忍冬藤提取物抗结肠癌作用研究

目的 研究忍冬藤提取物的体外抗结肠癌作用。方法 体外培养结肠癌HCT116和SW480细胞并用忍冬藤提取物干预,采用CCK8法检测忍冬藤提取物对HCT116和SW480细胞增殖的影响;采用Hochest法和Annexin V/PI法检测忍冬藤提取物及p53抑制剂PFT-α对HCT116和SW480细胞凋亡的影响;JC-1法检测忍冬藤提取物对HCT116细胞线粒体膜电位的影响;Western Blot检测忍冬藤提取物对HCT116细胞相关蛋白表达水平的影响。结果 相较于空白组,忍冬藤提取物可明显抑制结肠癌HCT116和SW480细胞增殖,并促进细胞凋亡(P<0.01),促进HCT116细胞线粒体膜电位坍塌,明显上调HCT116细胞Bax/Bcl-2的比值(P<0.01)以及Cleaved PARP、Cyt-C、Cleaved Caspase-3和p53蛋白表达(P<0.05,P<0.01);相较于忍冬藤提取物组,忍冬藤提取物+PFT-α明显抑制HCT116细胞凋亡(P<0.01)。结论 忍冬藤提取物可通过诱导p53依赖的线粒体凋亡发挥抗结肠癌作用。     

miR-126-5p靶向抑制ERCC1增加结肠癌细胞对奥沙利铂化疗的敏感性

目的 探讨miR-126-5p通过靶向抑制切除修复交叉互补基因1(excision repair cross complement 1,ERCC1)促进结肠癌细胞对奥沙利铂(oxaliplatin,L-OHP)的敏感性及其分子机制。方法 利用L-OHP浓度梯度递增法处理结肠癌细胞HCT116,建立耐L-OHP的人结肠癌细胞株HCT116/L-OHP。CCK-8法检测并计算L-OHP作用后的半数抑制浓度(half maximal inhibitory concentration,IC₅₀)值。qRT-PCR法检测miR-126-5p和ERCC1在HCT116和HCT116/L-OHP细胞株中的表达。利用Lipofectamine^? 2000转染miR-126-5p mimics(miR-126-5p mimics组)和miR-NC质粒(miR-NC组)后,CCK-8法检测HCT116/L-OHP细胞对L-OHP的敏感性的变化,流式细胞术检测细胞凋亡率。利用双荧光素酶报告实验验证miR-126-5p和ERCC1的相互作用机制。Western bl...     

辛二酰苯胺异羟肟酸对结肠癌HCT116和SW480细胞增殖、周期和凋亡的影响

目的:研究辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)对结肠癌细胞系HCT116和SW480增殖、周期和凋亡的影响,并对其分子作用机制进行初步探讨.方法:将不同浓度SAHA分别处理结肠癌HCT116和SW480细胞后,MTT法检测SAHA对HCT116和SW480细胞增殖的影响,流式细胞仪检测HCT116和SW480细胞周期和细胞凋亡率,罗丹明(rhodamine) 123和二氯二氢荧光素二乙酸酯(DCFH-DA)法检测HCT116和SW480细胞线粒体跨膜电位(Aψm)和活性氧(ROS)水平,Real-time PCR和Western blotting法检测乙酰化组蛋白3(Ac-H3)、p21、CyclinD1、Bax和Bcl-2的mRNA和蛋白的表达水平.结果:SAHA作用于HCT116和SW480细胞48 h后,细胞增殖被抑制、细胞周期G1期比率升高、凋亡率升高(均P＜0.05),线粒体跨膜电位显著下降、细胞内ROS产生增多(均P＜0.05).与对照组比较,SAHA处理组p21和Bax mRNA增多、Cyclin D1和Bcl-2 mRNA表达量减少(均P＜0.05),相关蛋白Ac-H3、p21和Bax增多,CyclinD1和Bcl-2减少(均P＜0.05).结论:SAHA抑制结肠癌HCT116和SW480细胞增殖、阻滞细胞周期并诱导细胞凋亡,其机可能与调节p21、CyclinD1和Bcl-2家族基因的表达、促进组蛋白乙酰化有关.     

人结肠癌奥沙利铂耐药细胞HCT116/L-OHP的建立及其耐药机制初探

目的:建立人结肠癌奥沙利铂(oxaliplatin,L-OHP)耐药细胞HCT116/L-OHP,并初步探讨其可能的耐药机制。方法:通过逐步增加作用于亲代细胞HCT116的L-OHP浓度与间断大剂量L-OHP作用,建立耐药细胞HCT116/L-OHP;MTT法检测L-OHP、5-氟尿嘧啶(5-fluorouracil,5-FU)、顺铂(cisplatin)和7-乙基-10-羟基喜树碱(7-ethyl-10-hydrol-camptothecin,HCPT)对HCT116和HCT116/L-OHP细胞的细胞毒性;蛋白质印迹法检测相关耐药蛋白的表达;基因芯片检测信号通路的改变。结果:成功构建了稳定耐药的耐药细胞HCT116/L-OHP,耐药倍数为17倍,与5-FU、DDP和HCPT有不同程度的交叉耐药性。与HCT116细胞比较,HCT116/L-OHP细胞中P糖蛋白(P-glycoprotein,P-gp)、肺耐药蛋白(lung resistance protein,LRP)和谷胱甘肽S转移酶(glutathione S-transferase,GST)的表达上调(P<0.01),9条信号通路上调(P<0.05),其中细胞周期信号通路上调最明显,p53信号通路次之。结论:HCT116/L-OHP细胞具有稳定的耐药性,其耐药机制可能与细胞耐药蛋白表达、细胞周期和p53信号通路表达上调有关。     

阿西替尼通过调控自噬对结肠癌HCT-116细胞增殖、凋亡的影响研究

目的研究阿西替尼(AXI)对结肠癌HCT 116细胞增殖、凋亡及自噬的影响。方法体外培养结肠癌HCT 116细胞,分为AXI 1组(150 μmol／L)、AXI 2组(300 μmol／L)、AXI 3组(600 μmol／L)及无任何添加的正常对照组(NC组)。四甲基偶氮唑蓝法(MTT)检测各组结肠癌HCT 116细胞增殖情况;AnnexinV FITC／PI法检测各组结肠癌HCT 116细胞凋亡情况;Western Blot检测结肠癌HCT 116细胞自噬相关蛋白LC3 Ⅱ、beclin1,雷帕霉素靶蛋白(mTOR)信号通路中p mTOR、p P70S6K蛋白水平、增殖相关蛋白CyclinD1、c Myc,以及凋亡相关cleaved caspase3、B细胞淋巴瘤 2(Bcl 2)、Bax蛋白表达水平。结果与NC组比较,AXI 1组和AXI 2组结肠癌HCT 116细胞增殖抑制率、凋亡率、LC3 Ⅱ、beclin1、Bax、cleaved Caspase3蛋白表达水平依次增加(P<005),p mTOR、p P70S6K、Cyclin D1、c Myc、Bcl 2蛋白表达水平依次减少(P<005)。AXI 2组和AXI 3组结肠癌HCT 116细胞增殖抑制率、凋亡率、LC3 Ⅱ、beclin1、p mTOR、p P70S6K、CyclinD1、c Myc、cleaved caspase3、Bcl 2、Bax蛋白表达水平比较,差异无统计学意义(P>005)。结论AXI可通过抑制mTOR通路激活,诱导结肠癌HCT 116细胞自噬,抑制其增殖,促进其凋亡,初步揭示了AXI对结肠癌细胞的作用机制,为结肠癌的靶向治疗提供了新思路。     

UGCG通过调控MDR1/P-gp参与人结肠癌奥沙利铂耐药的初步研究

目的 建立人结肠癌耐奥沙利铂（L-OHP）细胞株,检测该细胞株的多药耐药性并初步探讨其可能的耐药机制。方法 以人结肠癌细胞HCT116为对象,采用药物浓度梯度递增诱导法建立人结肠癌耐奥沙利铂细胞株HCT-116/L-OHP。CCK-8法检测L-OHP、顺铂（DDP）、5-氟尿嘧啶（5-Fu）对亲本细胞和耐药细胞株的半数抑制浓度（IC50）。使用UDP-葡萄糖神经酰胺糖基转移酶（UGCG）siRNA转染HCT-116/L-OHP细胞,实时荧光定量 PCR和Western blot检测干扰前后UGCG基因和多药耐药基因1（MDR1）mRNA及其编码的蛋白表达水平。结果 HCT-116/L-OHP对L-OHP的耐药指数为10.5,与DDP有一定程度的交叉耐药,耐药指数为4.61,但对5-Fu无交叉耐药。耐药细胞HCT-116/L-OHP中UGCG、MDR1 mRNA和UGCG、P-糖蛋白（P-gp, MDR1编码的蛋白）表达均增加,相比HCT-116细胞,差异具有统计学意义（P<0.05）。UGCG siRNA成功抑制HCT-116/L-OHP细胞中UGCG的表达,各干扰组MDR1 mRNA、P-gp表达减少,与对照组相比差异具有统计学意义（P<0.05）。结论 成功构建了人结肠癌耐药细胞株;UGCG基因通过调控MDR1/P-gp的表达参与人结肠癌奥沙利铂的耐药机制的形成。     

Ku70乙酰化修饰介导曲古霉素A（TSA）促结肠癌细胞凋亡的作用机制研究

目的:探讨Ku70乙酰化修饰介导曲古霉素A（TSA）促结肠癌细胞凋亡的作用和机制。方法:选取结肠癌HCT116细胞和SW620细胞,体外培养并采用浓度梯度TSA处理。MTT法观察TSA对细胞的IC50和活力的影响;Western blot和免疫荧光染色观察TSA对结肠癌HCT116细胞和SW620细胞中Ku70、acetyl-Ku70蛋白水平及胞内分布的影响;流式细胞术检测TSA对结肠癌HCT116细胞和SW620细胞凋亡的影响;Western blot检测TSA对结肠癌HCT116细胞和SW620细胞中凋亡相关蛋白Caspase-3、Bax和Bcl-2表达的影响。结果:MTT结果显示TSA可浓度依赖性抑制结肠癌HCT116细胞和SW620细胞活力且其IC50值分别为1.023 μmol/L和1.076 μmol/L（P<0.05）;Western blot和免疫荧光染色结果显示TSA可显著上调结肠癌HCT116细胞和SW620细胞中acetyl-Ku70的蛋白水平并促进其核转入（P<0.05）;流式细胞术结果表明TSA可促进结肠癌HCT116细胞和SW620细胞凋亡（P<0.05）;此外,Western blot结果显示TSA可明显上调结肠癌HCT116细胞和SW620细胞中凋亡蛋白Caspase-3和Bax的表达,下调抗凋亡蛋白Bcl-2的表达（P<0.05）。结论:结肠癌HCT116细胞和SW620细胞中,TSA可能通过增强Ku70乙酰化并促进其核转入,发挥促凋亡作用,为以Ku70翻译后修饰调控为靶点的肿瘤治疗提供新策略和理论依据。     

茯苓酸通过调控AKT/MDM2/p53通路影响结直肠癌HCT116细胞的恶性生物学行为

目的：探究茯苓酸（PA）是否通过AKT/MDM2/p53通路影响结直肠癌HCT116细胞的恶性生物学行为。方法：常规培养HCT116细胞,并将其分为对照组、MK-2206(AKT抑制剂)组、PA低浓度（PA-L）组、PA高浓度（PA-H）组、PA-H+SC79(AKT激活剂)组。CCK-8法、细胞克隆形成实验、流式细胞术、Transwell、qPCR法和WB法实验分别检测各组HCT116细胞的增殖活力,克隆形成能力,细胞凋亡,迁移、侵袭能力,E-cadherin、N-cadherin和vimentin mRNA表达以及AKT/MDM2/p53通路相关蛋白的表达。结果：PA可明显抑制HCT116细胞的增殖活力（P<0.05）、克隆形成能力（P<0.05）、迁移和侵袭能力（P<0.05）,诱导其凋亡（P<0.05）,抑制N-cadherin、vimentin mRNA的表达（P<0.05）,促进E-cadherin mRNA的表达（P<0.05）,抑制AKT、MDM2的磷酸化水平（P<0.05）,促进p53蛋白的表达（P<0.05）;AKT抑...     

CDX1对结直肠癌细胞增殖凋亡及p38MAPK磷酸化水平的影响

目的:探讨尾侧同源盒转录因子1（CDX1）对结直肠癌细胞增殖凋亡及p38MAPK磷酸化水平的影响。方法:用荧光定量PCR和Western blot检测正常肠上皮细胞FHC和结直肠癌细胞SW480、HCT116、Caco2中CDX1 mRNA和蛋白表达水平。在SW480细胞中转染pIRES-CDX1和pIRES记为CDX1组和载体组,以不做转染的细胞为对照组,荧光定量PCR和Western blot测定CDX1 mRNA和蛋白表达水平,MTT检测细胞增殖,流式细胞术检测细胞凋亡,Western blot测定细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）、B细胞淋巴瘤/白血病-2（Bcl-2）、信号转导与转录因子3(STAT3)、磷酸化的STAT3（p-STAT3）、p38丝裂原活化蛋白激酶（p38MAPK）、磷酸化的p38MAPK（p-p38MAPK）蛋白水平。结果:结直肠癌细胞SW480、HCT116、Caco2中CDX1 mRNA和蛋白表达水平均明显低于正常肠上皮细胞FHC（P<0.05）,SW480细胞中CDX1 mRNA和蛋白表达水平明显低于HCT116、Caco2（P<0.05）。CDX1组细胞中CDX1 mRNA和蛋白水平明显高于对照组（P<0.05）,载体组细胞中CDX1 mRNA和蛋白水平与对照组相比没有统计学差异（P>0.05）。CDX1组细胞存活率明显降低,细胞凋亡率明显升高,细胞中Cleaved Caspase-3、p-p38MAPK水平升高,细胞中Bcl-2、p-STAT3水平降低,与对照组相比差异具有统计学意义（P<0.05）。载体组细胞存活率、凋亡率及细胞中Cleaved Caspase-3、Bcl-2、STAT3、p-STAT3、p38MAPK、p-p38MAPK蛋白水平与对照组相比没有明显变化（P>0.05）。结论:CDX1抑制结直肠癌细胞增殖,诱导Caspase-3介导的细胞凋亡,促进细胞中p38MAPK磷酸化,抑制细胞中STAT3磷酸化。     

滋肾固髓汤通过调控p38MAPK/p53信号通路诱导结直肠癌HCT-116细胞凋亡

目的:探讨滋肾固髓汤对结直肠癌HCT-116细胞凋亡的影响及其分子机制。方法:制备滋肾固髓汤含药血清,首先采用不同体积分数滋肾固髓汤含药血清处理HCT-116细胞,噻唑蓝(MTT)检测细胞增殖活性;再将HCT-116细胞分为空白组、滋肾固髓汤低、中、高剂量组、SB203580(p38MAPK抑制剂)组和高剂量滋肾固髓汤+SB203580组,分别加入相应药物进行干预后,Hoechst 33258染色观察细胞凋亡的形态学变化;Annexin V-FITC流式细胞术检测细胞的凋亡水平;Western blot检测细胞中p38MAPK、磷酸化(p)-p38MAPK(Thr180/Tyr182)、鼠双微染色体2(MDM2)、p53、Cleaved caspase-3、Bax和Bcl-2等蛋白表达水平。结果:滋肾固髓汤可抑制HCT-116细胞增殖活性,并呈时间和浓度依赖性。不同浓度滋肾固髓汤处理可诱导HCT-116细胞呈现核聚集、皱缩或碎裂等凋亡形态变化,促进细胞凋亡,上调p-p38MAPK、p53、Bax和Cleaved caspase-3等蛋白表达水平,下调MDM2和Bcl-2蛋白表达水平。S...     

Emodin-Provoked Oxidative Stress Induces Apoptosis in Human Colon Cancer HCT116 Cells through a p53-Mitochondrial Apoptotic Pathway

Emodin, a natural anthraquinone isolated from the traditional Chinese medicine Radix rhizoma Rhei, can induce apoptosis in many kinds of cancer cells. This study demonstrated that emodin induces apoptosis in human colon cancer HCT116 cells by provoking oxidative stress, which subsequently triggers a p53-mitochondrial apoptotic pathway. Emodin induced mitochondrial transmembrane potential loss, increase in Bax and decrease in Bcl-2 expression and mitochondrial translocation and release of cytochrome c to cytosol in HCT116 cells. In response to emodin-treatment, ROS increased rapidly, and subsequently p53 was overexpressed. Pretreatment with the antioxidant NAC diminished apoptosis and p53 overexpression induced by emodin. Transfecting p53 siRNA also attenuated apoptosis induced by emodin, Bax expression and mitochondrial translocation being reduced compared to treatment with emodin alone. Taken together, these results indicate that ROS is a trigger of emodin-induced apoptosis in HCT116 cells, and p53 expression increases under oxidative stress, leading toBax-mediated mitochondrial apoptosis.     

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and apoptosis in colorectal cancer cells via p53-dependent and -independent pathways

Many chemotherapeutic agents induce apoptosis via a p53-dependent pathway. However, up to 50% of human cancers have p53 mutation and loss of p53 function. Histone deacetylase inhibitors (HDACIs) are emerging as a potentially important new class of anticancer agents. Here, we report that, Trichostatin A (TSA), a pan-HDAC inhibitor, could induce G2/M cell cycle arrest and apoptosis in both colorectal cancer cell lines with wild-type p53 (HT116 cells) and mutant p53 (HT29 cells), although HCT116 cells had more apoptotic cells than HT29 cells. TSA induces apoptosis in both cell lines via the mitochondrial pathway as indicated by decrease of the mitochondrial membrane potential (MMP) and activation of caspase-3. Additionally, TSA induces expression of the pro-apoptotic protein Bax and decreases the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL in both cell lines. Bax knockdown by siRNA significantly impaired TSA-induced apoptosis in both cell lines. These data suggest that TSA induces G2/M cell cycle arrest and Bax-dependent apoptosis in colorectal cancer cells (HCT116 cells and HT29 cells) by both p53-dependent and -independent mechanisms. However, cells with normal p53 function are more sensitive to TSA-induced apoptosis.     

Resistance of colon cancer cells to long-term 5-fluorouracil exposure is correlated to the relative level of Bcl-2 and Bcl-X(L) in addition to Bax and p53 status

Defects in apoptosis have been implicated in chemoresistance of colon cancer cells. We report here the ability to resist to 5-fluorouracil-induced apoptosis of 8 colon cancer cell lines differing in p53 and bax status: p53(-/0)bax(+/+) for TC7, SW480, HT-29; p53(+/+)bax(-/-) for LS174T, LoVo; p53(+/+) bax(+/-) for HCT116; p53(+/+) or p53(+/0)bax(+/+) for LS513 or HCT-EB, respectively. To approximate to the in vivo therapy, the cell lines were exposed to a long-term treatment of 5-FU. The analysis of proteins implicated in the apoptotic pathway has shown that the independent analysis of p53 or bax status was not sufficient to predict the extent of drug-resistance of all cell lines. In p53(+/+) cell lines but not in p53(-/0) cell lines, a low level of the pro-apoptotic Bax protein was correlated with a greater resistance of cells to 5-FU. In addition, we found that high levels of anti-apoptotic Bcl-2 and Bcl-x(L) proteins combined with a low level of Bax were correlated to high 5-FU resistance of wild-type p53 cell lines. The same correlation was obtained for 2 out of 3 mutated p53 cell lines. In conclusion, the relative levels of Bcl-2, Bcl-x(L) and Bax may altogether contribute to determine the resistance of a majority of colon tumor cells to long-term 5-FU treatment, whatever their p53 status.     

Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.     

Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.     

Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity.

Paclitaxel (PTX), a microtubule-active agent, blocks cell proliferation by inhibiting mitotic progression leading to mitotic and postmitotic arrest and cell death. Here we demonstrate for the first time that very low concentrations of PTX (3-6 nM) can completely inhibit cell proliferation without arresting cells at mitosis. At these low concentrations that are insufficient to inhibit mitotic progression, PTX induced both p53 and p21 causing G1 and G2 arrest in A549. In contrast, low PTX concentrations failed to induce G1 and G2 arrest in A549/E6 cells, that do not express p53. Furthermore, we observed that the levels of p53 and p21 induced by adriamycin and by low concentrations of PTX in A549 cells were comparable. This observation led us to conclude that low concentrations of PTX can induce p53 and p21 sufficiently to cause G1 and G2. Many other cell lines, including HCT116 cells, do not readily upregulate p53 in response to PTX, and therefore undergo exclusively mitotic and postmitotic arrest after PTX treatment. At low concentrations that do not cause mitotic arrest, PTX did not significantly inhibit proliferation of these cells. In HCT116 cells, loss of p53 (HCT/p53(-/-)) or p21 (HCT/p21(-/-)) affects both Bax and Bcl-2 expression. In cells lacking p53, levels of Bax and p21 were decreased. In cells lacking p21, levels of wt p53 were highly increased to compensate for the loss of p21. This in turn results in upregulation of Bax and downregulation of Bcl-2 resulting in an increase of the apoptotic Bax/Bcl2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. High levels of p53 and Bax/Bcl-2 ratio can also explain why loss of p21 is rarely found in human cancer.     

Bax plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer HCT116 cells by controlling Smac/Diablo and Omi/HtrA2 release from mitochondria

Bax is a crucial mediator of the mitochondrial pathway for apoptosis, and loss of this proapoptotic Bcl-2 family protein contributes to drug resistance in human cancers. We report here that the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (THG) induces apoptosis of human colon cancer HCT116 cells through a Bax-dependent signaling pathway controlling the cytosolic release of mitochondrial apoptogenic molecules. Treating HCT116 cells with THG results in caspase-8 activation; Bid cleavage; Bax conformational change and mitochondrial translocation; the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 into the cytosol; caspase-3 activation; and apoptosis. In contrast, knockout of Bax completely abrogates the full processing/activation of caspase-3 but has no effect on the processing of caspase-8 and the initial cleavage of caspase-3 to p24 fragment after THG treatment. The caspase-8-specific inhibitor z-IETD-fmk, as well as pan-caspase inhibitor z-VAD-fmk, but not the calpain inhibitor E-64d, prevents Bid cleavage, Bax conformational change, and subsequent caspase-3 processing and apoptosis. Caspase-8 processing is dependent on de novo protein synthesis; DR5 expression is strongly up-regulated by THG treatment. Moreover, the absence of Bax blocks THG-induced Omi and Smac release from mitochondria, and expression of cytosolic Omi (GFP-IETD-Omi) or Smac (GFP-IETD-Smac) restores the sensitivity of Bax-knockout HCT116 cells to apoptosis in response to THG treatment. Taken together, our results indicate that Bax-dependent Smac and Omi release plays an essential role in caspase-3 activation and apoptosis induced by THG in human colon cancer HCT116 cells.