丹参多酚酸盐通过线粒体途径诱导人肝癌SMMC-7721细胞的凋亡

目的：研究丹参多酚酸盐(salvianolate)体外诱导人肝癌细胞SMMC-7721凋亡作用及其可能机制。方法：不同质量浓度丹参多酚酸盐(0.5、1、2 mg/ml)与肝癌细胞共培养24 h后,流式细胞仪检测肝癌细胞凋亡,线粒体膜电位试剂盒(JC-1)检测线粒体膜电位变化;比色法测定1.0 mg/ml丹参多酚酸盐作用后肝癌细胞内caspase8、caspase9 及caspase3的活性,流式细胞仪检测培养体系内加入caspase9抑制剂(zLEHDfmk)或caspase3抑制剂(zDEVDfmk)后细胞凋亡率的变化,Western blotting检测肝癌细胞内线粒体凋亡途径相关蛋白Bax、Bcl2表达水平。结果：丹参多酚酸盐显著诱导肝癌细胞SMMC7721凋亡(P<0.05),同时线粒体膜电位随着药物浓度的升高而加剧下降(P<0.05)。1.0 mg/ml 丹参多酚酸盐处理肝癌细胞24 h后caspase-9与caspase-3的活性明显升高(P<0.05),而caspase-8的活性无明显变化(P>0.05);当培养体系内加入caspase-9或caspase3活性抑制剂后,丹参多酚酸盐诱导肿瘤细胞凋亡的作用明显降低(P<0.05)。Western blotting检测显示,丹参多酚酸盐处理组前凋亡蛋白Bax表达明显升高,抗凋亡蛋白Bcl2表达降低。结论：丹参多酚酸盐(0.5~2.0 mg/ml)剂量具有促进肝癌细胞凋亡的作用,且有剂量依赖的趋势,其机制与线粒体凋亡途径有关。     

SERPINB3 protects from oxidative damage by chemotherapeutics through inhibition of mitochondrial respiratory complex I

SERPINB3 (SB3) is a serine protease inhibitor overexpressed in several malignancies of epithelial origin, including primary liver cancer, where it inhibits apoptosis through poorly defined mechanisms. In the present study we analyze the effect of SB3 on hepatoma cell death elicited by a panel of chemotherapeutic agents. We report that SB3 shields cells from the toxicity of drugs with a pro-oxidant action such as doxorubicin, cisplatin and EM20-25. The rapid rise in ROS levels prompted by these compounds causes opening of the mitochondrial permeability transition pore (PTP), irreversibly committing cells to death. We find that a fraction of SB3 locates in mitochondrial inner compartments, and that this mitochondrial fraction increases under conditions of oxidative stress. Mitochondrial SB3 inhibits ROS generation and the ensuing PTP induction and cell death through an inhibitory interaction with respiratory Complex I. These findings identify a novel mechanism of action of SB3 that contributes to tumor cell resistance to anti-neoplastic drugs     

Dlk/ZIP kinase-induced apoptosis in human medulloblastoma cells: requirement of the mitochondrial apoptosis pathway.

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours.     

Dulxanthone A induces cell cycle arrest and apoptosis via up-regulation of p53 through mitochondrial pathway in HepG2 cells

Natural products derived from plants provide a rich source for development of new anticancer drugs. Dulxanthone A was found to be an active cytotoxic component in Garcinia cowa by bioactivity-directed isolation. Studies to elucidate the cytotoxic mechanisms of dulxanthone A showed that dulxanthone A consistently induced S phase arrest and apoptosis in the most sensitive cell line HepG2. Furthermore, p53 was dramatically up-regulated, leading to altered expression of downstream proteins upon dulxanthone A treatment. Cell cycle related proteins, such as cyclin A, cyclin B, cyclin E, cdc-2, p21 and p27 were down-regulated. Some apoptosis correlated proteins were also altered following the drug treatment. Bcl-2 family members PUMA was up-regulated while Bcl-2 and Bax were down-regulated. However, the expression ratio of Bax/Bcl-2 was increased. This resulted in the release of cytochrome C from the mitochondria to the cytosol. Concurrently, Apaf-1 was stimulated with p53 by dulxanthone A. In result, cytochrome C, Apaf-1 and procaspase-9 form an apoptosome, which in turn triggered the activation of caspase-9, caspase-3 and downstream caspase substrates. Lamin A/C and PARP were down-regulated or cleaved, respectively. Moreover, cell cycle arrest and apoptosis in HepG2 cells induced by dulxanthone A were markedly inhibited by siRNA knockdown of p53. In summary, dulxanthone A is an active cytotoxic component of G. cowa. It induces cell cycle arrest at lower concentrations and triggers apoptosis at higher concentrations via up-regulation of p53 through the intrinsic mitochondrial pathway in HepG2 cells. Dulxanthone A is therefore likely a promising preventive and/or therapeutic agent against Hepatoma.     

PUMA通过线粒体途径诱导骨肉瘤细胞凋亡的研究

目的:研究p53上调凋亡调控因子(PUMA)对骨肉瘤细胞凋亡的影响及机制。方法:在骨肉瘤细胞F5M2中转染PUMA过表达载体,同时转染对照阴性载体,以qRT-PCR和Western blot法测定转染后细胞中PUMA表达情况,以流式细胞术检测细胞凋亡情况,用JC-1法检测细胞线粒体膜电位,以Western blot法测定线粒体和胞浆中细胞色素C（Cyt C）蛋白表达情况,同时检测细胞中剪切的含半胱氨酸的天冬氨酸蛋白水 解酶3（Cleaved Caspase-3）、剪切的含半胱氨酸的天冬氨酸蛋白水解酶9（Cleaved Caspase-9）蛋白水平。结果:转染PUMA过表达载体后的F5M2细胞中PUMA mRNA和蛋白水平升高,而转染对照阴性载体对F5M2细胞中PUMA mRNA和蛋白水平没有影响。过表达PUMA后的F5M2细胞凋亡率升高,细胞线粒体膜电位下降,线粒体中Cyt C蛋白水平降低,胞浆中Cyt C蛋白水平升高,同时细胞中Cleaved Caspase-3、Cleaved Caspase-9蛋白水平也升高,与未做转染的F5M2细胞比,差异有统计学意义（P<0.05）。转染对照阴性载体后的细胞凋亡率、线粒体膜电位、线粒体和胞浆中Cyt C蛋白水平、Cleaved Caspase-3蛋白水平、Cleaved Caspase-9蛋白水平与未做转染的F5M2细胞相比没有明显变化（P>0.05）。结论:PUMA通过促进线粒体中Cyt C释放,降低线粒体膜电位,激活Caspase-3诱导骨肉瘤细胞凋亡。     

TRAIL蛋白与顺铂协同诱导横纹肌肉瘤细胞凋亡的实验研究

目的探讨肿瘤坏死因子（INF）相关凋亡诱导配体（TRAIL）蛋白和顺铂联合应用对人横纹肌肉瘤细胞生长抑制和诱导凋亡作用。方法将不同浓度的TRAIL作用于培养的人RD横纹肌肉瘤细胞，通过MTT比色法、流式细胞仪检测细胞凋亡和Fas蛋白表达的方法，观察其对横纹肌肉瘤细胞的作用，及其与顺铂协同作用的效果和机制。结果TRAIL浓度为1．0、10．0、100．0ng／ml时，细胞毒性指数分别为18．9％、20．8％和43．5％；顺铂浓度为1．0、5．0、10．0μg／ml时，细胞毒性指数分别为9．8％、23．4％和37．1％。而浓度为100ng／ml的TRAIL与浓度为5μg／ml的顺铂联合作用时，细胞毒性指数明显增加达66．4％，结合FCM分析显示联合应用提高了Fas蛋白的表达，与细胞凋亡率增加相一致。结论TRAIL和顺铂均能有效诱导横纹肌肉瘤细胞凋亡，联合应用具有明显的协同效果。     

Caspase-8 levels affect necessity for mitochondrial amplification in death ligand-induced glioma cell apoptosis

Fifty percent of high-grade glioma patients die within a year of diagnosis and less than two percent survive five years postdiagnosis. Elucidating apoptosis signaling pathways may assist in designing better adjuvant therapies. Preliminary characterizations suggested that glioma cells may either employ mitochondrial-independent or -dependent death receptor-induced apoptotic pathways, characteristic of cells termed type I and type II, respectively. In the present study, we generated panels of clonal transfectants overexpressing various levels of Bcl-2, in two parental glioma cell lines. These cells were used to explore molecular factors determining the necessity for mitochondrial amplification of death receptor signaling. Moderate Bcl-2 expression was sufficient to render one glioma cell line (D270) resistant to apoptosis induced by Fas ligand or TRAIL, consistent with these cells being type II. However, expression of even very high levels of Bcl-2 in a second line (D645) did not affect death ligand sensitivity, indicative of a type I phenotype. D270 cells expressed much less caspase-8 protein than D645 cells. Enforced overexpression of caspase-8 (or cytoplasmic Diablo/Smac) in D270 cells overcame Bcl-2 inhibition of death ligand-induced apoptosis, converting them from type II to type I. This indicates that caspase-8 levels can influence the requirement for mitochondrial involvement in death receptor apoptotic signaling in glioma cells.     

青藤碱通过线粒体途径诱导肺癌NCI-H460细胞凋亡的初步研究

目的:研究青藤碱(sinomenine,SIN)对人肺癌NCI-H460细胞株的生长抑制及诱导凋亡作用及其机制。方法:四甲基偶氮噻蓝（MTT）法检测细胞增殖的抑制作用,流式细胞仪AnnexinV/PI双染法检测细胞凋亡,TdT酶介导的dUTP缺口末端标记（TUNNEL）方法观察细胞的凋亡,罗丹明123(Rhodamine123)染色流式细胞仪检测线粒体膜电位(ΔΨm)。结果:SIN对NCI-H460细胞株生长具有抑制作用并诱导凋亡。AnnexinV/PI双染检测细胞的凋亡可见随SIN浓度增加,细胞凋亡增加呈浓度依赖性。TUNNEL阳性的凋亡细胞呈棕黄色,细胞核片段化改变。罗丹明染色检测线粒体膜电位的结果提示在SIN作用于NCI-H460细胞48h后,线粒体膜电位下降,SIN浓度越高,膜电位下降越显著。结论:SIN具有明显的细胞毒作用,能诱导NCI-H460细胞凋亡,SIN通过线粒体途径诱导NCI-H460细胞凋亡。     

榄香烯乳诱导线粒体凋亡途径逆转肺癌A549/DDP细胞株耐药

目的：探讨榄香烯乳（ elemene,ELE）逆转人肺腺癌耐顺铂（ cisplatin,DDP）细胞A549/DDP的耐药性及作用机制。方法：采用MTT法检测榄香烯乳单用的细胞毒作用及与DDP合用时耐药逆转作用。荧光探针JC-1结合激光共聚焦显微镜检测线粒体膜电位的变化。DCFH-DA荧光探针结合流式细胞仪检测细胞内活性氧（ reactive oxygen species,ROS）水平。用谷胱甘肽试剂盒结合分光光度法检测计算GSH/（ GSSG﹢GSH）比值。蛋白质印迹法检测胞质中Cyto C、Pro-caspase-3、Caspase-3和Bcl-2家族蛋白表达情况。结果：不同浓度榄香烯乳抑制A549/DDP细胞株生长,呈时间-剂量依赖性效应,联合顺铂能提高A549/DDP细胞株对顺铂的敏感性而逆转耐药。不同浓度榄香烯乳联合顺铂使A549/DDP细胞株线粒体膜电位下降,ROS浓度增加,GSH/（ GSSG﹢GSH）比值降低,上调胞质中Cyto C、Caspase-3、Bad蛋白表达,下调Pro-caspase-3、Bcl-2蛋白表达。结论：榄香烯乳逆转A549/DDP细胞株耐药性可能与其损伤线粒体膜,活化胞内氧化还原体系,诱导线粒体凋亡路径有关。     

过表达OMA1通过促进线粒体释放细胞色素C诱导肺腺癌A549细胞凋亡

目的:探讨OMA1在肺腺癌中的表达水平及其对肺腺癌A549细胞凋亡的影响及机制。方法:收集2018年01月至2019年10月在本院保存的42例经病理确诊为肺腺癌患者的癌组织样本及其癌旁组织(距离肿瘤边缘≥5 cm),免疫组化染色法检测癌组织及癌旁组织中OMA1表达情况。体外培养人肺腺癌细胞系NCI-H2009、Calu-3、SPC-A-1、A549及人正常肺上皮细胞BEAS-2B,qRT-PCR和Western blotting检测细胞中OMA1 mRNA和蛋白表达水平。采用OMA1过表达慢病毒及其空载慢病毒感染A549细胞,分为OMA1过表达慢病毒感染组(OMA1组)和空载慢病毒感染组(Vector组),另设置空白对照组(Blank组)。采用MTT检测细胞增殖活性;流式细胞术检测细胞凋亡水平;JC-10染色法检测细胞线粒体膜电位变化;qRT-PCR法检测细胞中OMA1 mRNA表达水平;Western blotting法检测细胞中OMA1、cleaved caspase-3、OPA1以及细胞线粒体和胞浆中Cyt C蛋白表达水平。结果:肺腺癌患者癌组织中OMA1表达水平显著低于癌旁组织...     

MI-63: A novel small-molecule inhibitor targets MDM2 and induces apoptosis in embryonal and alveolar rhabdomyosarcoma cells with wild-type p53

Background:

Interruption of the role of p53s as a tumour suppressor by MDM2 may be one of the mechanisms by which cancer cells evade current therapy. Blocking the inhibition of wild-type p53 by MDM2 in cancer cells should reactivate p53''s tumour suppressor functions and enhance current cancer treatments. MI-63 is a novel non-peptide small molecule that has shown strong binding affinity (K_i=3 nM) for MDM2; however, its effects on paediatric cancer cells and the specific mechanism of tumour suppressor reactivation have not been evaluated.

Methods:

Rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, expresses either wild-type or mutant p53 protein. We examined the inhibitory effects of MI-63 in embryonal RMS (ERMS) and alveolar RMS (ARMS) cell lines expressing wild-type or mutated p53.

Results:

Treatment with MI-63 reduced cell viability by 13.4% and by <1%, respectively, at 72 h in both RH36 and RH18 cell lines expressing wild-type p53. In contrast, RH30 and RD2 cells expressing p53 mutants are resistant to MI-63 treatment. An increased expression of p53, p21^WAF1, and Bax protein was observed after treatment with MI-63 in RMS cells with wild-type p53, and apoptosis was confirmed by cleaved PARP and caspase-3 expression. However, RD2 and RH30 RMS cells, as well as human normal skeletal muscle cells, showed a minimal increase in p53 signalling and no induction of cleaved PARP and caspase-3. MI-63 was compared with Nutlin-3, a known MDM2 inhibitor, and was found to be more potent in the inhibition of cell proliferation/viability. Further, synergy was observed when MI-63 was used in combination with doxorubicin.

Conclusion:

These results indicate that MI-63 is a potent therapeutic agent for RMS cells expressing wild-type p53 protein.     

JNJ-7706621对人乳腺癌细胞周期及凋亡的影响

  目的   探讨周期素依赖性蛋白激酶（cyclin-dependent kinases,CDKs）抑制剂JNJ-7706621对人乳腺癌细胞周期及凋亡的影响。  方法  常规培养乳腺癌T47D、MD-MB-231细胞,MTT法检测不同浓度JNJ-7706621（0、1、2、4 μM）对乳腺癌细胞增殖的影响;流式细胞术检测JNJ-7706621对细胞周期的影响,Annexin Ⅴ-FITC/PI双染法检测JNJ-7706621对细胞凋亡的影响;West. ern blot检测JNJ-7706621对凋亡及周期相关蛋白表达的影响。  结果  JNJ-7706621能够抑制乳腺癌T47D、MDA-MB-231细胞增殖,具有浓度和时间依赖性;流式细胞术显示,JNJ-7706621可将T47D、MDA-MB-231细胞的周期阻滞于G2/M期,具有浓度依赖性,与对照组相比差异有统计学意义（P < 0.05）;Annexin Ⅴ-FITC/PI双染法示随着JNJ-7706621浓度增加或作用时间的延长,T47D、MDA-MB-231细胞凋亡所占比例逐渐增加,与对照组相比,差异有统计学意义（P < 0.05）;Western blot结果显示,随JNJ-7706621浓度增加,Bcl-2的表达明显下调,Caspase3、PARP的剪切明显增高,而p53的表达无明显变化;周期相关蛋白CDK1的表达无明显变化;CDK1在Thr161位点的磷酸化水平降低,而在Tyr15位点的磷酸化水平增高;  结论   在体外条件下,JNJ-7706621可通过阻滞细胞周期与诱导细胞发生凋亡的方式,有效抑制乳腺癌细胞增殖,可能与抑制CDK1在Thr161位点的磷酸化水平有关。      

1,4-苯醌致K562细胞线粒体功能障碍与凋亡作用

目的：研究1,4-苯醌对人慢性髓系白血病细胞（K562细胞）线粒体功能及凋亡的影响。方法：分别用终浓度为0、10、20 μmol/L的1,4-苯醌处理K562细胞24 h,用CCK-8法检测细胞活力,通过流式细胞仪检测活性氧（ROS）生成量;用线粒体膜电位、三磷酸腺苷（ATP） 生成量来评价线粒体功能;用PI-Annexin V双染法检测细胞的凋亡,采用分光光度法检测caspase-3酶的活性。结果：与对照组比较,1,4-苯醌10和20 μmol/L染毒组K562细胞相对增殖率均降低（P均<0.05）;随着1,4-苯醌浓度的增加,ROS生成量逐渐上升、线粒体膜电位和ATP生成量均逐渐降低、细胞的凋亡率逐渐增高,其中1,4-苯醌20 μmol/L染毒组与对照组间的差异均有统计学意义（P < 0.05或P < 0.01）;caspase-3活性逐渐升高,1,4-苯醌10和20 μmol/L染毒组与对照组相比差异均有统计学意义（P均<0.01）。结论：1,4-苯醌可以诱导K562细胞ROS升高,抑制K562细胞增殖,造成线粒体功能障碍,诱导细胞凋亡升高,提示线粒体障碍在1,4-苯醌诱导K562细胞凋亡的过程中发挥了重要作用。     

过表达PRRX1 通过p53 介导的线粒体凋亡途径诱导肝癌SMMC7721 细胞凋亡

[摘要] 目的：探讨配对相关同源框1 蛋白（PRRX1）过表达对肝癌SMMC7721 细胞凋亡的影响及其分子机制。方法：分别用慢病毒介导PRRX1 过表达载体（pGMLV-PRRX1）、空载质粒（Vector）感染人肝癌SMMC7721 细胞,用qPCR和WB实验检测慢病毒感染后细胞中PRRX1 mRNA和蛋白的表达变化,用CCK-8 法、Annexin-V FITC/PI 染色流式细胞术分别检测PRRX1 过表达对SMMC7721 细胞增殖、凋亡的影响,用线粒体膜电位检测试剂盒（JC-10 染色法）检测细胞线粒体膜电位变化,用caspase 活性检测试剂盒（分光光度法）测定细胞中caspase-8 和caspase-9 酶活性,用WB实验检测细胞中p53、Bcl-2、Bax、Fas、Cleaved-caspase-3以及线粒体和细胞质中细胞色素C（Cty C）蛋白的表达。结果：成功构建PRRX1 过表达的SMMC7721 细胞株,感染细胞中PRRX1 mRNA和蛋白的表达水平显著升高（均P<0.01）。与对照组和空载组比较,PRRX1 过表达组SMMC7721 细胞的增殖能力显著下降、细胞凋亡率显著增高、Cleaved-caspase-3 剪切水平显著升高、线粒体膜电位显著下降、线粒体中Cty C蛋白表达下调、胞质中Cty C蛋白表达上调以及caspase-9 酶活性升高（P<0.05 或P<0.01）,同时p53 和Bax 蛋白表达增加而Bcl-2 蛋白表达降低（均P<0.05）,但Fas 蛋白表达及caspase-8 酶活性无显著变化（均P>0.05）。结论: PRRX1 过表达可诱导肝癌SMMC7721 细胞凋亡,其机制可能与p53介导的线粒体凋亡途径被激活有关。     

三七皂苷R1通过线粒体相关通路促进白血病细胞株HL-60凋亡

目的:观察三七皂苷(notoginsenoside) R1对白血病细胞株HL-60凋亡的影响,探讨其可能的作用机制.方法:采用MTT法和流式细胞术(Annexin V-FITC双染法)分别检测三七皂苷R1(10、20、40及80 μmol/L)处理HL-60细胞12、24、36及48 h后HL-60细胞的凋亡情况,Western blotting检测HL-60细胞内Bcl-2、Bax及细胞色素C(cytochrome-C,Cyt C)蛋白的表达水平,JC-1染色法观察HL-60线粒体膜电位的变化.结果:MTT和流式细胞术检测显示三七皂苷R1浓度依赖性诱导HL-60细胞的凋亡,且细胞存活率随处理时间的增加而降低;与空白对照组相比,加入三七皂苷R1后细胞中Bcl-2蛋白表达显著减少[(0.45 ±0.03) vs (1.00 ±0.00),P＜0.05]、Bax蛋白表达显著增加[(1.72 ±0.08) vs (1.00±0.00),P＜0.05];Bcl-2/Bax比值减小[(0.21±0.01) vs (1.00±0.00),P＜0.05];线粒体膜电位降低[(0.56±0.09) vs (1.00±0.00),P＜0.05];胞质(cyto)中Cyt-C蛋白表达水平显著下降[(0.42±0.03) vs (1.00±0.00),P＜0.05].结论:三七皂苷R1可显著诱导HL-60细胞凋亡,其作用机制可能是通过线粒体通路促进细胞的凋亡;本实验可为三七皂苷R1用于临床治疗白血病提供实验依据.     

白藜芦醇通过线粒体凋亡通路调节肺癌H460细胞增殖与凋亡的研究

目的:探讨白藜芦醇(Resveratrol,Res)对肺癌H460细胞增殖与凋亡的影响并探讨其可能的机制.方法:分别用浓度为0、25、100、200 μmol/L的Res作用于H460细胞48 h后,采用CCK-8法检测Res对H460细胞增殖的影响;流式细胞术检测细胞凋亡率;JC-1试剂盒检测细胞内线粒体膜电位水平;ATP试剂盒检测细胞内ATP水平;Western blot法检测细胞内Bcl-2、Bax和Cytochrome c蛋白表达情况.结果:与对照组比较,CCK-8及流式结果显示Res可抑制H460细胞的增殖,并使其凋亡率升高(P＜0.01);JC-1及ATP检测结果显示Res可降低H460细胞内线粒体膜电位及ATP水平(P＜0.01),且在该浓度范围内呈剂量相关性;Western blot检测发现随着Res浓度的增加,Bcl-2蛋白的表达水平下降,而Bax和Cytochrome c蛋白的表达水平升高.结论:实验浓度的Res可抑制H460细胞的增殖并诱导其凋亡,这一作用的机制可能与Res激活了线粒体凋亡通路进而诱发H460细胞凋亡有关.     

Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells

Over the coming years, skin cancer could become a significant public health problem. Previous results indicate that ursolic acid (UA), a pentacyclic triterpene acid, has pleiotropic biologic activities such as antiinflammatory and antiproliferative activities on cancer cells. As UA represents a promising chemical entity for the protection of human skin, in agreement with tests done by the cosmetic industry, we investigated its effects on the M4Beu human melanoma cell line. In this report, we demonstrated for the first time that UA had a significant antiproliferative effect on M4Beu, associated with the induction of an apoptotic process, characterized by caspase-3 activation, the downstream central effector of apoptosis. We demonstrated that UA-induced apoptosis was dependent on the mitochondrial intrinsic pathway, as shown by transmembrane potential collapse (DeltaPsim) and by alteration of the Bax-Bcl-2 balance, with a concomitant increase in Bax expression and decrease in Bcl-2 expression. We also showed that UA-induced DeltaPsim was associated with apoptosis-inducing factor leakage from mitochondria. Taken together, our results suggest that UA-induced apoptosis on M4Beu cells is accomplished via triggering of mitochondrial pathway. In conclusion, UA could be an encouraging compound in the treatment or prevention of skin cancer and may represent a new promising anticancer agent in the treatment of melanoma.     

The endoplasmic reticulum mitochondrial calcium cross talk is downregulated in malignant pleural mesothelioma cells and plays a critical role in apoptosis inhibition

The failure of apoptosis may contribute to the formation of cancer and to its resistance to therapy. Malignant pleural mesothelioma (MPM) is an aggressive tumor that responds poorly to standard chemo- and radio-therapies. Several studies have demonstrated that a plethora of oncogenes and tumor suppressors contribute to MPM onset/progression. Importantly, most of these genes are involved in the regulation of calcium (Ca²⁺)-handling. Cellular Ca²⁺ signaling is an important regulator of many physiological processes, and it has been widely reported to participate in the regulation of apoptotic cell death in cancer cells and tissues. However, in MPM the role of cellular Ca²⁺ has been poorly investigated. Therefore, we examined whether Ca²⁺ is involved in MPM. We found that mesothelioma cell lines and short-term cultures obtained from MPM-affected patients exhibited a critical dysregulation in Ca²⁺ signaling. We determined that this characteristic was associated with resistance to apoptotic stimuli and that correction of intracellular Ca²⁺ signaling resulted in the rescue of efficient apoptotic responses. In addition, we discovered that mitochondrial Ca²⁺-uptake plays a pivotal role as an inducer of apoptosis in MPM. Altogether, these findings suggest the identification of new MPM markers, which in turn could be potential targets for new therapeutic approaches.     

阻断STAT3信号通路对恶性淋巴瘤细胞凋亡的影响

目的探讨阻断信号转导与转录激活因子3(STAT3)信号通路对恶性淋巴瘤细胞凋亡的影响。方法用0、200、400、800、1600 nmol/L的STAT3信号通路特异性阻断剂JSI-124作用于恶性淋巴瘤细胞株Raji,噻唑蓝(MTT)法检测细胞增殖水平,计算半数抑制浓度。用半数抑制浓度的JSI-124作用于Raji细胞,流式细胞术检测细胞凋亡和细胞周期,Western blot法检测细胞周期蛋白D1(cyclin D1)、STAT3、磷酸化的STAT3(p-STAT3)、活化的半胱氨酸天冬氨酸蛋白水解酶3(cleaved caspase 3)的蛋白相对表达水平。结果随着JSI-124作用浓度的升高,细胞存活率逐渐下降。计算半数抑制浓度为(615.62±52.98)nmol/L,故后续选用600 nm/L的JSI-124作用于恶性淋巴瘤细胞。600 nm/L作用组细胞凋亡率明显高于0 nm/L作用组(P﹤0.01)。600 nm/L作用组G0/G1期细胞所占比例明显低于0 nm/L作用组,G2/M期细胞所占比例明显高于0 nm/L作用组,差异均有统计学意义(P﹤0.01);两组S期细胞所占比例比较,差异无统计学意义(P﹥0.05)。600 nm/L作用组p-STAT3、cyclin D1蛋白相对表达水平明显低于0 nm/L作用组,cleaved caspase 3蛋白相对表达水平明显高于0 nm/L作用组,差异均有统计学意义(P﹤0.01)。结论阻断STAT3信号通路可促进恶性淋巴瘤细胞凋亡,抑制细胞增殖,将细胞周期阻滞在G2/M期。