Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway

We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy.     

慢病毒介导的细胞分裂周期相关蛋白 2基因沉默对乳腺癌细胞增殖和凋亡的影响

目的 探讨应用慢病毒介导小干扰核糖核酸(siRNA)沉默细胞分裂周期相关蛋白2(CDCA2)基因对乳腺癌细胞增殖和凋亡的影响.方法 采用实时荧光定量逆转录聚合酶链反应(qRT-PCR)与蛋白质印迹法(Western blotting)法分别检测人正常乳腺上皮细胞株MCF-10A与乳腺癌细胞MCF-7、MDA-MB-231、BT549中CDCA2 mRNA及蛋白表达.构建慢病毒表达载体转染乳腺癌MCF-7细胞,实验分为对照组、Lv-NC组、Lv-siRNA-CDCA2组.采用噻唑蓝(MTT)实验及克隆形成实验检测沉默CDCA2表达对细胞增殖能力的影响;采用流式细胞仪检测沉默CDCA2表达对细胞凋亡能力的影响.Western blotting法检测细胞周期素D1(Cyclin D1)、CDK4、P21、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X(Bax)蛋白表达.采用裸鼠移植瘤实验观察Lv-siRNA-CDCA2对移植瘤体积及体质量的影响.结果 乳腺癌细胞MCF-7、MDA-MB-231、BT549中CDCA2 mRNA[(2.18±0.13)、(1.56±0.09)、(1.83±0.10)比(1.02±0.06)]及蛋白表达水平[(1.13±0.15)、(0.56±0.08)、(0.79±0.11)比(0.23±0.04)]与MCF-10A相比显著升高(P<0.05),乳腺癌MCF-7细胞中CDCA2的表达水平升高最为显著;与Lv-NC组相比,Lv-siRNA-CDCA2组MCF-7细胞增殖活性与克隆形成率显著降低(P<0.05),而细胞凋亡率显著升高(P<0.05),可促进P21、Bax表达(P<0.05),而抑制Cyclin D1、CDK4、Bcl-2表达(P<0.05);与Lv-NC组比较,Lv-siRNA-CDCA2组移植瘤体积和体质量均显著降低(P<0.05).结论 慢病毒介导的CDCA2基因沉默可乳腺癌细胞细胞增殖,诱导细胞凋亡,并抑制肿瘤生长.     

Bcl-2 siRNA induced apoptosis and increased sensitivity to 5-fluorouracil and HCPT in HepG2 cells

To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.     

Bcl-2 siRNA induced apoptosis and increased sensitivity to 5-fluorouracil and HCPT in HepG2 cells

To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.     

Inhibition of insulin-like growth factor 1 receptor signaling enhanced silibinin-induced activation of death receptor and mitochondrial apoptotic pathways in human breast cancer MCF-7 cells

Silibinin, which had been used as a hepatoprotectant, was shown to have anticancer activity. In this study we investigated the mechanisms of silibinin-induced apoptosis in human breast cancer MCF-7 cells. Expressions of Fas ligand (FasL), Fas-associated death domain protein (FADD), and Bax were significantly up-regulated in silibinin-treated cells, whilst silibinin induced a conspicuous translocation of Bax to mitochondria and release of cytochrome c to the cytosol. Therefore, both the extrinsic Fas death receptor and intrinsic mitochondrial death pathways played essential roles in silibinin-induced apoptosis. It was also found that silibinin markedly decreased protein expression of SIRT1, a mammalian homologue of yeast Sir2, which was proved to have a role in sequestering Bax away from mitochondria. Insulin-like growth factor 1 receptor (IGF-1R), a receptor tyrosine kinase with a crucial role in malignancy development, is expressed in most human primary breast carcinomas. Our results showed that silibinin-induced apoptosis was significantly reinforced by blocking IGF-1R signaling with tyrphostin AG1024, a specific inhibitor of IGF-1R autophosphorylation. Up-regulation of FADD, down-regulation of SIRT1 expression, and activation of the mitochondrial death pathway were apparently enhanced by AG1024 in the silibinin-treated MCF-7 cells.     

消瘤1号诱导MCF-7人乳腺癌细胞凋亡的研究

目的:研究中成药消瘤1号诱导MCF-7人乳腺癌细胞的凋亡作用,并通过分析其作用后MCF-7细胞Bax和Bcl-2蛋白表达的变化,探讨其诱导MCF-7细胞凋亡的分子机制。方法:采用细胞培养技术,用1、10、100、1000μg·mL-1的消瘤1号处理MCF-7细胞24h,PI染细胞,流式细胞仪测定细胞周期的变化,Western Blot法检测Bax和Bcl-2蛋白的表达。结果:流式细胞仪检测结果表明细胞阻滞在G0/G1期;Weston Blot法测定结果表明消瘤1号能够增加细胞中Bax蛋白表达,减少Bcl-2蛋白表达,且该2种蛋白的表达依赖于消瘤1号的浓度。结论:消瘤1号可通过调节Bax/Bcl-2蛋白表达的变化诱导MCF-7细胞凋亡。     

siRNA抑制人乳腺癌SK-BR-3细胞株VEGF基因表达的实验研究

目的研究siRNA(small interfering,RNA)对乳腺癌细胞SK-BR-3的VEGF基因表达的抑制作用,为RNAi技术在肿瘤生物治疗中的应用提供实验基础。方法体外合成一条针对VEGF基因的siRNA,使用脂质体转染的方法导入细胞,观察转染后乳腺癌细胞SK-BR-3的增殖变化,MTT法检测细胞存活率,RT-PCR检测转染后VEGFmRNA表达水平的变化,ELISA检测蛋白表达的下降效果。结果所设计的siRNA能有效抑制乳腺癌细胞的生长;降低了VEGFmRNA的表达;蛋白表达水平也显著降低。作为阴性对照的错义序列组siRNA则没有这种效果,不起作用。结论 siRNA可以有效抑制细胞株SK-BR-3中VEGF的表达,从而抑制细胞生长。应用RNA干扰技术可以有效抑制肿瘤细胞的增殖。     

siRNA沉默β-arrestin2促进肝星状细胞凋亡

目的研究小干扰RNA(small interfering RNA,siRNA)干扰β-arrestin2表达对大鼠肝星状细胞株(hepatic stellatecell T6,HSC-T6)凋亡的影响。方法将化学合成的siRNAβ-arrestin2以Lipofectamine包裹,转染HSC-T6细胞,设阴性对照和空白对照;采用逆转录-聚合酶链反应和Westernblot检测细胞β-arrestin2表达;采用Western blot检测细胞Bcl-2、Bax的表达;采用流式细胞仪检测细胞凋亡。结果转染siRNA的HSC-T6细胞β-arrestin2基因及蛋白表达水平明显下调,β-arrestin2 mRNA水平和蛋白表达水平比对照组分别下调了70%±1.76%和68.43%±2.88%(P<0.01);Bcl-2蛋白被抑制了32.58%±3.46%(P<0.01),而Bax表达增加38.00%±3.72%(P<0.01);转染β-arrestin2 siRNA的HSC-T6细胞凋亡率为37.5%,明显高于正常HSC对照组。结论 siRNAβ-arrestin2能高效抑制HSC-T6细胞β-ar-restin2表达明显减少Bcl-2的表达,增加Bax的表达,从而促进HSC-T6凋亡,具有预防及治疗肝纤维化的潜力。     

Downregulation of MDM2 by small interference RNA induces apoptosis and sensitizes MCF-7 breast cells to resveratrol

Recent studies have demonstrated the mouse double minute gene (MDM2), a main oncogene, as a novel and interesting therapeutic target for cancer therapy. The aim of this study was to investigate the involvement of MDM2 in antiproliferative and antimetastatic effects of resveratrol in breast cancer cells. MCF-7 cells were transfected with siRNA against MDM2 and resveratrol. Proliferation and apoptosis were evaluated by MTT assay and cell death ELISA assay, respectively. MDM2, p53, Bax, Bcl-2, caspase-3, MMP-2, and MMP9 expressions were determined by qRT-PCR and Western blotting. Transfection with si-MDM2 significantly suppressed the expression of MDM2 expression, resulting in MCF-7 cell growth inhibition and spontaneous apoptosis. Pretreatment with Si-MDM2 synergically increased antiproliferation and antimetatstatic effects of resveratrol. No significant anticancer effects were detected with negative control siRNA treatment. Our findings suggest that silencing of MDM2 by specific siRNA effectively induce apoptosis and also enhanced anticancer effects of resveratrol. Therefore, siMDM2 may be a potent combination in breast therapy.     

姜黄素通过调控miR-7641/PTPN14分子轴抑制乳腺癌发展进程的分子机制研究

目的 探讨姜黄素通过调控miR-7641/PTPN14分子轴抑制乳腺癌发展进程的分子机制。方法 采用实时荧光定量PCR（qRT-PCR）检测乳腺癌患者癌组织及细胞系中miR-7641表达情况;使用Kaplan-Meier方法作乳腺癌患者生存曲线;采用不同浓度的姜黄素处理细胞,或转染miR-7641 mimic、Anti-miR-7641及pcDNA-PTPN14载体,采用qRT-PCR检测miR-7641表达情况,MTT实验及克隆形成实验检测细胞增殖能力,Transwell小室法检测细胞迁移及侵袭,western blotting检测Ki67、pcDNA、CyclinD1、Bax、Bcl-2、caspase-3、caspase-8蛋白表达水平,采用双荧光素酶报告基因系统检测miR-7641与PTPN14靶向调控关系。结果 与癌旁组织或乳腺正常上皮细胞比较,miR-7641在乳腺癌患者癌组织及乳腺癌细胞系中高表达（P<0.01、0.001）,且miR-7641能够明显促进乳腺癌细胞的增殖、迁移及侵袭（P<0.05、0.01、0.001）,并促进Ki67、pcDNA、CyclinD1、Bcl-2蛋白表达,抑制Bax、caspase-3、caspase-8蛋白表达;miR-7641与PTPN14 3''-UTR靶向结合,姜黄素通过miR-7641/PTPN14分子轴抑制乳腺癌细胞的增殖、迁移及侵袭（P<0.01、0.001）,并抑制Ki67、pcDNA、CyclinD1、Bax蛋白表达,促进Bcl-2、caspase-3、caspase-8蛋白表达。结论 姜黄素可通过下调miR-7641促进PTPN14表达,进而抑制乳腺癌细胞的增殖、迁移及侵袭。     

1-oxoeudesm-11(13)-ene-12,8α-lactone-induced apoptosis via ROS generation and mitochondria activation in MCF-7 cells

A novel eudesmane-type sesquiterpene compound, 1-oxoeudesm-11(13)-eno-12,8a-lactone (OEL), was isolated from Aster himalaicus. Its effect on apoptosis in human breast adenocarcinoma (MCF-7) cells was investigated. MTT assay showed that OEL substantially reduced the viability of KB, MCF-7, U87, A172, and MG-63 cells. MCF-7 cells were used to further evaluate the antitumor effects and anticancer mechanisms of OEL. OEL-induced apoptosis was characterized by chromatin condensation, formation of apoptotic bodies, and phosphatidylserine on extracellular surface; these effects were confirmed by DAPI nuclear staining and flow cytometry. Increased expression of Bax and deceased expression of Bcl-2 were also observed in OELtreated MCF-7 cells. Moreover, OEL induced the loss of mitochondria membrane potential, release of cytochrome C, activation of caspase-9, and generation of reactive oxygen species. These findings indicate that reactive oxygen species generation and mitochondria activation were involved in apoptosis induced by OEL in MCF-7 cells. The results from our study demonstrated that OEL may be a promising pro-apoptotic compound that could be used to develop novel anticancer drugs.     

Methylanthraquinone from Hedyotis diffusa WILLD induces Ca2+-mediated apoptosis in human breast cancer cells

Methylanthraquinone from Hedyotis diffusa WILLD exhibited potent anticancer activity in many kinds of cancer cells. However, the exact mechanism and signaling pathway involved in methylanthraquinone-induced apoptosis have not been fully elucidated. Therefore, we explored the mechanisms of methylanthraquinone-mediated apoptosis in MCF-7 human breast cancer cells. When MCF-7 cells were co-incubated with methylanthraquinone, the percentage of apoptotic cell and S phase of cell cycle was markedly increased. In addition, a rise in intracellular calcium levels, phosphorylation of JNK and activation of calpain were found in MCF-7 cells after exposure to methylanthraquinone. With the methylanthraquinone-mediated reduction of mitochondrial membrane potential, cytochrome c was released from mitochondria to cytosol. Moreover, methylanthraquinone strongly induced cleavage of caspase-4, caspase-9 and caspase-7 in MCF-7 cells. These results suggested that methylanthraquinone from Hedyotis diffusa WILLD induced MCF-7 cells apoptosis via Ca²⁺/calpain/caspase-4 pathway.     

透骨草提取物通过Bcl-2和Bax蛋白诱导人乳腺癌细胞株MCF-7凋亡

目的探讨Bax和Bcl-2蛋白在透骨草提取物诱导人乳腺癌细胞株MCF-7细胞凋亡过程中的作用,为透骨草提取物治疗乳腺癌提供实验依据。方法采用吖啶橙染色方法透骨草提取物对MCF-7细胞形态的影响;采用流式细胞术检测透骨草提取物对MCF-7细胞凋亡率的影响;采用蛋白印迹法检测MCF-7细胞Bcl-2和Bax蛋白的表达。结果 30.5μg/ml透骨草提取物可诱导MCF-7细胞胞体皱缩,胞核固缩、呈新月状且边集,出现凋亡小体。30.5μg/ml透骨草提取物处理的MCF-7细胞凋亡率（22.3±1.2）%明显高于对照组（3.2±1.0）%（P〈0.05）。30.5μg/ml透骨草提取物可使MCF-7细胞Bcl-2蛋白表达明显低于对照组（P〈0.05）,而Bax蛋白表达明显高于对照组（P〈0.05）。结论透骨草提取物对MCF-7细胞有诱导凋亡作用,其作用机制可能与下调Bcl-2蛋白表达、上调Bax蛋白表达有关。     

Isoalantolactone induces apoptosis in human breast cancer cells via ROS-mediated mitochondrial pathway and downregulation of SIRT1

Isoalantolactone possessed various biological activities. However, whether it could treat breast cancer and its underlying mechanism remained largely unknown. This study was designed to evaluate the anticancer effects of isoalantolactone on breast cancer and explored the molecular mechanism. Two human breast cancer cell lines (MDA-MB-231 and MCF-7) and one normal breast cell line (MCF-10A) were applied. Our data suggested that isoalantolactone decreased breast cancer cell viability in a dose-dependent manner, but showed almost no toxicity to MCF-10A cells. The anticancer effects of isoalantolactone were related to the overexpression of reactive oxygen species. Isoalantolactone significantly induced breast cancer cell apoptosis by activating caspase cascade, cleaving poly (ADP-ribose) polymerase. Increase of Bax/Bcl-2 ratio, depolarization of mitochondrial membrane potential, release of cytochrome c from mitochondria to cytoplasm and cell cycle arrest at G2/M phase were associated to the apoptosis induction. Additionally, isoalantolactone increased the protein expression of p38 MAPK and JNK. The apoptosis-induction of isoalantolactone could be abrogated by co-treatment with SB203580 (inhibitor of p38 MAPK) or SP600125 (inhibitor of JNK). Furthermore, isoalantolactone induced breast cancer cells apoptosis in a caspase-independent pathway, which was downregulation of SIRT1. Therefore, isoalantolactone may serve as a chemotherapeutic agent for the treatment of human breast cancer.     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

Celastrol isolated from Tripterygium regelii induces apoptosis through both caspase-dependent and -independent pathways in human breast cancer cells

The aim of the present study was to evaluate the underlying apoptotic mechanisms of celastrol, a major biologically active component of Tripterygium regelii, in human breast adenocarcinoma MCF-7 cells. Celastrol was isolated from T. regelii chloroform extract by silica gel column chromatography, and its chemical structure was identified via ¹H NMR and ¹³C NMR. Celastrol significantly inhibited cell growth in dose- and time-dependent manners. Celastrol induced sub-G1 DNA accumulation, formation of apoptotic bodies, nuclear condensation, and a DNA ladder in MCF-7 cells. Celastrol triggered the activation of caspase family proteins. Celastrol caused activation of caspase-7, -8, and -9, PARP cleavage, caspase-8-mediated bid cleavage, and release of cytochrome c and AIF. In addition, celastrol decreased the expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein. These results suggest that celastrol inhibits the proliferation of MCF-7 cells through induction of apoptosis, which is mediated by a mitochondrial-dependent caspase pathway.     

Tetrahydrocurcumin induces G2/M cell cycle arrest and apoptosis involving p38 MAPK activation in human breast cancer cells

Curcumin (CUR) is a major naturally-occurring polyphenol of Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. In recent years, it has been reported that CUR exhibits significant anti-tumor activity in vivo. However, the pharmacokinetic features of CUR have indicated poor oral bioavailability, which may be related to its extensive metabolism. The CUR metabolites might be responsible for the antitumor pharmacological effects in vivo. Tetrahydrocurcumin (THC) is one of the major metabolites of CUR. In the present study, we examined the efficacy and associated mechanism of action of THC in human breast cancer MCF-7 cells for the first time. Here, THC exhibited significant cell growth inhibition by inducing MCF-7 cells to undergo mitochondrial apoptosis and G2/M arrest. Moreover, co-treatment of MCF-7 cells with THC and p38 MAPK inhibitor, SB203580, effectively reversed the dissipation in mitochondrial membrane potential (Δψm), and blocked THC-mediated Bax up-regulation, Bcl-2 down-regulation, caspase-3 activation as well as p21 up-regulation, suggesting p38 MAPK might mediate THC-induced apoptosis and G2/M arrest. Taken together, these results indicate THC might be an active antitumor form of CUR in vivo, and it might be selected as a potentially effective agent for treatment of human breast cancer.     

Germacrone inhibits the proliferation of breast cancer cell lines by inducing cell cycle arrest and promoting apoptosis

Traditional medicinal herbs are an untapped source of potential pharmaceutical compounds. This study aims to determine whether the proliferation of breast cancer cell lines could be inhibited by germacrone, a natural product isolated from Rhizoma curcuma. Germacrone treatment significantly inhibited cell proliferation, increased lactate dehydrogenase (LDH) release, and induced mitochondrial membrane potential (ΔΨm) depolarization in both MCF-7 and MDA-MB-231 cells in a dose-dependent manner. Germacrone induced MDA-MB-231 and MCF-7 cell cycle arrest at the G0/G1 and G2/M phases respectively and induced MDA-MB-231 cell apoptosis. Furthermore, germacrone treatment significantly increased Bok expression and cytochrome c release from mitochondria without affecting Bcl-2, Bcl-xL, Bax, and Bim protein expressions. In addition, germacrone treatment induced caspase-3, 7, 9, PARP cleavage. We concluded that germacrone inhibited the proliferation of breast cancer cell lines by inducing cell cycle arrest and apoptosis through mitochondria-mediated caspase pathway. These results might provide some molecular basis for the anti-tumor activity of Rhizoma curcuma.     

Curcumin synergistically increases effects of β-interferon and retinoic acid on breast cancer cells in vitro and in vivo by up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways

Objective: The study aimed to investigate the effects of combination treatment of curcumin and β-interferon (IFN-β)/retinoic acid (RA) on breast cancer cells, including cell viability, apoptosis and migration, and to determine the mechanisms related to GRIM-19 through STAT3-dependent and STAT3-independent pathways.

Methods: The following groups were used for the in vitro experiment: control siRNA, GRIM-19 siRNA, IFN-β/RA and IFN-β/RA?+?curcumin. Cell viability is by the MTT method, cell apoptosis by flow cytometry and cell migration by wound healing experiment; GRIM-19, STAT3, survivin, Bcl-2, GADD153 and COX-2 expression was measured by Western blot. In vivo experiment, MCF-7 cells were subcutaneously injected into nude mice.

Results: GRIM-19 siRNA promoted MCF-7 cell proliferation and migration; inhibited cell apoptosis; and promoted the expression of STAT3, survivin, Bcl-2 and MMP-9. IFN-β/RA inhibited cell proliferation and migration; promoted cell apoptosis; up-regulated GRIM-19; and inhibited the expression of STAT3, survivin, Bcl-2 and MMP-9. Combination treatment of curcumin and IFN-β/RA had a stronger effect than that of the IFN-β/RA group. In addition, curcumin and IFN-β/RA combination inhibited the expression of COX-2 and up-regulated GADD153.

Conclusion: Curcumin synergistically increases the effects of IFN-β/RA on breast cancer cells. The mechanism may be related to the up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways.