon cancer cell lines, SW-480 and HT-29. For comparison, we included the compound sulindac sulfide (SS), because sulindac compounds are well-established colon cancer chemopreventive agents. We found that SAMC, but not SAC, inhibited the growth of both cell lines at doses similar to that of SS. SAMC also induced apoptosis, and this was associated with an increase in caspase3-like activity. These affects of SAMC were accompanied by induction of jun kinase activity and a marked increase in endogenous levels of reduced glutathione. Although SS caused inhibition of cell cycle progression from G1 to S, SAMC inhibited progression at G2-M, and a fraction of the SW-480 and HT-29 cells were specifically arrested in mitosis. Coadministration of SS with SAMC enhanced the growth inhibitory and apoptotic effects of SS. These findings suggest that SAMC may be useful in colon cancer prevention when used alone or in combination with SS or other chemopreventive agents.     

Identification of one exon deletion of intestinal alkaline sphingomyelinase in colon cancer HT-29 cells and a differentiation-related expression of the wild-type enzyme in Caco-2 cells

Sphingomyelin (SM) metabolism in the gut has been implicated in colonic tumorigenesis. Intestinal alkaline sphingomyelinase (alk-SMase) hydrolyses SM in the intestinal content and at the brush border. The enzyme activity is decreased in the tissues of human colorectal tumours. This study examines whether site or chain-mutation of alk-SMase occurs in colon cancer HT-29 cells and Caco-2 cells. Total RNA was isolated and the cDNA of alk-SMase was amplified by RT-PCR. The size of the cDNA from HT-29 cells was smaller than that of the wild-type cDNA. DNA sequencing identified a deletion of exon 4 in alk-SMase cDNA in HT-29 cells. No mutation in genomic alk-SMase DNA from exon 3 to 5 was identified. The exon 4 deletion was caused by a shift of RNA splice site in chromosome 17q25. In Caco-2 cells, no mutation of alk-SMase cDNA was identified. Transient expression in COS-7 cells showed that the enzyme from the cDNA in HT-29 cells had little alk-SMase activity whereas that in Caco-2 cells was as active as the wild-type alk-SMase. The deleted region included residue His353, which is predicted to form a substrate-binding site of alk-SMase. H353A substitution resulted in a protein with no alk-SMase activity. In monolayer cultured Caco-2 cells and HT-29 cells the alk-SMase activities were low. However, to culture the cells under polarizing conditions increased alk-SMase activity and reduced SM level in Caco-2 cells. The alk-SMase activity varied in parallel with alkaline phosphatase activity. In conclusion, we identified an inactive deletion in alk-SMase in HT-29 cells, and a differentiation-related expression of the enzyme in Caco-2 cells. The results provide a molecular mechanism related to previous findings of reduced alk-SMase activity in human colon cancers.     

Aspirin induces cell death and caspase-dependent phosphatidylserine externalization in HT-29 human colon adenocarcinoma cells.

The induction of cell death by aspirin was analysed in HT-29 colon carcinoma cells. Aspirin induced two hallmarks of apoptosis: nuclear chromatin condensation and increase in phosphatidylserine externalization. However, aspirin did not induce either oligonucleosomal fragmentation of DNA, decrease in DNA content or nuclear fragmentation. The effect of aspirin on Annexin V binding was inhibited by the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases in the apoptotic action of aspirin. However, aspirin did not induce proteolysis of PARP, suggesting that aspirin does not increase nuclear caspase 3-like activity in HT-29 cells. This finding may be related with the 'atypical' features of aspirin-induced apoptosis in HT-29 cells.     

Imatinib inhibits colorectal cancer cell growth and suppresses stromal-induced growth stimulation, MT1-MMP expression and pro-MMP2 activation

Tumor progress depends on the proliferation of cancer cells, their interactions with stroma and the proteolytic action of enzymes. Colon cancer is c-kit positive and responsive to the specific tyrosine kinase inhibitor imatinib. We investigated the effect of imatinib on the proliferation of a panel of epithelial colon cancer cell lines in presence and absence of the antimetabolite 5-FU, and the effect of conditioned media (CM) derived from colon stromal fibroblasts with and without previous exposure to imatinib. The effects of imatinib on gene expression of MMPs and TIMPs were also studied. Imatinib effectively inhibited the proliferation of all cell lines, showing IC(50) from 0.3 to 3 microM. Its combination with 5-FU significantly enhances the growth inhibition of the highly tumourigenic HT-29 cells. CM derived from stromal fibroblasts induced the proliferation of the HT-29 cells; this stimulatory effect was abolished upon treatment with CM obtained after exposure of fibroblasts to imatinib. Gene expression of MT1-, MT2-MMP and MMP-7 was also inhibited depending on the cell line, whereas that of TIMP-2 was not affected. CM stimulated MT1-MMP protein expression by HT-29; this stimulatory effect was suppressed in the presence of imatinib. Activation of pro-MMP2 to MMP2 in culture medium of HT-29 treated with CM was increased and this activity was inhibited in presence of imatinib. The obtained data showed that imatinib is a powerful inhibitor of human colon cancer cell growth and effectively suppresses the stromal-induced stimulation of cancer cell growth and activation of proMMP2. Further studies are warranted to evaluate the in vivo effects.     

Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells

Epigallocatechin-3-gallate (EGCG), a major component in green tea polyphenols, has been proven to suppress colonic tumorigenesis in animal models and epidemiological studies. As EGCG is retained in the gastrointestinal tract after oral administration, this pharmacokinetics property gives it the potential to function as a chemopreventive agent against colon cancer. In this study, human colorectal carcinoma HT-29 cells were treated with EGCG to examine the anti-proliferative and pro-apoptotic effects of EGCG, as well as the molecular mechanism underlying these effects. Cell viability assay, nuclear staining, DNA fragmentation, caspase assay, cytochrome c release, DiOC6(3) staining, mitogen-activated protein kinases (MAPK) phosphorylation and trypan blue exclusion assays, were utilized to dissect the signaling pathways induced by EGCG. After 36 h treatment, EGCG inhibited HT-29 cell growth with an IC50 of approximately 100 microM. HT-29 cells treated with doses higher than 100 microM showed apparent nuclear condensation and fragmentation, which was confirmed by DNA laddering. Caspase-3 and -9 activation was detected after 12 h treatment, accompanied by mitochondrial transmembrane potential transition and cytochrome c release. Activation of MAPKs was detected as early signaling event elicited by EGCG. Inhibition of c-Jun N-terminal kinase (JNK) pathway showed the involvement of JNK in EGCG-induced cytochrome c release and cell death. EGCG-induced JNK activation was blocked by the antioxidants glutathione and N-acetyl-l-cysteine, suggesting that the cell death signaling was potentially triggered by oxidative stress. In summary, our results from this study suggest that in HT-29 human colon cancer cells (i) EGCG treatment causes damage to mitochondria, and (ii) JNK mediates EGCG-induced apoptotic cell death.     

Anti Cancer Effects of Cnidium officinale Makino Extract Mediated through Apoptosis and Cell Cycle Arrest in the HT-29 Human Colorectal Cancer Cell Line

The anti cancer properties and underlying cell death mechanism induced by the extract of the roots of Cnidium officinale Makino(COM) were investigated. The ethanolic extract of COM inhibited the proliferation of human colon cancer cells (HT-29) in both dose-dependent and time-dependent manners. Analysis of the cell cycle after treatment of HT-29 cells with various concentrations of COM showed that COM extract inhibited the cellular proliferation of HT-29 cells via G1 phase arrest of the cell cycle. The apoptotic effect of COM on HT-29 cells was confirmed by Annexin V- propidium iodide apoptosis test. RT-PCR and Western blot analysis both revealed that COM extract dose-dependently increased the expressions of p53, p21, Bax and Caspase-3. Anti-apoptotic factor Bcl-2 expression was down regulated as well as cyclin D1 and CDK4. These data suggest that COM has anti cancer properties by inducing apoptosis and cell cycle arrest in HT-29 cells and could have possible therapeutic potential against human colon adenocarcinoma.     

TNF-alpha modulates the differentiation induced by butyrate in the HT-29 human colon adenocarcinoma cell line

The aim of this study was to determine whether and how tumour necrosis factor alpha (TNF-alpha) modulates butyrate effects. After the treatment of human colon adenocarcinoma HT-29 cells with sodium butyrate (NaBt), TNF-alpha or with their combinations we detected cell cycle (flow cytometry), cell proliferation (amidoblack and MTT assays), the amount of dead (floating) and apoptotic cells (flow cytometry and fluorescence microscopy), and the level of differentiation by alkaline phosphatase (ALP) activity (spectrophotometry), relative F-actin content (confocal laser scanning microscopy analysis) and E-cadherin expression (Western blot analysis). Both TNF-alpha and NaBt decreased cell growth in a dose-dependent manner. After combined treatment of the cells with both agents used, either none or additive effects were observed as compared with NaBt treatment alone. The level of dead and apoptotic cells was dose-dependently increased after this combined treatment. In contrast, TNF-alpha suppressed ALP activity and F-actin accumulation induced by NaBt. The results suggest that TNF-alpha does not influence significantly the antiproliferative effects of NaBt but, contrary to its potentiation of apoptosis, it markedly reduces NaBt-induced differentiation of HT-29 colon adenocarcinoma cells.     

An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells

Anti-proliferative and pro-apoptotic activities of fractions of Pleurotus ostreatus were examined using HT-29 colon cancer cells in vitro. A hot-water-soluble fraction of the mycelium of the liquid cultured mushroom was partially isolated and chemically characterized as a low-molecular-weight alpha-glucan. HT-29 cells were exposed to the different isolates and significant inhibition of proliferation was obtained in a dose-dependent manner. Proliferation inhibition was shown to be the result of apoptotic induction because the pro-apoptotic molecules Bax and cytosolic cytochrome-c were upregulated. Fluorescence-activated cell sorter analyses of polysaccharide-treated HT-29 cells showed a high percentage of Annexin-positive cells. Here, we describe a newly identified low-molecular-weight alpha-glucan with promising anti-tumorigenic properties, and demonstrate its direct effect on colon cancer cell proliferation via induction of programmed cell death.     

Boswellic acids trigger apoptosis via a pathway dependent on caspase-8 activation but independent on Fas/Fas ligand interaction in colon cancer HT-29 cells

Boswellic acids are the effective components of gum resin of Boswellia serrata, which has anti-inflammatory properties. Recent studies on brain tumors and leukemic cells indicate that boswellic acids may have antiproliferative and apoptotic effects with the mechanisms being not studied in detail. We studied their antiproliferative and apoptotic effects on colon cancer cells and the pathway leading to apoptosis. HT-29 cells were treated with beta-boswellic acid (BA), keto-beta-boswellic acid (K-BA) and acetyl-keto-beta-boswellic acid (AK-BA), respectively. Apoptosis was determined by flow cytometry, by cytoplasmic DNA-histone complex and the activity of caspase-3. The cleavage of poly-(ADP-ribose)-polymerase (PARP) and expression of Fas were examined by western blot. Specific caspase inhibitors, polyclonal Fas antibody, and antagonistic Fas antibody ZB4 were employed to elucidate apoptotic pathways. DNA synthesis and cell viability were examined. Both K-BA and AK-BA increased cytoplasmic DNA-histone complex dose-dependently and increased pre-G(1) peak in flow cytometer analysis, with the effects of AK-BA being stronger than K-BA. BA only increased the formation of DNA-histone complex at a high concentration. K-BA and AK-BA increased caspase-8, caspase-9 and caspase-3 activities accompanied by cleavage of PARP. The effects of AK-BA on formation of cytoplasmic DNA histone and on caspase-3 activation were 3.7- and 3.4-fold, respectively, more effective than those induced by camptothecin. The apoptosis induced by AK-BA was inhibited completely by caspase-3 or caspase-8 inhibitor and partially by caspase-9 inhibitor. ZB4 blocked exogenous Fas ligand-induced apoptosis, but had no effect on AK-BA-induced apoptosis. AK-BA had no significant effect on expression of Fas. Apart from apoptotic effect, these acids also inhibited [(3)H]thymidine incorporation and cell viability to different extent. In conclusion, boswellic acids, particularly AK-BA and K-BA have antiproliferative and apoptotic effects in human HT-29 cells. The apoptotic effect is mediated via a pathway dependent on caspase-8 activation but independent of Fas/FasL interaction.     

Autophagy inhibition sensitizes WYE-354-induced anti-colon cancer activity in vitro and in vivo

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 are frequently dysregulated in human colon cancers. In the present study, we evaluated the potential anti-colon cancer cell activity by a novel mTORC1/2 dual inhibitor WYE-354. We showed that WYE-354 was anti-survival and anti-proliferative when adding to primary (patient-derived) and established (HCT-116, HT-29, Caco-2, LoVo, and DLD-1 lines) colon cancer cells. In addition, WYE-354 treatment activated caspase-dependent apoptosis in the colon cancer cells. Mechanistically, WYE-354 blocked mTORC1 and mTORC2 activation. Meanwhile, it also induced autophagy activation in the colon cancer cells. Autophagy inhibitors (bafilomycin A1 and 3-methyladenine), or shRNA-mediated knockdown of autophagy elements (Beclin-1 and ATG-5), remarkably sensitized WYE-354-mediated anti-colon cancer cell activity in vitro. Further studies showed that WYE-354 administration inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Importantly, its activity in vivo was further potentiated with co-administration of the autophagy inhibitor 3-MA. Phosphorylations of Akt (Ser-473) and S6 were also decreased in WYE-354-treated HT-29 xenografts. Together, these pre-clinical results demonstrate the potent anti-colon cancer cell activity by WYE-354, and its activity may be further augmented with autophagy inhibition.     

Docosahexaenoic acid sensitizes colon cancer cells to sulindac sulfide-induced apoptosis

Sulindac analogs represent one of the most efficacious groups of NSAIDs reducing the risk of colon cancer. Recent studies have shown that sulindac sulfide, a sulindac analog effective at lower doses compared to its parent compound, triggers the death receptor (DR)5-dependent extrinsic apoptotic pathway. Induction of apoptosis via activation of the DR-mediated pathway would be an ideal therapeutic strategy to eliminate cancer cells. In this study, we investigated the possibility that colon cancer cells are sensitized to sulindac sulfide-induced apoptosis by docosahexaenoic acid (DHA), via activation of the DR/extrinsic apoptotic pathway. Our data demonstrated that DHA combination sensitized colon cancer cells to sulindac sulfide-induced apoptosis, leading to enhanced growth suppression of human colon cancer xenografts. The combination effect was primarily attributed to increased cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-8 activation. Moreover, pretreatment with z-IETD-FMK (caspase-8 inhibitor) or stable expression of dominant negative caspase-8 genes blocked DHA/sulindac sulfide cotreatment-induced apoptosis. In view of the finding that DR5 silencing abrogated the combination-stimulated apoptosis, we propose that apoptotic synergy induced by sulindac sulfide plus DHA is mediated via DR5. Our findings collectively support the utility of a combination of sulindac sulfide and DHA in the effective prevention and treatment of colon cancer.     

Expression of protein kinase C beta1 confers resistance to TNFalpha- and paclitaxel-induced apoptosis in HT-29 colon carcinoma cells.

The expression of different protein kinase C (PKC) isoenzymes has been shown to vary with proliferation rates, differentiation or apoptosis in normal colon crypts. In addition, the activity of some PKC isoenzymes appears to be reduced in colorectal cancer. The aim of the present work was to determine whether modulation of PKC expression would affect the susceptibility of a p53-defective colon carcinoma cell line to different apoptotic treatments. HT-29 cells exhibited sensitivity to paclitaxel (Taxol) and tumor necrosis factor alpha (TNFalpha) in a dose- and time-dependent manner but were relatively resistant to etoposide. Inhibition of PKC activity augmented the susceptibility of HT-29 cells to apoptosis, and phorbol ester induction of PKC reduced such susceptibility. Transfected HT-29(PKC) cells, hyper-expressing the beta1 isoform of PKC, were less sensitive to TNFalpha and paclitaxel than the normal counterpart. The present data 1) indicate that the expression of PKC influences the susceptibility of HT-29 colon cancer cells to apoptotic drugs apparently regardless of their mechanism of action, and 2) suggest paclitaxel as a potential candidate for the treatment of colon cancer, possibly in association with inhibitors of PKC (alpha and beta) at doses not cytotoxic per se.     

Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft

Astragalus memebranaceus is used as immunomodulating agent in treating immunodeficiency diseases and to alleviate the adverse effects of chemotherapeutic drugs. In recent years, it has been proposed that Astragalus may possess anti-tumorigenic potential in certain cancer cell types. In this study, the anti-carcinogenic effects of Astragalus saponin extract were investigated in HT-29 human colon cancer cells and tumor xenograft. Our findings have shown that Astragalus saponins (AST) inhibit cell proliferation through accumulation in S phase and G2/M arrest, with concomitant suppression of p21 expression and inhibition of cyclin-dependent kinase activity. Besides, AST promotes apoptosis in HT-29 cells through caspase 3 activation and poly(ADP-ribose) polymerase cleavage, which is indicated by DNA fragmentation and nuclear chromatin condensation. Nevertheless, we also demonstrate the anti-tumorigenic effects of AST in vivo, of which the reduction of tumor volume as well as pro-apoptotic and anti-proliferative effects in HT-29 nude mice xenograft are comparable with that produced by the conventional chemotherapeutic drug 5-fluorouracil (5-FU). In addition, the side effects (body weight drop and mortality) associated with the drug combo 5-FU and oxaliplatin are not induced by AST. These results indicate that AST could be an effective chemotherapeutic agent in colon cancer treatment, which might also be used as an adjuvant in combination with other orthodox chemotherapeutic drugs to reduce the side effects of the latter compounds.     

NGX6基因联用5-Fu对人结肠癌细胞凋亡的影响

目的:探讨候选抑瘤基因NGX6联用5-Fu对结肠癌细胞凋亡的影响.方法:以稳定转染并表达NGX6基因的HT-29细胞与5-Fu联用作为实验组.以PDTC与5-Fu联用的HT-29细胞作为对照组.通过EMSA检测各组结肠癌HT-29细胞核转录因子-κB(NF-κB)的激活情况,利用MTT比色法检测各组细胞增殖的情况.吖啶橙(AO)/溴化乙啶(EB)双染法显微镜观测以及PI/Annexin-V双染流式细胞仪检测各组细胞凋亡情况.结果:稳定转染并表达NGX6基因的HT-29细胞以及应用了PDTC的HT-29细胞NF-κB的激活均明显受到抑制;与5-Fu作用的HT-29细胞组比较,5-Fu联合PDTC作用于HT-29细胞后,HT-29细胞增殖受到明显抑制,5-Fu诱导HT-29细胞凋亡作用增强;与5-Fu联合PDTC作用于HT-29细胞的对照组比较,在诱导细胞凋亡以及抑制细胞增殖方面,稳定转染并表达NGX6基因的HT-29细胞与5-Fu联用组和对照组所取得一致的效果,NGX6基因增强5-Fu对HT-29细胞增殖抑制的能力及诱导HT-29细胞凋亡的能力.结论:NGX6基因抑制了肿瘤细胞NF-κB的激活,具有增强5-Fu诱导结肠癌细胞凋亡的能力,其机制可能是抑制肿瘤细胞NF-κB的激活,NGX6基因对肿瘤的治疗及预后起积极作用.     

改变细胞膜脂肪酸组成防治大肠癌的基因治疗研究*

目的:利用脂肪酸去饱和酶基因fat- 1 改变细胞膜脂肪酸组成,进行大肠癌的基因治疗研究。方法:将fat- 1 基因插入腺病毒载体中,与骨架载体同源重组,构建腺病毒重组载体（Ad-GFP-fat1）,通过包装细胞系（293）产生的腺病毒,感染人大肠癌株HT- 29细胞。提取细胞的总RNA,以fat- 1 基因的反义mRNA 作探针,用Northern Blot检测fat- 1 基因在HT- 29细胞内的表达。以流式细胞仪对HT- 29细胞G0/G1 期、S 期、G2/M期所占比例进行检测,分析fat- 1 基因对HT- 29细胞增殖和凋亡的影响。以气相色谱分析仪分析fat- 1 基因对HT- 29细胞细胞膜n-6 PUFAs 和n-3 PUFAs 含量及n-6/n- 3PUFAs 比例的影响。将HT- 29细胞皮下接种于裸鼠右前肢腋下,建立裸鼠HT- 29大肠癌细胞皮下移植瘤模型。成瘤后进行治疗实验,经连续5 次治疗,于最后一次治疗后第3 天处死小鼠,取肿瘤称重。分析fat- 1 基因裸鼠体内抗肿瘤效果。结果:通过基因重组技术,得到高滴度的含fat- 1 基因的重组病毒;腺病毒介导的fat- 1 基因能够在HT- 29细胞中有效表达;fat- 1 基因的表达可降低HT- 29细胞膜n-6/n- 3PUFAs 的比例,有效抑制HT- 29细胞增殖,促进细胞凋亡并能抑制裸鼠移植瘤的发展。结论:fat- 1 基因的表达,可抑制HT- 29细胞的体内外增殖并诱导细胞凋亡,在大肠癌基因治疗中可能具有良好利用价值。      

鞘磷脂水解产物对结肠癌细胞中碱性鞘磷脂酶的影响

背景与目的： 碱性鞘磷脂酶(alk-SMase)是鞘磷脂(SM)新陈代谢和发挥抑制结肠癌作用的关键酶。通过体外实验探讨SM的水解产物的神经酰胺(Cer)和鞘氨醇(Sph)对人结肠癌细胞HT-29中alk-SMase表达的影响。 材料与方法： 分别用12.5、25、50 μmol/L的C2-Cer和Sph处理HT-29细胞12、24、48 h后,分别用Western blot法和RT-PCR检测细胞中alk-SMase的蛋白水平和mRNA表达水平的变化。实验并设DMSO溶剂对照组。 结果： C2-Cer和Sph作用HT-29细胞后,其alk-SMase蛋白及其mRNA的表达下调。 结论： Cer和Sph对HT-29细胞中alk-SMase的表达具有负反馈调节作用。     

Downregulation of Mcl-1 synergizes the apoptotic response to combined treatment with cisplatin and a novel fiber chimeric oncolytic adenovirus

The aim of this study was to examine the effects of SG511, a novel fiber chimeric oncolytic adenovirus with E1B 55-kDa deleted, combined with cisplatin on cancer cells and to identify their underlying mechanisms. The combined effect of SG511 and cisplatin on HeLa and HT-29 cells was assessed by a crystal violet assay and an MTT assay, followed by combination index analysis. Cell apoptosis was evaluated by DAPI staining and visualized by fluorescein-mediated signal detection. Mitochondrial membrane potential was detected by flow cytometric analysis of Rhodamine 123 accumulation. The activation of the caspase pathway and the expression of Bcl-2 family proteins were examined by western blotting. Results show that SG511 vector infected various human cancer cell lines and induced growth inhibition effectively. Of note, SG511 synergistically enhanced the anti-proliferative activity of cisplatin, a DNA-damaging agent, against HeLa and HT-29 cells in?vitro, concomitantly with increased apoptosis and activation of the mitochondrial pathway. Furthermore, treatment with SG511 alone or in combination with cisplatin resulted in reduced expression the anti-apoptotic Bcl-2 family member Mcl-1 in HeLa and HT-29 cells. Importantly, this combination did not increase the growth inhibitory effects of cisplatin on human normal liver cells. Collectively, SG511, a novel fiber chimeric oncolytic adenovirus, sensitizes cancer cells to apoptosis by reducing anti-apoptotic Mcl-1 protein levels.     

Sulindac sulfide-induced apoptosis involves death receptor 5 and the caspase 8-dependent pathway in human colon and prostate cancer cells

Sulindac is the most extensively investigated clinically relevant chemopreventive nonsteroidal anti-inflammatory drug. Sulindac sulfide is one of the major metabolites of sulindac that is believed to mediate its antitumorigenic effects by inducing apoptosis. Recent evidence suggests that sulindac sulfide engages the mitochondrial pathway involving caspase 9 and Bax to mediate its apoptotic effects [Zhang et al., Science (Wash. DC), 290: 989-992, 2000]. In this report, we demonstrate that sulindac sulfide also engaged the membrane death receptor (DR) pathway to mediate apoptosis. Sulindac sulfide up-regulated DR5 and activated the proximal caspase 8 in various different colon and prostate cancer cell lines. Sulindac sulfide specifically up-regulated the DR5 levels but had no effect on the levels of other DRs including DR4, Fas, and tumor necrosis factor receptor 1. To further delineate the role of DR5 in sulindac sulfide-induced apoptosis, we used JCA-1 prostate cancer cells that are deficient in mounting a Fas and tumor necrosis factor receptor 1-dependent apoptotic response but are proficient in mediating DR5-dependent apoptosis. JCA-1 cells were stably transfected with dominant-negative Fas-associated death domain to block the flow of apoptotic signals originating from the endogenous DR5, and sulindac sulfide-induced apoptosis was investigated. Our results indicated that by blocking the DR5-dependent apoptotic pathway, dominant-negative Fas-associated death domain did indeed inhibit sulindac sulfide-induced apoptosis. Furthermore, exogenous tumor necrosis factor-related apoptosis-inducing ligand, the ligand for DR5, also potentiated sulindac sulfide-induced apoptosis in all of the cell lines tested, thereby further supporting the involvement of DR5 in sulindac sulfide-induced apoptosis. Thus, our results demonstrate that sulindac sulfide also engages the membrane DR pathway involving DR5 and proximal caspase 8 to induce apoptosis.