阿霉素诱导SACC-83细胞凋亡的试验研究

目的：探讨阿霉素诱导腺样囊性癌细胞凋亡的最佳剂量。方法：采用SACC-83细胞系，于体外分别以含有5μg／ml、10μg／ml、20μg／ml阿霉素的培养液处理12小时后，进行凋亡DNA电泳梯状图谱、流式细胞仪凋亡率的检测和细胞形态学分析。结果：5μg／ml、10μg／ml组细胞检测到凋亡带，而20μg／ml组可见明显的坏死带。形态学上看到20μg／ml组主要表现为坏死；10μg／ml凋亡与坏死同时存在，；5μg／ml时表现为单纯的细胞凋亡。流式细胞仪检测结果发现，在5μg／ml浓度阿霉素作用下，凋亡细胞可达68．46％与其它组相比有显著性差异（P＜0．01）。结论：通过阿霉素诱导SACC-83细胞建立凋亡模型，检测发现阿霉素5μg／ml作用下可得到较高含量的凋亡细胞。     

新藤黄酸诱导人结肠癌HCT116细胞凋亡的作用机制研究

目的:探讨新藤黄酸诱导人结肠癌HCT116细胞凋亡的作用及其可能的分子机制。方法:采用MTT法检测新藤黄酸对人结肠癌HCT116细胞增殖的抑制作用;DAPI染色及荧光显微镜观察细胞凋亡情况;FCM法检测细胞周期分布;蛋白质印迹法检测新藤黄酸对cyclin D1、cyclin E、P21、P27和多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]蛋白表达的影响。结果:新藤黄酸对HCT116细胞增殖有明显的抑制作用,并呈浓度和时间依赖性。DAPI染色后荧光显微镜观察发现,新藤黄酸能明显诱导HCT116细胞凋亡。FCM法检测发现,新藤黄酸可以使HCT116细胞G0/G1期比例显著升高,S期比例相应降低,表明细胞周期阻滞于G0/G1期。蛋白质印迹法检测结果表明,新藤黄酸下调了细胞周期蛋白cyclin D1和cyclin E的表达,上调了P21和P27的蛋白表达,诱导了PARP蛋白的剪切。结论:新藤黄酸通过下调细胞周期蛋白cyclin D1、cyclin E的表达和上调P21、P27的表达,使HCT116细胞阻滞在G0/G1期,进而显著抑制HCT116细胞的增殖,并诱导其凋亡。     

中药诱导腺样囊性癌细胞凋亡的研究进展

从中药中寻找毒副反应小、安全有效的抗肿瘤药物为近年的研究热点，其诱导肿瘤细胞凋亡将成为抗肿瘤治疗的新靶标。中药在抑制、杀伤肿瘤细胞、改善症状与体征以及减轻放化疗不良反应等方面发挥着重要作用。中药可以通过多种途径诱导癌细胞凋亡。本文综述了近年来中药及其提取物如紫杉醇、黄芪、苦参碱、染料木黄酮、红景天甙、中华眼镜蛇蛇毒等诱导腺样囊性癌细胞（salivary adenoid cystic carcinoma，SACC）凋亡的研究近况及其作用机制。     

LncRNA ANRIL靶向miR-195对HCT116细胞及裸鼠移植瘤放射增敏实验研究

目的 探究LncRNA ANRIL对结直肠癌HCT116细胞体外体内放射增敏作用及机制。方法 qPCR检测ANRIL表达。将阴性对照siRNA、ANRIL siRNA、miR-NC mimic、miR-195 mimic、miR-NC inhibitor、miR-195 inhibitor转染至HCT116细胞中分别记为阴性对照、沉默ANRIL、过表达miR-NC、过表达miR-195、抑制miR-NC、抑制miR-195组,以不做任何处理的HCT116细胞为空白对照组。克隆形成实验检测放射敏感性,流式细胞术检测细胞凋亡,StarBase预测ANRIL下游miRNAs,双荧光素酶报告基因实验进一步验证。裸鼠皮下移植瘤实验检测ANRIL对照射后移植瘤生长影响。结果 沉默ANRIL组细胞存活分数较阴性对照组降低(P<0.05),其放射增敏比为1.52。沉默ANRIL+4Gy组细胞凋亡率较阴性对照+4Gy组增加[(27.86±2.78)%︰(12.06±1.46)%,P<0.05]。裸鼠皮下移植瘤实验结果显示在13、16、19、22、25天时阴性对照组肿瘤体积比沉默ANRIL组降低[(234±66)、(273±63)、(296±72)、(321±85)、(403±94) mm³与(357±79)、(485±124)、(617±143)、(764±174)、(985±221) mm³,P<0.05]。miR-195是ANRIL靶基因,抑制miR-195可逆转沉默ANRIL对HCT116细胞放射增敏和凋亡促进作用及移植瘤生长抑制作用。结论 LncRNA ANRIL通过调控miR-195表达来调节HCT116细胞放射敏感性,可能为临床结直肠癌放疗提供一个新的增敏靶点。     

hTERT启动子靶向性驱动Bax诱导腺样囊性癌细胞凋亡机制的探讨

目的:探讨人端粒酶逆转录酶(hTERT)启动子调控Bax基因在端粒酶阳性的腺样囊性癌细胞-SACC-83中表达的特异性,以及Bax对SACC-83细胞增殖的影响.方法:脂质体介导pACTERT-Bax质粒载体转染SACC-83和端粒酶阴性的人胚肺成纤维细胞(HEL),通过RT-PCR检测Bax表达,MTT法检测Bax基因表达对细胞增殖的影响,流式细胞仪检测细胞凋亡率.结果:hTERT启动子调控Bax基因在SACC-83细胞中明显表达,对该肿瘤细胞的增殖有明显的抑制作用(相对活性58.90%),且诱导细胞凋亡比例增加(11.62%);而在HEL细胞中hTERT启动子未能诱导Bax基因表达,对该细胞的增殖和凋亡无明显影响.结论:hTERT启动子可以诱导Bax基因在SACC-83中特异性表达及诱导细胞凋亡,同时还能消除Bax基因对正常细胞的潜在毒性.     

下调REV7基因对于人结肠癌HCT116细胞放射敏感性影响及机制研究

目的 研究下调REV7基因表达对人结肠癌HCT116细胞放射敏感性影响及其机制。方法 对HCT116细胞进行培养并运用RNA干扰技术实现REV7基因下调,将细胞分为空白组、转染阴性RNA oligo片段阴性对照组、转染REV7 RNA oligo的REV7基因下调组。克隆形成实验反映细胞增殖水平,蛋白印迹法检测相关基因表达水平、细胞凋亡发生水平和非同源末端连接途径发生水平。结果 6Gy照后REV7 siRNA组细胞克隆形成率降低(P<0.05)。REV7 siRNA组REV7基因下调效率>60%。REV7 siRNA组γH2AX、Caspase9表达升高(P<0.05),Ku80、XRCC4表达降低(P<0.05)。结论 下调REV7基因能提高HCT116细胞放射敏感性,其机制可能与下调REV7后非同源末端连接的发生被削弱有关。     

下调REV7基因对于人结肠癌HCT116细胞放射敏感性影响及机制研究

目的 研究下调REV7基因表达对人结肠癌HCT116细胞放射敏感性影响及其机制。方法 对HCT116细胞进行培养并运用RNA干扰技术实现REV7基因下调,将细胞分为空白组、转染阴性RNA oligo片段阴性对照组、转染REV7 RNA oligo的REV7基因下调组。克隆形成实验反映细胞增殖水平,蛋白印迹法检测相关基因表达水平、细胞凋亡发生水平和非同源末端连接途径发生水平。结果 6Gy照后REV7 siRNA组细胞克隆形成率降低(P<0.05)。REV7 siRNA组REV7基因下调效率>60%。REV7 siRNA组γH2AX、Caspase9表达升高(P<0.05),Ku80、XRCC4表达降低(P<0.05)。结论 下调REV7基因能提高HCT116细胞放射敏感性,其机制可能与下调REV7后非同源末端连接的发生被削弱有关。     

下调PRPS2基因表达对HCT116细胞增殖和凋亡的影响

目的:探讨下调磷酸核糖焦磷酸合成酶亚基II(PRPS2)基因表达对人结肠癌HCT116细胞增殖和凋亡的影响。方法:构建PRPS2基因干扰载体sh-PRPS2,正常对照组不转染载体,阴性对照组转染阴性对照载体sh-PRPS2-0,3个实验组分别转染干扰载体sh-PRPS2-1、sh-PRPS2-2和sh-PRPS2-3,经DNA测序鉴定后转染人结肠癌HCT116细胞,分别采用RT-PCR和Western blot法检测各载体转染后细胞PRPS2 mRNA和蛋白水平的表达变化,CCK8试剂盒检测细胞增殖能力,流式细胞术检测细胞周期及凋亡的变化。结果:经DNA测序证实成功构建sh-PRPS2干扰载体3个,分别为sh-PRPS2-1、sh-PRPS2-2和sh-PRPS2-3。将上述3个干扰载体分别转染HCT116细胞72 h后,RT-PCR结果显示,PRPS2 mRNA相对表达量依次为0.61±0.03、0.89±0.02、0.27±0.05,与对照组比较,均显著下降(P<0.05)。Western blot结果显示,PRPS2蛋白相对表达量依次为0.37±0.06、0.84±0.05、0.30±0.04,与对照组比较均显著下降(P<0.05)。转染sh-PRPS2-3载体72 h后细胞增殖抑制率达19.8%±2.4%,凋亡率上升至68.4%±4.6%,G0/G1期细胞比例上升为12.9%±3.8%。结论:PRPS2的低表达可以有效抑制细胞增殖,促进细胞凋亡,细胞阻滞于G0/G1期,本研究为肿瘤的靶向治疗提供了研究基础。     

EZH2蛋白对人结肠癌细胞HCT116生物学行为的影响

目的:探讨泛素连接酶EZH2蛋白对人结肠癌细胞增殖、凋亡、侵袭、转移等生物学行为的影响。方法:设计合成特异性干扰EZH2蛋白表达的小干扰RNA,通过QPCR、Western blot技术检测转染小干扰RNA后的干扰效果;MTT法检测沉默EZH2蛋白表达对结肠癌细胞系增殖的影响;流式细胞仪测定EZH2蛋白对结肠癌细胞凋亡的影响;Transwell实验检测EZH2蛋白对结肠癌细胞侵袭转移的影响。结果:设计合成的小干扰RNA能有效沉默EZH2蛋白的表达,EZH2蛋白被沉默后,人结肠癌细胞HCT116的增殖明显受到抑制,细胞的凋亡明显增加,侵袭与转移能力降低。结论:EZH2蛋白参与人结肠癌细胞HCT116生物学行为的调控,可能在结肠癌的发生发展中发挥着重要的作用,有望成为结肠癌治疗的新靶点。     

肠癌组织中miR-141的表达及其对HCT116细胞增殖和迁移的影响

目的:探讨miR-141在肠癌组织中的表达情况以及其对HCT116细胞生物学功能的影响及其作用机制。方法:选取2016年05月至2018年05月在中国医科大学附属第一人民医院手术切除的30对肠癌组织以及癌旁组织进行miRNA芯片筛查。逆转录定量聚合酶链反应分析其中异常表达miRNA情况,评估miR-141的表达与肿瘤相关信息的相关性。通过TargetScan软件分析miR-141可能靶向的蛋白,在HCT116细胞中通过荧光素酶报告基因实验检测miR-141对DEK蛋白的靶向作用。HCT116细胞中分别过度表达和沉默表达miR-141后,通过MTT实验检测细胞增殖情况,Transwell实验检测细胞的迁移情况。结果:芯片分析和逆转录定量聚合酶链反应指出miR-141在肠癌组织中表达低于癌旁组织,miR-141与肿瘤的进程具有相关性,TargetScan软件指出miR-141可以靶向作用于DEK,荧光素酶报告基因实验印证了miR-141对DEK的靶向作用。MTT实验指出miR-141过度表达显著抑制细胞增殖,miR-141沉默表达显著促进细胞增殖,与对照组相比,差异有统计学意义（P＜0.05）。Transwell实验显示,miR-141过度表达可以抑制HCT116细胞的迁移,miR-141沉默表达后HCT116细胞的迁移得到促进。结论:miR-141通过靶向DEK蛋白能够抑制HCT116细胞的增殖和迁移。     

Bone morphogenetic protein-4 inhibits heat-induced apoptosis by modulating MAPK pathways in human colon cancer HCT116 cells

Cancer thermotherapy and radiofrequency ablation (RFA) have been adopted as modalities for treating various kinds of cancer. We have previously demonstrated that bone morphogenetic protein-4 (BMP-4) is up-regulated in colonic adenocarcinoma. Here, we investigated whether an increase of BMP-4 expression changes cellular response to heat treatment in human colon cancer HCT116 cells. BMP-4 overexpressing HCT116 cells generated by stable transfection showed a significantly increased survival rate and a decreased apoptotic rate in comparison to empty vector controls after heat treatment at 45 degrees C for 20min. The expression levels and pattern of HSP90, HSP70, and HSP27 after heat treatment were similar between these two cell lines. There was no difference in expression levels of Bcl-2 and Bax in these two cell lines and their expression remained unchanged after heat treatment. Both activities of the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were stimulated by heat in these cells. Comparatively, BMP-4 overexpressing cells had an intense and prolonged ERK activation, while a less intense and short JNK activation. Correspondingly, treatment of BMP-4 overexpressing cells with noggin, a BMP-4 antagonist, resulted in a reduction of heat-activated ERK but an increase of heat-activated JNK and significantly increased heat-induced apoptotic rate. These results indicate that BMP-4 can protect colon cancer cells from heat-induced apoptosis through enhancing the activation of ERK as well as inhibiting the activation of JNK.     

Overexpression of p65/RelA potentiates curcumin-induced apoptosis in HCT116 human colon cancer cells

Curcumin, the yellow pigment in the spice turmeric, has potent chemopreventive activities that involve diverse molecular pathways. It is widely believed that curcumin pro-apoptotic properties are mediated by downregulation of NF kappa B (NFkappaB). The p65/RelA subunit of NFkappaB may influence cell death, in part by activation of NFkappaB anti-apoptotic target genes including X-linked inhibitor of apoptosis (XIAP), A20, bcl-xL and inhibition of sustained activation of c-Jun N-terminal kinase (JNK). We have shown previously that curcumin inhibits NFkappaB, activates JNK and promotes apoptosis in HCT116 colorectal cancer cells. Here, we show that forced overexpression of p65 does not affect curcumin-induced JNK activation. Indeed, overexpression of p65 enhanced curcumin-mediated apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and poly(ADP-ribose) polymerase (PARP) cleavage. This potentiating effect of p65 upon curcumin-mediated apoptosis was reversed by transfection of cells with an IkappaB super-repressor (DeltaNIkappaB). Curcumin treatment inhibited expression of NFkappaB anti-apoptotic target genes in mock-transfected and in p65-overexpressing HCT116 cells, although expression levels remained higher in the latter. Taken together, these results show that curcumin-mediated activation of JNK or induction of apoptosis does not require inhibition of p65. Furthermore, curcumin/p65 synergy in promotion of apoptosis cannot be attributed to active repression of NFkappaB anti-apoptotic genes.     

Cellular response and activation of apoptosis by mithramycin SK in p21-deficient HCT116 human colon carcinoma cells

HCT116 (p21^−/−) human colon carcinoma cells treated with mithramycin SK (MSK), a novel analog of the antitumor antibiotic mithramycin A (MTA), were transiently arrested in G2/M, with some cells entering a faulty mitotic cycle without cytokinesis that resulted in G1-like cell arrest, which consisted of post-mitotic aneuploid G1 cells. Some of these cells synthesized DNA and elicited an apoptotic response. The absence of p21^WAF1 made HCT116 cells more sensitive to MSK than to the related MTA. MSK also showed higher antiproliferative activity than MTA on HCT116 cells with different genetic backgrounds, including those lacking the p53 gene. Apoptosis in MSK-treated p21^−/− cells involved caspase 2 rather than caspase 3. Untreated HCT116 (p21^−/−) cells presented a little caspase 3 activity, which increased slightly after treatment with MSK. The apoptotic response in p21^−/− cells comprised caspase 2 acting as an executor caspase together with a loss of mitochondrial membrane potential that may be initiated by caspase 2. In contrast, caspase 3 was activated in wild-type HCT116 after treatment with MSK.     

Sodium salicylate induces apoptosis in HCT116 colorectal cancer cells through activation of p38MAPK

Sodium salicylate is known to induce apoptosis in a variety of cancer cells. However, the molecular mechanism for salicylate-induced apoptosis is yet unclear. Here we show that in HCT116 colon carcinoma cells, 10 mM sodium salicylate induces caspase-3 activation and degradation of its substrates, poly(ADP-ribose) polymerase (PARP), beta-catenin, and retinoblastoma (Rb). In contrast, sodium salicylate did not exert any significant effects on the expression of Fas L that is implicated in extrinsic apoptotic pathway and the levels of Bcl-2 family proteins, Bcl-2, Bcl-xsl, and Bad, which are involved in intrinsic apoptotic pathway, and anti-apoptotic molecules, c-IAP1 and HSP73. In addition, 10 mM salicylate induced p53 tumor suppressor protein that plays an important role in cell cycle arrest or apoptosis and the induction seemed to be linked to its phosphorylation at Set 15. To investigate the signal pathways for salicylate-induced apoptosis, we examined the effects of sodium salicylate on protein kinase activities. Sodium salicylate activated p38MAPK through phosphorylation at Thr 180/Tyr 182 and Akt/PKB at Ser 473, whereas it partially activated ERK1/2 through its phosphorylation at Thr 202/Tyr 204. We also show that SB203580 (a specific p38MAPK inhibitor), but not other protein kinase inhibitors (PD98059, LY294002, and wortmannin), significantly prevented salicylate-induced apoptosis. These results suggest that sodium salicylate-induced apoptosis in HCT116 colorectal cancer cells is mediated by p38MAPK.     

Recombinant mistletoe lectin induces p53-independent apoptosis in tumour cells and cooperates with ionising radiation

Mistletoe extracts are used as alternative cancer treatment in addition to standard chemotherapy and radiation treatment and have an immunostimulatory and pain-relieving effect. A direct antitumour effect of mistletoe extracts against tumour cells of lymphoid origin has been linked to the D-galactoside-specific mistletoe lectin I. In this study, we investigated the cellular effect of bacterially expressed, recombinant mistletoe lectin alone or in combination with ionising radiation in a genetically defined p53-wild-type and p53-deficient E1A/ras-transformed murine tumour cells system. Downregulation of the proliferative activity and cell killing by recombinant mistletoe lectin occurred in a clear dose response (0.1-1 ng ml(-1)). Induction of apoptosis was p53-independent, but apoptosis-associated factor-1-dependent. Cellular treatment with lectin in combination with ionising radiation resulted in both p53-wild-type and p53-deficient tumour cells in an at least additive, antiproliferative effect and enhanced activation of caspase-3. Combined treatment with ionising radiation and lectin revealed a similar cytotoxic effect in human, p53-mutated adenocarcinoma cells. Thus, recombinant mistletoe lectin alone and in combination with ionising radiation bypasses often prevalent apoptotic deficiencies in treatment-resistant tumour cells.     

Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak

The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax (Bcl2-associated X protein) and Bak (Bcl2-antagonist/killer 1). Although Bax and Bak seem to have redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for fluorouracil-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax, but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant, but they are counteracted by distinct anti-apoptotic Bcl-2 family proteins in different species.