Bcl-2 gene prevents induction of apoptosis in l1210 murine leukemia-cells by sn-38, a metabolite of the camptothecin derivative cpt-11

New camptothecin (CPT) derivatives have recently been synthesized following the finding that CPT has strong antitumor activity due to its inhibition of topoisomerase I through the formation of stable topoisomerase I-DNA cleavable complexes, but has not been clinically used due to its pronounced toxicity. 7-ethyl-10-hydroxy-CPT (SN-38), a metabolite of the CPT derivative 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-CPT(CPT-11), plays an essential role in mediating the antitumor effect of CPT-11. However, the reasons for the cytotoxicity of SN-38 remain unclear. In this study, we demonstrated using results of DNA fragmentation assay and cell cycle analysis that SN-38 and CPT both induce apoptosis in L1210 murine leukemia cells. We demonstrated in addition that enforced expression of the bcl-2 gene in L1210 cells by MPZenNeo (bcl-2) retroviral gene transfer increased resistance to the apoptosis induced by SN-38 and CPT. These findings suggest the possibility that the bcl-2 gene impedes the activity of a common pathway for apoptosis induced by SN-38 and CPT.     

Taurolidine induces apoptosis of murine melanoma cells in vitro and in vivo by modulation of the Bcl-2 family proteins

BACKGROUND: This study evaluates whether taurolidine, a novel antibiotic agent, induces murine melanoma cell apoptosis in vitro and in vivo. METHODS: Murine melanoma cells (B16 4A5 and B16 F10) were treated with taurolidine (0-100 microM) for 12 and 24 hr. Cell viability and apoptosis were assessed by MTT assay and FACScan analysis. Expression of the Bcl-2 family proteins was detected by Western blot analysis. In vivo, taurolidine-induced anti-tumor cytotoxicity was assessed in C57BL/6 mice. Therapeutic effectiveness, by intraperitoneal injection of taurolidine (15 mg/mouse) on alternate days for 2 weeks, was evaluated in mice bearing B16 4A5 tumor xenografts. Primary and metastatic tumor growth and intra-tumor apoptotic index were measured. RESULTS: Taurolidine induced cell apoptosis and reduced cell viability in murine melanoma cells. The pro-apoptotic protein Bax was enhanced, whereas the anti-apoptotic protein Bcl-2 was inhibited by taurolidine treatment. In vivo, systemic injection of 15-mg taurolidine was identified as the maximally tolerated dose. Administration of taurolidine at 15 mg/mouse significantly inhibited primary and metastatic tumor growth, which was mirrored by a significantly increased intra-tumor apoptotic index. CONCLUSIONS: These results demonstrate that taurolidine significantly attenuated melanoma tumor growth, which may result from taurolidine-induced apoptosis by modulation of the Bcl-2 family proteins.     

Bcl-2过表达对多柔比星诱导的膀胱癌细胞凋亡和NF-KB活化影响的观察

目的：探讨凋亡相关基因Bcl2过表达对多柔比星（ADM）诱导的膀胱癌细胞凋亡和NF—KB活化的影响。方法：采用脂质体转染法将Bcl-2基因转入膀胱癌细胞,G418筛选获得抗性亚克隆细胞株,RT—PCR检测Bcl-2基因的表达。用6．25、12．5和25μg／mL的ADM分别作用于细胞24h,流式细胞术检测细胞凋亡,蛋白质印迹法检测NF-kB活化情况。结果：建立分别稳定过表达Bcl-2基因和新霉素抗性基因（neo）的膀胱癌亚克隆株BIU87／Bcl-2和RIU87／neo,RT-PCR结果显示,H1U87／Bcl-2细胞的Bcl-2mRNA水平明显高于BIU87／neo和BIU87细胞,F=63．107,P〈0．0l。经6．25、12．5和25μg／mLADM作用24h后,BIU87／Bci-2细胞凋亡率较BIU87／neo细胞明显降低,t=11．216,JP〈0．01。与BIU87／neo细胞相比,ADM作用24h后BIU87／Bcl-2细胞胞质IKB明显减少,t=-O．255,P=0．018;而胞核NF—KBp65明显增多,t=2．088,P=O．049。结论：Bel-2基因能够抑制ADM诱导的膀胱癌BIU87细胞凋亡,其抗凋亡作用涉及NF—KB活化。     

苦参碱对小鼠H22肝癌细胞凋亡作用的实验研究

目的：研究中药苦参碱在体内外对小鼠H22肝癌细胞的诱导凋亡作用，探讨其可能的抗肿瘤作用机制。方法：Annexin V-FITC／PI双标记法检测苦参碱对H22细胞的早期促凋亡作用；免疫组织化学法检测苦参碱作用后H22细胞内Bcl-2、Bax两种凋亡相关蛋白的表达。建立H22肝癌移植瘤小鼠模型，观察苦参碱对荷瘤小鼠肿瘤生长情况的影响及肿瘤抑制率；电镜观察荷瘤小鼠肿瘤组织的超微结构改变；免疫组化方法检测小鼠肿瘤组织内Bcl-2、Bax的表达情况。结果：AnnexinV法检测到1．0mg／mL和1．5mg／mL苦参碱作用48h后H22细胞有早期凋亡改变，凋亡细胞百分率分别为11．71％和17．86％，与对照组相比差异有统计学意义（P〈0．05）。苦参碱对荷瘤小鼠肿瘤的抑制率达60％以上；免疫组化显示，苦参碱作用后H22细胞内及小鼠肿瘤组织内Bcl-2蛋白的表达水平下降，Bax蛋白表达增强。电镜证实苦参碱治疗后荷瘤小鼠肿瘤组织内有凋亡细胞和凋亡小体的存在。结论：苦参碱在体内体外都对小鼠H22肝癌细胞表现出较强的抗肿瘤作用和诱导凋亡作用。促凋亡作用与其上调细胞内Bax表达，抑制Bcl-2表达有关。     

Bcl-2反义肽核酸诱导HL60细胞凋亡

目的　探讨不同结构的反义药物对HL6 0细胞系生物学活性的影响。方法　应用细胞计数、细胞形态观察和流式细胞术观察并比较反义肽核酸和反义寡核苷酸对白血病细胞HL6 0生物学活性的影响。结果　 10 μmol/L靶向bcl mRNA蛋白编码区的反义肽核酸能有效地抑制HL6 0细胞的生长、下调bcl 2蛋白的水平及诱导细胞凋亡。 10μmol/L同样靶点的反义肽核酸和反义寡核苷酸作用HL6 0细胞 72小时 ,细胞凋亡的百分率分别为 17.8± 1.5 3 ,13.17± 1.12 ,统计学上有显著性差异。结论　反义Bcl 肽核酸能诱导HL6 0细胞的调亡 ,比反义寡核苷酸有更好的反义作用。     

雷帕霉素诱导人肝癌细胞BEL-7402凋亡中Bcl-2作用研究

目的探讨雷帕霉素(rapamycin,RAPA)体外对肝癌BEL-7402细胞生长抑制及诱导细胞凋亡中的作用及Bcl-2变化在凋亡机制中的意义。方法以5、10、20、30、40和50nmol/L不同浓度的RAPA作用于体外培养的BEL-7402细胞,MTT法检测细胞生长抑制率,应用流式细胞仪检测细胞凋亡,Hoechst33258荧光染色法观察细胞凋亡时的形态学变化,Westernblot观察Bcl-2、Bcl-xl和bax等凋亡相关表达变化。结果RAPA可显著抑制BEL-7402细胞的生长,诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系。RAPA作用肝癌细胞BEL-740248h后,在Hoechst33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征,凋亡过程中伴有抗凋亡蛋白Bcl-2、Bcl-xl表达的降低和促凋亡蛋白bax上调。结论RAPA可能通过诱导抗凋亡蛋白Bcl-2、Bcl-xl表达的降低和促凋亡蛋白bax表达上调而诱导凋亡发生,抑制BEL-7402细胞的生长。     

Bcl-2反义寡核苷酸诱导HL-60细胞凋亡的研究

目的：探讨bcl-2反义寡核苷酸诱导ＨＬ－６０细胞的凋亡。方法：将人工合成的bcl-2反义寡聚脱氧苷酸片段与ＨＬ－６０,细胞共培养,在作用的第０天、３天、５天通过免疫组化法（ＡＰＡＡＰ法）检测细胞内bcl-2蛋白表达水平的变化,通过流式细胞仪分析细胞凋亡的情况。结果：bcl-2反这寡核苷酸可下调ＨＬ－６０细胞内bcl-2的蛋白表达水平,其作用与其浓度和时间呈正相关,同时可诱导细胞凋亡,而bcl-2反义寡核苷酸及对照无此作用。     

Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用

目的:利用本实验室已建立PIG11蛋白稳定低表达的HepG2细胞株和PIG11蛋白稳定高表达HepG2细胞株,研究Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用,进一步探讨PIG11基因表达对HepG2细胞凋亡的机制。方法:细胞培养,分组:HepG2细胞、pLXSN-PIG11-HepG2(pLXSN-PIG11转染建立PIG11蛋白高表达HepG2细胞)、pLXSN-HepG2(pLXSN空载体转染HepG2细胞)、miR-PIG11-HepG2(miRNA干扰PIG11蛋白低表达HepG2细胞)、miR-HepG2(干扰空载体HepG2细胞)。Hoechst33342染色荧光和PI染色流式细胞仪检测各组细胞凋亡情况。Western blot检测Caspase-8蛋白、Bcl-2蛋白的表达情况。结果:Hoechst 33342染色荧光显微镜下pLXSN-PIG11-HepG2细胞组可见核染色质浓缩,核碎片,荧光增强,以及凋亡小体。PI染色流式细胞仪检测结果显示:HepG2细胞、miR-PIG11-HepG2细胞、miR-HepG2细胞、pLXSN-PIG11-HepG2细胞、pLXSN-HepG2细胞的凋亡率分别为5.72%±0.81%、1.34%±0.71%、5.10%±0.40%、34.83%±2.29%、5.34%±0.60%。PIG11高表达的pLXSN-PIG11-HepG2细胞组凋亡率比其它各组增高(P<0.01),PIG11低表达的miR-PIG11-HepG2细胞组凋亡率降低(P<0.01)。Western blot检测结果显示PIG11高表达的pLXSN-PIG11-HepG2细胞Caspase-8蛋白表达上调,Bcl-2蛋白表达下调(P<0.01),PIG11低表达的miR-PIG11-HepG2细胞Caspase-8蛋白的表达下调,Bcl-2蛋白表达上调(P<0.01)。结论:PIG11蛋白高表达能诱导HepG2细胞凋亡,其机制可能与Caspase-8蛋白及Bcl-2蛋白表达相关。     

Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用

目的：利用本实验室已建立PIG11蛋白稳定低表达的HepG2细胞株和PIG11蛋白稳定高表达HepG2细胞株,研究Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用,进一步探讨PIG11基因表达对HepG2细胞凋亡的机制。方法：细胞培养,分组：HepG2细胞、pLXSN-PIG11-HepG2（pLXSN-PIG11转染建立PIG11蛋白高表达HepG2细胞）、pLXSN-HepG2（pLXSN空载体转染HepG2细胞）、miR-PIG11-HepG2（miRNA干扰PIG11蛋白低表达HepG2细胞）、miR-HepG2（干扰空载体HepG2细胞）。Hoechst33342染色荧光和PI染色流式细胞仪检测各组细胞凋亡情况。Western blot检测Caspase-8蛋白、Bcl-2蛋白的表达情况。结果：Hoechst 33342染色荧光显微镜下pLXSN-PIG11-HepG2细胞组可见核染色质浓缩,核碎片,荧光增强,以及凋亡小体。PI染色流式细胞仪检测结果显示：HepG2细胞、miR-PIG11-HepG2细胞、miR-HepG2细胞、pLXSN-PIG11-HepG2细胞、pLXSN-HepG2细胞的凋亡率分别为5.72%±0.81%、1.34%±0.71%、5.10%±0.40%、34.83%±2.29%、5.34%±0.60%。PIG11高表达的pLXSN-PIG11-HepG2细胞组凋亡率比其它各组增高（P〈0.01）,PIG11低表达的miR-PIG11-HepG2细胞组凋亡率降低（P〈0.01）。Western blot检测结果显示PIG11高表达的pLXSN-PIG11-HepG2细胞Caspase-8蛋白表达上调,Bcl-2蛋白表达下调（P〈0.01）,PIG11低表达的miR-PIG11-HepG2细胞Caspase-8蛋白的表达下调,Bcl-2蛋白表达上调（P〈0.01）。结论：PIG11蛋白高表达能诱导HepG2细胞凋亡,其机制可能与Caspase-8蛋白及Bcl-2蛋白表达相关。     

Photorepair prevents ultraviolet-induced apoptosis in human cells expressing the marsupial photolyase gene

Photolyase absorbs blue light and employs the energy to remove UV-induced DNA damage, cyclobutane pyrimidine dimers, or pyrimidine pyrimidone (6-4) lesions. These enzymes have been found in many living organisms ranging from bacteria to aplacental mammals, but their photoreactivation effect, such as survival increase of UV-irradiated cells by light-illumination, has not been identified in placental mammals, including humans. Therefore, we introduced a photolyase gene derived from the marsupial rat kangaroo, Potorous tridactylus, into HeLa cells and established the first human cell line capable of photorepairing UV-induced pyrimidine dimers. Several clones were found to increase cell survival after UV irradiation when illuminated by fluorescent light. The induction of apoptosis by UV irradiation was investigated in these photoreactivation-proficient cells. Several typical features of the programmed cell death, such as internucleosomal DNA degradation, presence of subdiploid cells, loss of membrane integrity, and chromosomal condensation, were found to be induced by UV in the HeLa cells, but they can be reduced by photorepair. This implicates that cyclobutane pyrimidine dimers cause UV-induced apoptosis in human cells.     

Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression

Primary or acquired resistance to current treatment protocols remains a major concern in clinical oncology and may be caused by defects in apoptosis programs. Since recent data suggest that TRAIL can bypass apoptosis resistance caused by Bcl-2, we further investigated the role of Bcl-2 in TRAIL-induced apoptosis. Here we report that overexpression of Bcl-2 conferred protection against TRAIL in neuroblastoma, glioblastoma or breast carcinoma cell lines. Bcl-2 overexpression reduced TRAIL-induced cleavage of caspase-8 and Bid indicating that caspase-8 was activated upstream and also downstream of mitochondria in a feedback amplification loop. Importantly, Bcl-2 blocked cleavage of caspases-9, -7 and -3 into active subunits and cleavage of the caspase substrates DFF45 or PARP. Also, Bcl-2 blocked cleavage of XIAP and overexpression of XIAP conferred resistance against TRAIL indicating that apoptosis was also amplified through a feedforward loop between caspases and XIAP. In contrast, in SKW lymphoblastoid cells, TRAIL-induced activation of caspase-8 directly translated into full activation of caspases, cleavage of XIAP, DFF45 or PARP and apoptosis independent of Bcl-2 overexpression, although Bcl-2 similarly inhibited loss of mitochondrial membrane potential and the release of cytochrome c, AIF and Smac from mitochondria in all cell types. By demonstrating a cell type dependent regulation of the TRAIL signaling pathway at different level, e.g. by Bcl-2 and by XIAP, these findings may have important clinical implication. Thus, strategies targeting the molecular basis of resistance towards TRAIL may be necessary in some tumors for cancer therapy with TRAIL.     

Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

Background  

Pancreatic ductal adenocarcinoma shows a distinct apoptosis resistance, which contributes significantly to the aggressive nature of this tumor and constrains the effectiveness of new therapeutic strategies. Apoptosis resistance is determined by the net balance of the cells pro-and anti-apoptotic "control mechanisms". Numerous dysregulated anti-apoptotic genes have been identified in pancreatic cancer and seem to contribute to the high anti-apoptotic buffering capacity. We aimed to compare the benefit of simultaneous gene silencing (SGS) of several candidate genes with conventional gene silencing of single genes.     

Phenethyl isothiocyanate triggers apoptosis in Jurkat cells made resistant by the overexpression of Bcl-2

Isothiocyanates are a class of naturally occurring chemopreventive agents known to be effective at triggering apoptosis. In this study, we show that whereas overexpression of the oncoprotein Bcl-2 renders Jurkat T-lymphoma cells resistant to a range of cytotoxic agents, phenethyl isothiocyanate is able to overcome the inhibitory action of Bcl-2 and trigger apoptosis. A 50-fold increase in Bcl-2 expression shifted the dose-response curve, with an increase in the phenethyl isothiocyanate LD(50) from 7 to 15 micromol/L, but there was still a complete loss in cell viability at doses in excess of 20 micromol/L. At these concentrations, cytotoxicity was strongly associated with caspase activation, phosphatidylserine exposure, and morphologic changes characteristic of apoptosis. Cytotoxicity was inhibited by treatment of the cells with a broad-spectrum caspase inhibitor. A structure-activity analysis showed that the phenethyl and benzyl isothiocyanates were most effective at triggering apoptosis in cells overexpressing Bcl-2 whereas phenyl isothiocyanate and benzyl thiocyanate had no proapoptotic activity. Allyl isothiocyanate also had limited efficacy despite its ability to trigger apoptosis in the parental Jurkat cell line. From this information, we propose that isothiocyanates modify a key cysteine residue in an apoptosis regulatory protein and that the aromatic side chain facilitates access to the target site. An in-depth investigation of the cellular targets of the aromatic isothiocyanates is warranted.     

UBE2T基因通过调控ROS抑制结直肠癌细胞凋亡

目的:通过结肠癌细胞实验检测UBE2T对结肠癌细胞凋亡的影响。方法:细胞计数盒（CCK-8）法检测UBE2T基因转染对结肠癌细胞活力的影响;流式细胞仪检测UBE2T转染后对结肠癌细胞凋亡的影响;DCFDA染色流式细胞仪检测UBE2T转染对结肠癌细胞中ROS水平的影响;CCK-8法检测H2O2（增加ROS）对UBE2T基因转染降低结肠癌细胞活力的影响;流式细胞仪检测H2O2对UBE2T基因转染增加结肠癌细胞凋亡率的影响。结果:UBE2T转染能够明显增加结肠癌细胞活力（P＜0.05）;UBE2T基因转染抑制结肠癌细胞凋亡率（P＜0.05）;UBE2T转染减少结肠癌细胞ROS水平;预处理H2O2减弱UBE2T基因转染对结肠癌细胞活力的增加作用（P＜0.05）;预处理H2O2逆转UBE2T基因转染对结肠癌细胞凋亡率的抑制作用（P＜0.05）。结论:UBE2T基因转染能够抑制结肠癌凋亡,其机制是通过调节结直肠癌细胞中ROS水平。     

Lipid raft disruption prevents apoptosis induced by 2-chloro-2'-deoxyadenosine (Cladribine) in leukemia cell lines

To clarify the role of lipid rafts in 2-chloro-2′-deoxyadenosine (2CdA; Cladribine)-induced apoptosis, the effects of disruption of lipid rafts by methyl-β-cyclodextrin (MβCD) and filipin on 2CdA-induced apoptosis were investigated in four human acute lymphoblastic leukemia (ALL) cell lines comprised of T cells (MOLT-4, Jurkat) and B cells (NALM, BALL-1). The disruption of lipid rafts significantly inhibited 2CdA-induced apoptosis, indicating the crucial role of lipid rafts in the induction of apoptosis in leukemia cells. These reagents significantly inhibited 2CdA-induced elevation of the intracellular calcium concentration ([Ca²⁺]_i) in MOLT-4 cells, and 2CdA-induced apoptosis was partly inhibited by the Ca²⁺ chelators BAPTA-AM and EGTA, and the L-type Ca²⁺ channel blocker nifedipine. On the other hand, they had no effects on the cellular uptake of 2CdA. These results indicated that lipid rafts partly contributed to 2CdA-induced apoptosis by regulating Ca²⁺ influx via the plasma membrane.     

Profile of gene expression induced by the tumour promotor TPA in murine epithelial cells

Malignant transformation of mouse skin by chemical carcinogens and tumour promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process that leads to squamous cell carcinoma (SCC) formation. In an effort to identify tumour-associated genes, we studied the influence of short-term TPA-treatment on the gene expression profile of murine skin. A comprehensive microarray with some 5,000 murine gene specific cDNA fragments was established and hybridised with pooled RNA derived from control and TPA-treated dorsal skin samples. Of these genes, 54 were up- and 35 were down-regulated upon TPA application. Additionally, we performed suppression subtractive hybridisation (SSH) with respective RNA pools to generate and analyse a cDNA library enriched for TPA-inducible genes. Expression data of selected genes were confirmed by quantitative real-time PCR and Northern blot analysis. Comparison of microarray and SSH data revealed that 26% of up-regulated genes identified by expression profiling matched with those present in the SSH library. Besides numerous known genes, we identified a large set of unknown cDNAs that represent previously unrecognised TPA-regulated genes in murine skin with potential function in tumour promotion. Additionally, some TPA-induced genes, such as Sprr1A, Saa3, JunB, Il4ralpha, Gp38, RalGDS and Slpi exhibit high basal level in advanced stages of skin carcinogenesis, suggesting that at least a subgroup of the identified TPA-regulated genes may contribute to tumour progression and metastasis.     

PrLZ protects prostate cancer cells from apoptosis induced by androgen deprivation via the activation of Stat3/Bcl-2 pathway

PrLZ/PC-1 is a newly identified, prostate-specific and androgen-inducible gene. Our previous study showed that PrLZ can enhance the proliferation and invasive capability of LNCaP cells, contributing to the development of prostate cancer. However, its potential role in androgen-independent processes remains elusive. In this study, we showed that PrLZ enhanced in vitro growth and colony formation of prostate cancer cells on androgen deprivation as well as tumorigenicity in castrated nude mice. In addition, PrLZ stabilized mitochondrial transmembrane potential, prevented release of cytochrome c from mitochondria to cytoplasm, and inhibited intrinsic apoptosis induced by androgen depletion. Mechanistically, PrLZ elevated the phosphorylation of Akt and Stat3 and upregulated Bcl-2 expression. Our data indicate that PrLZ protects prostate cancer cells from apoptosis and promotes tumor progression following androgen deprivation. In summary, we propose that PrLZ is a novel antiapoptotic gene that is specifically activated in prostate cancer cells escaping androgen deprivation may offer an appealing therapeutic target to prevent or treat advanced prostate malignancy.     

Decreased levels of p26-Bcl-2, but not p30 phosphorylated Bcl-2, precede TGFbeta1-induced apoptosis in colorectal adenoma cells

Bcl-2 expression is confined to the base of the colonic crypt, whereas transforming growth factor beta (TGFbeta) is expressed in the upper crypt, as are the apoptotic death promoters, Bak and Bax. In colonic adenoma cells, TGFbeta induces a growth arrest. In some adenoma cell lines, this is accompanied by apoptosis and in others it is not. In this study, we used two human colonic adenoma cell lines: RG/C2, in which TGFbeta induces a G1 arrest without apoptosis, and BH/C1, in which TGFbeta induces both a G1 arrest and apoptosis. TGFbeta does not induce apoptosis in RG/C2 cells even if hydrocortisone and insulin are removed from the culture medium. In BH/C1 cells, TGFbeta induces apoptosis in the presence of insulin and hydrocortisone. Apoptosis induced by TGFbeta is preceded by a reduction in p26-Bcl-2 protein levels. There was no change in the levels of the p30 phosphorylated form of Bcl-2 or in levels of the proapoptotic proteins Bax or Bak. RG/C2 cells did not show decreased Bcl-2 levels in response to TGFbeta- induced growth inhibition. Therefore, TGFbeta regulates Bcl-2 expression in colonic adenoma cells which undergo apoptosis in response to TGFbeta, but not in those which are growth inhibited, but resistant to TGFbeta-induced apoptosis. TGFbeta may play an important role in the colonic epithelium, not only in the inhibition of cell proliferation, but also in the regulation of apoptosis.