雷公藤红素对人前列腺癌细胞凋亡及SENP1基因表达的影响

目的：研究雷公藤红素对人前列腺癌（PCa）细胞生长、凋亡及SUMO特异蛋白酶1（SUMO-specificproteases 1,SENP1）基因表达的影响。方法：对前列腺癌细胞PC-3和LNCaP进行雷公藤红素处理,通过测定细胞生长曲线,荧光染色,荧光显微镜观察,流式细胞仪分析和实时定量PCR,检测雷公藤红素对前列腺癌细胞生长、凋亡及SENP1 mRNA表达水平的影响。结果：雷公藤红素能够显著抑制PCa细胞的生长,并且抑制作用呈剂量依赖性;雷公藤红素能够诱导PCa细胞凋亡,1μmol/L雷公藤红素处理24h诱导14.8%的PC-3细胞和23.2%的LNCaP细胞发生凋亡或死亡;雷公藤红素还能够降低PCa细胞中SENP1 mRNA水平,尤其是PC-3细胞。结论：雷公藤红素能够显著抑制PCa细胞的生长并诱导细胞凋亡,表明雷公藤红素具有抗前列腺癌作用,雷公藤红素能够降低PCa细胞中SENP1 mRNA水平,揭示雷公藤红素可能通过SENP1相关信号通路达到抗前列腺癌作用。     

雷公藤红素对人早幼粒细胞白血病NB4细胞增殖和凋亡的影响

目的:研究雷公藤红素对人早幼粒细胞白血病细胞系NB4的抑制作用和诱导凋亡作用以及对SU-MO依赖性特异泛素E3连接酶(RNF4)mRNA表达水平的影响.方法:用不同浓度的雷公藤红素处理NB4细胞,测定细胞生长曲线;CCK-8试剂盒检测细胞存活率;Annexin V/PI双染、荧光显微镜观察、流式细胞仪检测细胞凋亡情况;实时定量PCR检测RNF4 mRNA表达.结果:雷公藤红素能够显著抑制NB4细胞的增殖,雷公藤红素对NB4细胞处理48h的细胞半数抑制浓度为0.854±0.035 μmol/L;0.5 μmol/L和1μmol/L雷公藤红素处理NB4细胞48小时后均发生明显的细胞凋亡;0.1μmol/L雷公藤红素可使NB4细胞中的RNF4mRNA水平上调约60％.结论:雷公藤红素对急性早幼粒细胞白血病NB4细胞有明显的抗增殖和促凋亡作用,可能通过与RNF4相关的信号通路达到抗白血病作用,这为临床应用雷公藤红素治疗急性早幼粒细胞白血病提供了实验依据.     

雷公藤红素体外抑制人类急性髓系白血病细胞的实验研究

 目的　研究雷公藤红素体外对人类急性髓系白血病（AML）细胞的体外抑制作用。方法　应用四甲基偶氮唑蓝（MTT）法和流式细胞术（FCM）等方法研究雷公藤红素对人类AML细胞的影响。结果　MTT实验显示,与空白组比较,培养不同时间后不同浓度组雷公藤红素的细胞增殖抑制率均明显升高,差异有统计学意义（P＜0.01）。FCM检测结果显示,1 μmol／L以上浓度雷公藤红素作用不同时间,AML细胞均可出现凋亡现象,凋亡率明显高于不加药物的对照组（P＜0.01）。结论　雷公藤红素对AML有明显的抑制作用,其作用可能与其诱导细胞凋亡有关。     

雷公藤红素上调lncRNA MEG3表达抑制肝癌细胞增殖及促进细胞凋亡

目的:探讨MEG3在雷公藤红素诱导的肝癌HepG2细胞凋亡和细胞增殖抑制过程中的作用。方法:采用CCK-8法检测HepG2细胞增殖情况,流式细胞仪检测细胞凋亡情况,Western blot检测cleaved caspase-3、Bax和Bcl-2蛋白的表达,real-time PCR检测MEG3的表达。结果:雷公藤红素抑制HepG2细胞增殖、促进细胞凋亡并上调MEG3的表达;敲低MEG3的表达后显著逆转了雷公藤红素诱导的HepG2细胞增殖抑制和细胞凋亡。结论:雷公藤红素通过上调MEG3的表达抑制HepG2细胞增殖、促进细胞凋亡。     

雷公藤单体体外抑制胶质瘤细胞的实验研究

背景与目的：已有研究发现雷公藤具有抗肿瘤作用,本研究旨在探讨雷公藤单体对胶质瘤细胞的体外抑制作用。方法：通过MTT法测定3种雷公藤单体（甲素,红素和WilforolA)对胶质瘤细胞株SHG44,C6,U251的体外抑制作用;应用免疫组化法观察雷公藤甲素与雷公藤红素对SHG44胶质瘤细胞中Bax,Bcl-2蛋白表达的影响。结果：雷公藤二萜类单体雷公藤甲素对胶质瘤细胞有极显著的抑制作用;雷公藤三萜类单体中红素的抑制作用次之,两者均使SHG44细胞Bax表达增加,Bcl-2表达下降。结论：雷公藤甲素与雷公藤红素对胶质瘤细胞有明显的抗肿瘤作用,其作用与促进Bax表达,抑制Bcl-2表达,导致细胞凋亡有关。     

用RNAi沉默survivin基因表达对前列腺癌细胞系PC3的影响

目的：运用RNA干扰（RNA interference，RNAi）技术阻断前列腺癌细胞系PC3中survivin基因的表达，并研究survivin基因沉默后对PC3细胞产生的影响。方法：用真核转录载体pSilencer^TM3．1-H1 neo构建针对survivin基因的重组真核转录载体pSilencer3．1-SVVl、pSilencer3．1-SVV2和pSilencer3．1-SVV3，并用脂质体法转染前列腺癌细胞系PC3，通过RT—PCR、蛋白印迹实验检测survivin的表达变化，并用流式细胞术、MTT法检测转染后PC3细胞的凋亡、细胞周期、细胞生长速度、对顺铂的敏感性等方面的变化。结果：重组载体pSilencer3．1-SVV2和pSilencer3．1-SVV3有效地阻断了PC3细胞中survivin基因在mRNA和蛋白水平上的表达（P〈0．01）。重组载体转染PC3细胞后，与对照组相比，细胞的凋亡增加了10％～15％；细胞生长速度明显变慢，其细胞数在84h时与对照组相比减少约30％；G1期细胞增加了10％。G2期和S期细胞减少了5％以上。加入顺铂后，pSilencer3．1-SVV2、pSilencer3．1-SVV3重组质粒转染的PC3细胞的存活数下降了35％～45％，细胞凋亡则增加了10％-14％。结论：初步证明了survivin基因在前列腺癌细胞分化增殖、抗凋亡等方面所扮演的重要角色，为进一步阐明survivin基因与前列腺癌的关系以及以survivin基因为靶点的前列腺癌基因治疗研究奠定了基础。     

雷公藤单体体外抑制胶质瘤细胞的实验研究

目的 :研究雷公藤单体对胶质瘤细胞的体外抑制作用。方法 :通过 MTT法测定 3种雷公藤单体 (甲素、红素和 Wilforol A)对胶质瘤细胞株 SHG4 4、C6、U2 5 1的体外抑制作用 ;应用免疫组化法观察雷公藤甲素与雷公藤红素后 SHG4 4胶质瘤细胞 bax、bcl- 2蛋白表达的变化。结果 :雷公藤二萜类单体雷公藤甲素对胶质瘤细胞有极明显的抑制作用 ;雷公藤三萜类单体中红素的抑制作用次之。两者均使 SHG4 4细胞 bax表达增加 ,bcl- 2表达下降。结论 :雷公藤甲素与雷公藤红素对胶质瘤细胞有明显的抑制作用 ,其作用与促进 bax表达、抑制 bcl- 2表达 ,导致细胞凋亡有关。     

雷公藤多甙诱导HL-60细胞凋亡

目的：探讨雷公藤多甙对ＨＬ－６０细胞凋亡的作用。方法：应用细胞形态学检查,ＤＮＡ凝胶电泳及流式细胞仪分析检测。结果：ＨＬ－６０细胞加上雷公藤多甙１０ｍｇ／Ｌ孵育７２ｈ,流式细胞仪检测细胞凋亡率为２９．４％（与空白对照组比较,Ｐ〈０．０１）。电检查和光镜检查均可见细胞核固缩、碎裂等。ＤＮＡ电泳显示明显的梯状条带。结论：雷公藤多甙能诱导ＨＬ－６０细胞凋亡,提示雷公藤多甙可能具有抗白血病作用。     

白藜芦醇诱导白血病Molt-4细胞凋亡及对WAVE1基因表达的影响

 目的　探讨白藜芦醇诱导人类急性淋巴细胞白血病Molt-4细胞凋亡的作用机制。方法　噻唑蓝比色法（MTT）测定细胞生长抑制率;流式细胞术检测细胞周期分布及凋亡率;半定量反转录聚合酶链反应（RT-PCR）法检测WAVE1基因的表达。结果　MTT结果显示：12.5、25.0、50.0、100.0、200.0 μmol／L的白藜芦醇作用于Molt-4细胞24、48、72 h后,细胞的最大抑制率分别达到29.32 %、36.11 %、53.92 %、62.50 %、74.98 %,并呈时间-剂量依赖性（F＝33.614,P＜0.05）;流式细胞术检测结果显示：与对照组相比,50.0、100.0 μmol／L白藜芦醇作用于Molt-4细胞48 h后,S期细胞比例由42.2 %分别上升为68.6 %和78.1 %,细胞发生了S期阻滞（F＝19.453, P＜0.01）;PCR结果显示：50.0、100.0 μmol／L的白藜芦醇处理Molt-4细胞48 h后,WAVE1／GAPDH比值分别为0.356±0.03、0.382±0.05,与对照组 （0.586± 0.06）比较,差异有统计学意义（F＝8.950,P＜0.01）。结论　白藜芦醇可诱导Molt-4细胞发生凋亡,其作用机制可能与下调WAVE1基因表达有关。     

雷公藤内酯醇诱导肿瘤细胞凋亡的机制

雷公藤内酯醇对血液系统肿瘤和多种实体瘤细胞都有杀伤作用。其主要作用机制是通过抑制NF—κB反式激活作用、活化p53和caspases、阻断p21^WAF1/CIP1介导的生长阻抑及激活MAPK信号通路等多种途径来诱导肿瘤细胞凋亡。现对此加以综述，旨在为雷公藤内酯醇的临床抗肿瘤研究提供一些线索。     

DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis.     

Tumor‐suppressive microRNA‐145 induces growth arrest by targeting SENP1 in human prostate cancer cells

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer‐related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin‐like modifier (SUMO)‐specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA‐145 (miRNA‐145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3′‐untranslated region of SENP1 mRNA is a potential target of miR‐145. Here we found that low expression of miR‐145 was correlated with high expression of SENP1 in PCa cell line PC‐3. The transient introduction of miR‐145 caused cell cycle arrest in PC‐3 cells, and the opposite effect was observed when miR‐145 inhibitor was transfected. Further studies revealed that the SENP1 3′‐untranslated region was a regulative target of miR‐145 in vitro. MicroRNA‐145 also suppressed tumor formation in vivo in nude mice. Taken together, miR‐145 plays an important role in tumorigenesis of PCa through interfering SENP1.     

Down-regulation of SENP1 expression increases apoptosis of Burkitt lymphoma cells

Objective: To investigate the effect of down-regulation of Sentrin/SUMO-specific protease 1 (SENP1)expression on the apoptosis of human Burkitt lymphoma cells (Daudi cells) and potential mechanisms. Methods:Short hairpin RNA (shRNA) targeting SENP1 was designed and synthesized and then cloned into a lentiviralvector. A lentiviral packaging plasmid was used to transfect Daudi cells (sh-SENP1-Daudi group). Daudi cellswithout transfection (Daudi group) and Daudi cells transfected with blank plasmid (sh-NC-Daudi group) servedas control groups. Flow cytometry was performed to screen GFP positive cells and semiquantitative PCR andWestern blot assays were employed to detect the inference efficiency. The morphology of cells was observedunder a microscope before and after transfection. Fluorescence quantitative PCR and Western blot assays wereconducted to measure the mRNA and protein expression of apoptosis related molecules (caspase-3, 8 and 9). Aftertreatment with COCl2 for 24 h, the mRNA and protein expression of hypoxia inducible factor -1α (HIF-1α) wasdetermined. Results: Sequencing showed the expression vectors of shRNA targeting SENP1 to be successfullyconstructed. Following screening of GFP positive cells by FCM, semiqualitative PCR showed the interferenceefficiency was 79.2±0.026%. At 48 h after transfection, the Daudi cells became shrunken, had irregular edgesand presented apoptotic bodies. Western blot assay revealed increase in expression of caspase-3, 8 and 9 withprolongation of transfection (P<0.05). Following hypoxia treatment, mRNA expression of HIF-1α remainedunchanged in three groups (P>0.05) but the protein expression of HIF-1α markedly increased (P<0.05). However,in the sh-SENP1-Daudi group, the protein expression of HIF-1α remained unchanged Conclusion: SENP1-shRNAcan efficiently inhibit SENP1 expression in Daudi cells. SENP1 inhibition may promote cell apoptosis. Thesefindings suggest that SENP1 may serve as an important target in the gene therapy of Burkitts lymphoma.     

Downregulation of reticulocalbin‐1 differentially facilitates apoptosis and necroptosis in human prostate cancer cells

Reticulocalbin 1 (RCN1), an endoplasmic reticulum (ER)‐resident Ca²⁺‐binding protein, is dysregulated in cancers, but its pathophysiological roles are largely unclear. Here, we demonstrate that RCN1 is overexpressed in clinical prostate cancer (PCa) samples, associated with cyclin B, not cyclin D1 expression, compared to that of benign tissues in a Chinese Han population. Downregulation of endogenous RCN1 significantly suppresses PCa cell viability and arrests the cell cycles of DU145 and LNCaP cells at the S and G2/M phases, respectively. RCN1 depletion causes ER stress, which is evidenced by induction of GRP78, activation of PERK and phosphorylation of eIF2α in PCa cells. Remarkably, RCN1 loss triggers DU145 cell apoptosis in a caspase‐dependent manner but mainly causes necroptosis in LNCaP cells. An animal‐based analysis confirms that RCN1 depletion suppresses cell proliferation and promotes cell death. Further investigations reveal that RCN1 depletion leads to elevation of phosphatase and tensin homolog (PTEN) and inactivation of AKT in DU145 cells. Silencing of PTEN partially restores apoptotic cells upon RCN1 loss. In LNCaP cells, predominant activation of CaMKII is important for necroptosis in response to RCN1 depletion. Thus, RCN1 may promote cell survival and serve as a useful target for cancer therapy.     

siRNA-mediated silencing of Notch-1 enhances docetaxel induced mitotic arrest and apoptosis in prostate cancer cells

Purpose: Notch is an important signaling pathway that regulates cell fate, stem cell maintenance and theinitiation of differentiation in many tissues. It has been reported that activation of Notch-1 contributes totumorigenesis. However, whether Notch signaling might have a role in chemoresistance of prostate cancer isunclear. This study aimed to investigate the effects of Notch-1 silencing on the sensitivity of prostate cancercells to docetaxel treatment. Methods: siRNA against Notch-1 was transfected into PC-3 prostate cancer cells.Proliferation, apoptosis and cell cycle distribution were examined in the presence or absence of docetaxel byMTT and flow cytometry. Expression of p21waf1/cip1 and Akt as well as activation of Akt in PC-3 cells were detectedby Western blot and Real-time PCR. Results: Silencing of Notch-1 promoted docetaxel induced cell growthinhibition, apoptosis and cell cycle arrest in PC-3 cells. In addition, these effects were associated with increasedp21waf1/cip1 expression and decreased Akt expression and activation in PC-3 cells. Conclusion: Notch-1 promoteschemoresistance of prostate cancer and could be a potential therapeutic target.     

Regulation of apoptosis in prostate cancer

Transformation and malignant progression of prostate cancer is regulated by the inability of prostatic epithelial cells to undergo apoptosis rather than by increased cell proliferation. The basic apoptotic machinery of most prostate cancer cells is intact and the inability to undergo apoptosis is due to molecular alterations that result in failure to initiate or execute apoptotic pathways. This review discusses the role of anti-apoptotic proteins such as Bcl-2/Bcl_XL, NF-B, IGF, caveolin, and Akt, and pro-apoptotic molecules such as PTEN, p53, Bin1, TGF-, and Par-4 that can regulate progression of prostate cancer. In addition to highlighting the salient features of these molecules and their relevance in apoptosis, this review provides an appraisal of their therapeutic potential in prostate cancer. Molecular targeting of these proteins and/or their innate pro- or anti-apoptotic pathways, either singly or in combination, may be explored in conjunction with conventional and currently available experimental strategies for the treatment of both hormone-sensitive and hormone-resistant prostate cancer.     

microRNA-155在食管癌中的表达及其对食管癌细胞增殖凋亡的影响

目的 探讨miRNA-155在食管癌中的表达及其对食管癌EC9706细胞增殖及凋亡的影响.方法 采用TaqMan探针实时定量PCR(qRT-PCR)方法检测50例食管癌患者中新鲜食管癌组织和其相应癌旁正常组织中miRNA-155的表达情况;使用脂质体LipofectamineTM2000转染EC9706细胞上调miRNA-155的表达,利用四甲基偶氮唑盐比色法(MTT法)检测转染后细胞的增殖变化,流式细胞仪检测其细胞凋亡率的变化.结果 食管癌组织中miRNA-155的表达水平明显低于与其相应的癌旁正常组织(P=0.000);转染miRNA-155后,miRNA-155转染组EC9706细胞中miRNA-155的表达水平高于对照组和NC组(P﹤0.05);细胞的增殖明显受到抑制,且细胞的凋亡率明显升高.结论 miRNA-155在食管癌组织中呈低表达,上调其表达能够明显抑制食管癌EC9706细胞增殖和诱导其凋亡.