Growth inhibition and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780

Curcumin,aphenolicpigmentextractedfrom curcuma,isthemajoreffectivecomponentofthat traditionalChinesemedicineandusedasadietarycoloringagentandanaturalcondiment.Recentre searchreportshaveshownthatcurcumincouldse lectivelyinhibittheproliferationandinducetheap optosisofmanytumorcells,buttheinvolvedmech anismsstillunclear(1,2).Thephenomenonthatcurcu mininducesapoptosisoftumorcellscouldbeseen innumerouscancers,however,theeffectsofcurcu minonhumanovariancancercellshavenotbeenre portedyet.Inthisresear…     

HDAC1 expression and effect of curcumin on proliferation of Raji cells

Summary Histone deacetylase (HDAC₁) has a high expression in many cancer cells and curcumin can inhibit the growth of cancer cells. This paper was designed to investigate the expression of HDAC₁ of Raji cells and the effect of curcumin on their proliferation and apoptosis. Raji cells were treated with 3. 125–50 μmol/L curcumin for 8–48 h and the growth inhibition rates of Raji cells were measured by MTT. The expression of HDAC₁ on Raji cells were examined by mRNA, Western blot at 24 h various concertrations (1.6–50 μmol/L). Curcumin could selectively inhibit the proliferation of Raji cells in a dose and time dependent manner, with the inhibition rate being 52.47%–82.18% (P<0.01). The up-regulation of HDAC₁ expression was observed within 24 h after the treatment with curcumin as shown by RT-PCR and Western blot. With the increase of concentration, the expression was down-regulated in a dose dependent manner. It is concluded that the expression of HDAC₁ plays an important role in the proliferation and apoptosis of Raji cells and curcumin can inhibit the growth of Raji cells at various concentrations and promote the apoptosis of Raji cells. WU Qing, female, born in 1970, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30271672).     

牡荆苷对人食管癌EC-109细胞生长及凋亡的影响

目的 观察金莲花中提取的牡荆苷体外对人食管癌EC-109细胞生长及诱导EC-109细胞凋亡的作用,探讨p53及bcl-2蛋白表达在牡荆苷抗肿瘤细胞机制中的作用。方法 用不同浓度牡荆苷作用于对数生长期的EC-109细胞,通过CCK-8法检测其对EC-109细胞体外生长、增殖的抑制作用;Hoechst33258荧光染色观察细胞形态的变化;琼脂糖凝胶电泳检测DNA 条带;AnnexinV-FITC/PI双标法流式细胞术观察牡荆苷对EC-109细胞凋亡的诱导作用;流式细胞术检测EC-109细胞中抑癌基因p53和促癌基因bcl-2蛋白的表达。结果 牡荆苷对EC-109细胞生长、增殖具有明显的抑制作用,能够诱导EC-109细胞凋亡,且作用与牡荆苷浓度、作用时间呈正相关,可上调p53蛋白表达,下调bcl-2蛋白表达。结论 牡荆苷可能通过上调p53蛋白和下调bcl-2蛋白的表达,促进EC-109细胞凋亡,抑制EC-109细胞生长。     

Growth-inhibiting and apoptosis-inducing effects of Tanshinone II A on human gastric carcinoma cells

Summary To explore the effects of Tanshinone II A on the proliferation, apoptosis and gene expression of p53 and bcl-2 in human gastric carcinoma MKN-45 cells. Cell count and MTT assay were used to study the proliferation-inhibiting effect of Tanshinone II A on MKN-45 cells. The effect of Tanshinone II A on the cell cycle and apoptosis of MKN-45 cells were examined by propidium iodide (PI) staining and flow cytometry. Semi-quantitative RT-PCR was used to further verify the expression of p53 and bcl-2 gene after exposure to Tanshinone A in MKN-45 cells. The results showed that Tanshinone A significantly inhibited the growth and proliferation of MKN-45 cells in a dose-and time-dependent manner (P<0.05). Tanshinone A arrested MKN-45 cells in G₂/M phase which led to an obvious accumulation of G₂/M phase cells while decreased number of G₀/G₁ phase cells. This resulted in apoptosis of MKN-45 cells and the apoptosis rate was as high as 43.91% after treatment with 2.0 μg/mL Tanshinone II A for 96 h. It was also found that Tanshinone II A up-regulated expression of p53 gene and down-regulated expression of bcl-2 gene. The cytostatic and antiproliferative effect of Tanshinone II A makes it a promising anticancer agent for the treatment of gastric carcinoma.     

Preparation of curcumin prodrugs and theirin vitro anti-tumor activities

Summary The curcumin prodrugs, which could be selectively activated in tumor cells, were prepared to establish a basis for the targeted chemotherapy for cancer. On the basis of the molecular structure of curcumin, the N-maleoyl-L-valine-curcumin (NVC), N-maleoyl- glycine-curcumin (NGC) were chemically synthesized and identified by IR and NMR spectroscopy. After treatment with these two prodrugs for 6–24 h, the rates of growth inhibition on human bladder cancer EJ cells and renal tubular epithelial (HKC) cells were detected by MTT colorimetry. Our results showed that after the treatment with 20 μmol/L–40 μmol/L NVC and NGC for 6–24 h, the growth inhibitory effects on EJ cells were 6.71%–65.13% (P<0.05), 10.96%–73.01% (P<0.05), respectively, in both dose- and time-dependent manners. When compared with the curcumin of same concentrations, the growth inhibitory effects of these two prodrugs on HKC cells were significantly decreased (P<0.01). It is concluded that activation of curcumin prodrugs via hydrolysis functions of cellular esterase could inhibit the growth activities of tumor cells, and reduce the side effects on normal diploid cells. This provided a novel strategy for further exploration of tumor-targeted chemotherapeutic drugs. LU Peng, male, born in 1977, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30200284).     

姜黄素诱导宫颈癌Hela细胞凋亡及其作用机制的研究

目的:研究姜黄素在体外诱导宫颈癌H eL a细胞凋亡及其作用机制。方法:3H-脱氧胸苷掺入法检测细胞增殖,流式细胞仪检测细胞凋亡,RT-PCR检测HPV E 6的mRNA水平表达;W estern b lot检测细胞凋亡相关基因bcl-2、bax、P 53和P 21ras的蛋白水平表达。结果:姜黄素对H eL a细胞生长有抑制作用,并呈剂量依赖性;流式细胞仪和荧光双染法结果提示姜黄素可诱导H eL a细胞凋亡;RT-PCR提示姜黄素作用H eL a细胞后HPV E 6的mRNA水平表达逐渐降低;W estern b lot结果提示姜黄素能上调B ax、P 53和P 21ras的蛋白水平表达,而B cl-2的表达无明显影响。结论:姜黄素通过抑制HPV E 6的表达,恢复P 53的功能,引起细胞凋亡,起到杀伤肿瘤的作用。     

Construction of Three-dimensional In Vitro Culture Model of Ovarian Carcinoma and the Study of Its Multicellular Drug Resistance

The resistance to chemotherapeutic drugs is one of the main cause of failure of the treatment of solid tumors. In ovarian carcinoma, both single cell and multicellular aggregates are found in patients’ ascitic fluid[1]. As our previous study showed that, in contrast to monolayer cell (MC) cultured in vitro, the solid tumors had similar bio-logical behaviors as compared with multicellular aggre-gates and displayed resistant to a wide variety of cyto-toxic agents[2, 3]. The examination of mult…     

Inhibitory Effect of Curcumin on Proliferation of Human Pterygium Fibroblasts

In order to investigate the effect of curcumin on proliferation and apoptosis of human pterygium fibroblasts (HPF) in culture and search for a new method to prevent the recurrence after pterygium surgery, HPF was incubated with 0-160 μmol/L curcumin for 24-96 h. The MTT method was used to assay the biologic activities of curcumin at different time points and different doses. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by immunohistochemistry. The cell cycle distribution was detected by flow cytometry (FCM). Admini- stration of 20-80 μmol/L curcumin for 24-72 h could significantly inhibit HPF proliferation in a dose- and time-dependent manner (P<0.05). After treatment with curcumin at different concentrations of 20, 40, 80 and 160 μmol/L for 24 h, FCM revealed there was a significant sub-G1 peak at each concentration. The number of HPF in G0/G1 phase was increased, while in S phase, it was decreased (P<0.05). At the concentration of 20-80 μmol/L, curcumin, in a dose-dependent manner (P<0.05), could inhibit the expression of PCNA in HPF. It was suggesterd that curcumin could significantly in- hibit the proliferation of HPF, make HPF arrest in G0/G1 phase and induce the apoptosis of HPF in a dose- and time-dependent manner.     

Difference in Expression of Bcl-2 and Bcl-xl Genes in Cisplatin- sensitive and Cisplatin-resistant Human in Ovarian Cancer Cell Lines

Apoptosisisthemainmechanismofcelldeathandisregulatedbyalargenumberofgenes.Amongthem ,theBcl 2familiesareconsideredtoplayakeyroleintheexecutionorpreventionofapoptosisinducedbygenotoxicstressthroughthealterationofapoptoticsignals.Epithe lialovariancancerist…     

Dracorhodin perchlorate suppresses proliferation and induces apoptosis in human prostate cancer cell line PC-3

The growth inhibition and pro-apoptosis effects of dracorhodin perchlorate on human prostate cancer PC-3 cell line were examined. After administration of 10-80 μmol/L dracorhodin perchlorate for 12-48 h, cell viability of PC-3 cells was measured by MTT colorimetry. Cell proliferation ability was detected by colony formation assay. Cellular apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent staining, and flow cytometry (FCM) with annexin Ⅴ-FITC/propidium iodide dual staining. The results showed that dracorhodin perchlorate inhibited the growth of PC-3 in a dose- and time-dependent manner. IC50 of dracorhodin perchlorate on PC-3 cells at 24 h was 40.18 μmol/L. Cell clone formation rate was decreased by 86% after treatment with 20 μmol/L of dracorhodin perchlorate. Some cells presented the characteristic apoptotic changes. The cellular apoptotic rates induced by 10-40 μmol/L dracorhodin perchlorate for 24 h were 8.43% to 47.71% respectively. It was concluded that dracorhodin perchlorate significantly inhibited the growth of PC-3 cells by suppressing proliferation and inducing apoptosis of the cells.     

p53突变型胃癌细胞株细胞凋亡及基因调控机制的研究

目的研究顺铂作用于p53野生型AGS、p53突变型MGC-803、SGC-7901 3种胃癌细胞株后，引起细胞凋亡及p53下游基因表达的变化，探讨其发生机制。方法采用流式细胞技术(FCM)检测不同浓度顺铂（0、1、2μg/mL）作用24h后AGS、MGC-803、SGC-7901 3种胃癌细胞株，细胞周期和凋亡率的变化；RT-PCR法检测上述不同浓度顺铂分别作用于3种胃癌细胞株，p53下游基因p21、bax、puma、bcl-2、bcl-xl表达的变化。结果FCM显示，顺铂可引起3种细胞细胞周期发生改变：S+G2期比例均明显升高，同时3种细胞的凋亡率均明显增多（P<0.01）；RT-PCR结果显示，AGS细胞的p53、bax、p21表达均随顺铂剂量的增加而增加（P<0.05），puma变化不明显（P>0.05）；bcl-2、bcl-xl的表达随顺铂剂量的增加而降低(P<0.05)。MGC-803、SGC-7901细胞的bcl-2、bcl-xl表达均随顺铂剂量的增加而降低(P<0.05)，而bax、p21、puma的表达变化不明显。结论在p53突变细胞株中，DNA损伤可诱导细胞周期阻滞和细胞凋亡，凋亡调控因子的表达也发生改变。其机制可能与p53突变的具体位点有关，亦可能与p53非依赖性途径的信号传递有关。     

姜黄素对人乳腺癌MCF-7细胞增殖抑制作用及机制研究

目的:探讨姜黄素对人乳腺癌MCF-7细胞增殖抑制、诱导凋亡作用及其分子作用机制。方法:MTT法检测姜黄素对MCF-7细胞的增殖抑制作用;流式细胞术PI单染法检测细胞周期;Annexin V/PI双染法检测细胞凋亡;RT-PCR法检测凋亡相关基因Bcl-2和Bax的mRNA表达水平。结果:姜黄素对MCF-7细胞生长有明显抑制作用,并呈剂量、时间依赖性;FCM结果显示姜黄素能使MCF-7细胞阻滞在G1/S期;Annexin V/PI双染法验证姜黄素可以诱导细胞凋亡;RT-PCR结果显示Bax mRNA水平明显上调,而Bcl-2的mRNA表达水平降低。结论:姜黄素对人乳腺癌MCF-7细胞增殖具有显著的抑制作用并可诱导细胞凋亡,其作用机制可能与其上调Bax基因表达水平的同时,下调Bcl-2基因表达水平,从而诱导细胞凋亡有关。     

RNAi抑制Aurora B激酶对卵巢癌A2780细胞增殖和周期的影响

目的研究RNAi抑制Aurora B激酶对卵巢癌A2780细胞增殖和周期的影响。方法采用RNAi方法,将AURKB RNAi转入A2780细胞,RT-PCR、Western blot检测Aurora B mRNA和蛋白的表达,MTT检测A2780细胞增殖情况,流式细胞术检测A2780细胞周期改变。结果①通过RT-PCR、Western blot检测到人类5种卵巢癌细胞株A2780、SKOV3、CAOV3、A2780/taxol及AD6中均明显表达Aurora B mRNA和蛋白;②A2780细胞转染AURKBRNAi后,Aurora B mRNA和蛋白的表达明显下降,转染浓度至200 pmol/L RNAi 48 h后,Aurora B mRNA和蛋白的表达被完全抑制;③MTT检测和锥虫蓝染色结果显示,于转染200 pmol/L AURKB RNAi第4天开始,A2780细胞增殖明显受到抑制,吸光度值与空白对照组和阴性对照组相比,差异有统计学意义(P<0.05);④Flow cytometry检测显示,转染200 pmol/L AURKB RNAi 24 h,大量A2780细胞进入有丝分裂期,并形成多倍体细胞,于72 h多倍体细胞比例达到峰值20.54%,转染后96 h转染组细胞凋亡率(11.67%)显著高于对照组(0.57%);⑤Hoechst 33258染色观察细胞凋亡结果显示,与A2780对照和阴性对照相比,转染200 pmol/L浓度AURKB RNAi 96 h后A2780细胞凋亡明显增多。结论 Aurora B的抑制使A2780细胞增殖受到明显抑制;导致多倍体形成,使细胞基因组严重不稳定,最终导致细胞凋亡,Aurora B可能是卵巢癌治疗的有效分子靶点。     

Apoptosis inducing effects of arsenic trioxide on human bladder cancer cell line BIU-87

Objective　To explore the apoptosis inducing effects of arsenic trioxide (As2O3) on human bladder cancer cells and elucidate possible mechanisms. Methods　After treatment with As2O3, the growth inhibition rates of human bladder cancer cell line BIU-87 were studied by MTT and cell counts methods. DNA synthesis rates were detected by 3　H-TdR assay. The morphological changes of cancer cells were observed by light and electronic microscopy and cell apoptosis rates were detected by TdT-mediated dUTP nick end labeling (TUNEL). bcl-2 gene expression of BIU-87 cells was observed by strept avidin-biotin complex (SABC) immunohistochemical method. Results　As2O3 could effectively inhibit the growth of BIU-87 (P&lt;0.05), which were time and concentration dependent. The inhibition rate of 4.0?μmol/L As2O3 for DNA synthesis of cancer cells was 55.64% (P&lt;0.01). Partial cancer cells presented the characteristic morphological changes of apoptosis which depended on the time of exposure to drug (P&lt;0.05). bcl-2 expression of BIU-87 cells was decreased significantly (P&lt;0.05). Conclusion　As2O3 can significantly induce apoptosis in bladder cancer cells by down-regulating the expression of the bcl-2 gene and inhibiting DNA synthesis. This provides a potentially effective method for prevention and cure of human bladder cancer.     

生长抑素与大肠癌细胞凋亡及调控基因bcl-2、bax及p53关系的研究

目的　探讨生长抑素 (SS)与大肠癌细胞凋亡指数 (AI)和凋亡调控基因p5 3、bcl 2、bax的关系。方法　采用免疫组织化学SABC法和分子生物学细胞原位凋亡检测技术中的TUNEL法 ,检测 6 2例大肠癌组织中SS、p5 3、bcl 2、bax及细胞凋亡的表达情况。结果　在大肠癌组织SS高表达组、中表达组的AI明显高于SS低表达组 (P <0 .0 1) ;p5 3、bcl 2、bax阳性表达率在SS低表达组、中表达组、高表达组三组间相比较存在着明显差别 (P <0 .0 5 ) ,其中bax在SS高表达组、中表达组的阳性表达率明显高于低表达组 (P <0 .0 5 ) ;bcl 2与其相反。p5 3在SS高表达组的阳性表达率明显低于低表达组 (P <0 .0 5 ) ;而p5 3在SS中表达组表达的阳性表达率低于低表达组 ,但其统计无明显差别 (P >0 .0 5 )。结论　生长抑素对大肠癌细胞凋亡的调控可能与 p5 3、bcl 2、bax基因的异常表达有关     

外源性硫化氢通过ATP敏感钾通道诱导人子宫肌瘤细胞凋亡

目的探讨ATP敏感钾通道（K_ATP）在外源性硫化氢（H₂S）诱导人子宫肌瘤细胞凋亡中的作用。方法用硫氢化钠(NaHS)作为H₂S的供体处理原代培养的人子宫肌瘤细胞,K_ATP通道抑制剂格列本脲(Gly)预处理细胞。实验分为对照组、不同浓度NaHS组、ATP敏感性钾通道（K_ATP）抑制剂组和Gly + NaHS组。用流式细胞术检测细胞凋亡率,采用实时定量PCR和Western blot分别检测p53和bcl-2 mRNA和蛋白的表达。结果与对照组比较,NaHS（10^-4 和10^-3 mol/L）处理人子宫肌瘤细胞24和48 h后以剂量和时间依赖的方式增加了细胞的凋亡率（均P<0.05）。与对照组比较,NaHS（10^-4 mol/L）处理人子宫肌瘤细胞24 h后p53的表达显著增加（P＜0.05）,而bcl-2表达显著降低（P＜0.05）。Gly没有影响人子宫肌瘤细胞的凋亡率、p53和bcl-2的表达,但部分地拮抗NaHS的致凋亡作用以及NaHS诱导的子宫肌瘤细胞中p53表达的上调和bcl-2表达的下调（均P<0.05）。结论外源性H₂S诱导子宫肌瘤细胞凋亡,其机制与H₂S开放K_ATP通道有关。     

p53、bcl—2基因在喉鳞癌中的蛋白表达及其临床意义

目的 探讨细胞凋亡相关基因p53,bcl-2与喉鳞癌的关系。方法 应用免疫组化ABC法对60例喉鳞癌和8例声带息肉标本中的p53,bcl-2基因的蛋白表达进行检测。结果 60例喉鳞癌中p53,bcl-2基因蛋白的表达阳性率均较高,分别为61．7％（37／60）和43．3％（26／60）,并且其阳性表达率与喉鳞癌的病理分化程度和预淋巴结转移有关（P＜0．05）。而与喉鳞癌的临床分期和分型无关（P＞0．05）。8例声带息肉标本的p53,bcl-2基因蛋白的表达均为阴性。结论 本实验从蛋白水平上证实了喉鳞癌与p53,bcl-2基因的关系,提示此两种基因可能参与了喉鳞癌细胞凋亡的调节,对喉鳞癌的发生可能起着重要的作用。     

P53 Gene Mutation and Expression of MDM2, P53, P16 Protein and their Relationship in Human Glioma

To investigate the effect of P53 protein accumulation and p53 gene mutation in the pathogenesis of glioma and to study the role of MDM2, P53 and P16 protein in glioma formation and progression and their relationship with each other, LSAB immunohistochemical staining method and non-isotopic PCR-SSCP techniques were used to detect the expression of MDM2, P53 and P16 protein and p53 gene mutation in 48 cases of gliomas. The results showed that the positive expression rate of MDM2, P53 and the negative rate of P16 was 22.9 %, 41.7 % and 60.4 %, respectively. The latter two in high grade （grade Ⅲ , Ⅳ） gliomas had a significantly higher rate than in the low grade （grade Ⅱ ） gliomas. Moreover, the co-expression of MDM2 and P53 protein was confirmed in only 1 of 48 cases. No significant difference was found in the rate of the expression of MDM2 between high grade and low grade gliomas （P〉0.1） . PCR-SSCP results showed that mutation of 5 --8 exons of p53 gene was detected in 17 out of 48 cases （35.42 %） . Mutation was detected in 16 of 20 cases of positive p53 expression, and another one was detected in 28 cases of negative expression cases. The correlation between p53 mutation and p53 immunopositivity was observed in 89.6 % of the cases. P53 gene mutation and the level of MDM2, P53 and PI6 protein were not related to age, gender of the patients, tumor location and size. It is concluded that the mutation of p53 and deletion of p16 might play important roles in the tumorigenesis of gliomas and it was significantly associated with the grade of tumor differentiation. P53 protein accumulation can indirectly reflect p53 mutation. MDM2 amplification and overexpression might be an early event in the growth of human gliomas.     

Study on apoptosis and expression of P53, Bcl-2, bax in cardiac myocytys of congestive heart failure induced by ventricular pacing

A characteristic feature of heart failure is theprogressive worsening of ventricular function overmonths or years despite the absence of clinically ap-parent intercurrent advance events.The mechanismresponsible for this hemodynamic deterioration arenot known.Recently,study of electrograph showsloss of myocytes exist in failure heart of human anddogs[1] .Another study revealed thatlossof myocyteswas thoughtto be apoptotic[2— 5] .In this study,we used Td T- mediatedbi-otinylated- d UTP nick e…     

The relationship between p38MAPK and apoptosis during paclitaxel resistance of ovarian cancer cells

Summary To investigate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and cell apoptosis during the paclitaxel resistance of ovarian carcinoma cell lines, flow cytometry (FCM) and PI staining were employed to determine the effect of p38MAPK inhibitor SB203580 on the apoptosis of A2780/Taxol cells, a drug-resistant human ovarian carcinoma cell line. p38MAPK protein expression in SB203580-treated cells was immunochemically measured. The 50% inhibition concentration (IC₅₀) of paclitaxel on A2780/Taxol cells was determined by MTT assay. MDR-1 mRNA, and expression of p38MAPK and phospho-p53 protein were detected by RT-PCR and Western blotting, respectively. The apoptosis rate of A2780/Taxol cells was (19.7±1.04)% 24 h after SB203580 treatment. A significant difference in apoptosis rate was found among experiment group, control group and untreated group (P<0.05). The relative reversal rate of A2780/Taxol cells to paclitaxel was (57.18±2.01)%. As compared with the control group and the untreated group, p38MAPK protein and MDR-1 mRNA in SB203580-treated cells was substantially decreased. The expression of p53 protein was significantly increased. It is concluded that p38MAPK pathway is related to paclitaxel resistance of ovarian carcinoma, and blockade of this pathway can promote the apoptosis of the drug-resistant cells and reverse the drug-resistance. Moreover, p38MAPK-mediated apoptosis in paclitaxel-resistant ovarian carcinoma cells depends on the activation of p53. This project was supported by a grant from R&D program of Heilongjiang Province (No. GB05C402-11).