熊去氧胆酸对阿霉素诱导的H9c2心肌细胞的影响及机制研究

目的:探讨熊去氧胆酸(UDCA)对阿霉素(DOX)诱导的H9c2心肌细胞损伤的影响及机制.方法:体外培养H9c2细胞,1 μMDOX和不同浓度UDCA处理H9c2,CCK-8法测定细胞活力;实时定量聚合酶链反应检测心肌细胞凋亡分子Bax及炎症因子IL-1β、IL-6的表达;Western blotting检测UDCA对...     

Synthesis,Characterization and In Vitro Study of Biocompatible Cinnamaldehyde Functionalized Magnetite Nanoparticles (CPGF Nps) For Hyperthermia and Drug Delivery Applications in Breast Cancer

Cinnamaldehyde, the bioactive component of the spice cinnamon, and its derivatives have been shown to possess anti-cancer activity against various cancer cell lines. However, its hydrophobic nature invites attention for efficient drug delivery systems that would enhance the bioavailability of cinnamaldehyde without affecting its bioactivity. Here, we report the synthesis of stable aqueous suspension of cinnamaldehyde tagged Fe₃O₄ nanoparticles capped with glycine and pluronic polymer (CPGF NPs) for their potential application in drug delivery and hyperthermia in breast cancer. The monodispersed superparamagnetic NPs had an average particulate size of ∼20 nm. TGA data revealed the drug payload of ∼18%. Compared to the free cinnamaldehyde, CPGF NPs reduced the viability of breast cancer cell lines, MCF7 and MDAMB231, at lower doses of cinnamaldehyde suggesting its increased bioavailability and in turn its therapeutic efficacy in the cells. Interestingly, the NPs were non-toxic to the non-cancerous HEK293 and MCF10A cell lines compared to the free cinnamaldehyde. The novelty of CPGF nanoparticulate system was that it could induce cytotoxicity in both ER/PR positive/Her2 negative (MCF7) and ER/PR negative/Her2 negative (MDAMB231) breast cancer cells, the latter being insensitive to most of the chemotherapeutic drugs. The NPs decreased the growth of the breast cancer cells in a dose-dependent manner and altered their migration through reduction in MMP-2 expression. CPGF NPs also decreased the expression of VEGF, an important oncomarker of tumor angiogenesis. They induced apoptosis in breast cancer cells through loss of mitochondrial membrane potential and activation of caspase-3. Interestingly, upon exposure to the radiofrequency waves, the NPs heated up to 41.6°C within 1 min, suggesting their promise as a magnetic hyperthermia agent. All these findings indicate that CPGF NPs prove to be potential nano-chemotherapeutic agents in breast cancer.     

Effects of High Iron and Glucose Concentrations over the Relative Expression of Bcl2, Bax, and Mfn2 in MIN6 Cells

Type 2 diabetes is characterized by hyperglycemia and oxidative stress. Hyperglycemia is linked to mitochondrial dysfunction and reduced β-cell mass due to the reduced expression of genes such as Mfn2 as well as the participation of the Bcl2 gene family, responsible for increased apoptosis. The purpose of this study was to describe the effect of different iron and/or glucose concentrations over Mfn2, Bax, and Bcl2 expressions in a β-pancreatic cell line (MIN6 cells). MIN6 cells were pre-incubated with different iron and/or glucose concentrations, and the relative mRNA abundance of the Bcl2/Bax ratio and of Mfn2 genes was measured by qRT-PCR. Heme oxygenase (HO) activity, iron uptake, superoxide dismutase activity, and glutathione content were also determined. The Bcl2/Bax ratio increased and Mfn2 expression decreased in MIN6 cells after glucose stimulation. These effects were higher when glucose and iron were incubated together. Additionally, treatment with glucose/iron showed a higher HO activity. Our study revealed that high glucose/Fe concentrations in MIN6 cells induced an increase of the Bcl2/Bax ratio, an indicator of increased cell apoptosis.     

布雷菲德菌素A通过线粒体凋亡途径增强顺铂抗肺癌GLC-82细胞的作用

本研究证明了线粒体凋亡途径在布雷菲德菌素A（brefeldin A,BFA）联合顺铂（cis-dichlorodiamine platinum,CDDP）抗非小细胞肺癌（non-small cell lung cancer,NSCLC）中的作用。MTT结果显示,BFA对肺癌GLC-82和NCI-H1299细胞的半数有效抑制浓度（half maximal inhibitory concentration,IC50）分别是100 ng/mL和400 ng/mL,CDDP对GLC-82和NCI-H1299细胞的IC50分别是4 μg/mL和15 μg/mL;而分别采用半量的BFA和CDDP联合处理GLC-82或NCI-H1299细胞后,抑制作用均进一步加强。DAPI染色结果进一步证明了二者的协同作用——与单独用药组相比,细胞核染色质固缩加剧,核裂解碎片增多,乃至形成凋亡小体,表明细胞凋亡的发生。与单药组比较,联合用药导致肺癌GLC-82细胞线粒体膜电位显著下降;q-RT-PCR及Western印迹结果显示,在联合用药早期（24 h）,GLC-82细胞可能通过提高Bcl2表达以促进存活;而在联合用药晚期（48 h）,细胞已发生不可逆转的凋亡,Bcl2表达受抑制,同时二者通过促进Bax表达来诱导细胞色素C释放,使胱天蛋白酶 3发生剪切激活,最终诱导细胞凋亡发生。提示线粒体凋亡途径可能是BFA协同CDDP抗非小细胞肺癌的分子机制之一,为肺癌的临床治疗方案提供了更多的理论依据。     

蟾毒灵对人卵巢癌SKOV-3细胞增殖的影响

目的：探讨蟾毒灵对人卵巢癌SKOV-3细胞增殖抑制和凋亡的影响,为卵巢癌临床治疗提供依据和分子基础。方法：不同浓度蟾毒灵处理卵巢癌SKOV-3细胞后,MTT法检测细胞增殖抑制作用,细胞免疫化学染色法检测细胞的凋亡,Western Blot法检测Bax,Bcl-2,Caspase-3蛋白以及计算Bax／Bcl-2的比值。结果：蟾毒灵能够抑制SKOV-3细胞的增殖,且成时间和剂量依赖性,免疫荧光显示蟾毒灵对SKOV-3细胞具有凋亡作用,Western Blot检测发现蟾毒灵能够促进Caspase-3蛋白的活化,提高Bax／Bcl-2的比值。结论：蟾毒灵在体外能够抑制卵巢癌SKOV-3细胞的增殖和促进卵巢癌SKOV-3细胞的凋亡。     

miR-218 promoted the apoptosis of human ovarian carcinoma cells via suppression of the WNT/β-catenin signaling pathway

MicroRNA-218 (miR-218) is a short, noncoding RNA, with multiple biological functions. In this study, we aimed to investigate the potential effects of miR-218 on the apoptosis of human ovarian carcinoma cells and the underlying mechanisms by which miR-218 exerted its actions. After over-expressing miR-218 in human ovarian carcinoma (OVCAR3) cells, cell viability was determined by MTT method, cell apoptosis was observed by flow cytometry (FCM), mRNA expression of miR-218, Bcl2, Bax was measured by RT-PCR and protein expression levels of Wnt, tankyrase and β-catenin were quantified by Western blots. Over-expression of miR-218 potently suppressed cell viability and promoted the apoptosis of human ovarian carcinoma cells in a time-dependent manner. In addition, the down-regulation of tankyrase expression level was detected in miR-218-over-expressed cells. Following the block of the Wnt/β-catenin signaling pathway using the inhibitor XAV-939, the effects of miR-218 on the proliferation and apoptosis of human ovarian carcinoma cells were significantly suppressed. Augmenting expression of miR-218 and/or miRNA-218 mimicking therapeutics may provide viable avenue for the treatment of ovarian cancer.     

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.     

Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection

This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.     

Nampt及高糖高胰岛素微环境对人顺铂耐药肺癌细胞株增殖和转移的影响

摘要 目的：探究烟酰胺磷酸核糖转移酶（Nampt）及高糖高胰岛素（HG+HI）微环境对人顺铂耐药肺癌细胞增殖和转移的影响。方法：将人顺铂耐药细胞株A549/DDP分为6组（n=6）：对照组（control）、高糖高胰岛素干预组（HH，使用添加30 mmol/L的葡萄糖和500 mU/L的胰岛素的培养基培养72 h）、分别转染sh-NC和sh-Nampt组（sh-NC和sh-Nampt，使用Lipofectamine 2000将sh-NC和sh-Nampt分别转染到细胞中，转染时间为48 h）、HH干预sh-NC和sh-Nampt组（HH+sh-NC和HH+sh-Nampt）。每组6个重复样本。qRT-PCR检测转染效率，MTT法检测细胞增殖，流式细胞仪检测细胞凋亡，Transwell检测细胞迁移和侵袭，qRT-PCR检测Nampt mRNA，Western blot检测Nampt、Bcl-2、Bax、MMP-2、MMP-9、p-PI3K、PI3K、p-AKT和AKT蛋白表达。结果：与对照组和sh-NC组比较，sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，而细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。与对照组和sh-NC组比较，HH组和HH+sh-NC组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平升高，Bax蛋白表达水平降低（P<0.005）。与HH组和HH+sh-NC组比较，HH+sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。结论：高糖高胰岛素微环境可能通过上调Nampt/PI3K/AKT信号通路诱导人顺铂耐药肺癌细胞的增殖和转移。     

Targeted Anticancer Drug Delivery Using Chitosan,Carbon Quantum Dots,and Aptamers to Deliver Ganoderic Acid and 5-Fluorouracil

Breast cancer is a malignancy that affects mostly females and is among the most lethal types of cancer. The ligand-functionalized nanoparticles used in the nano-drug delivery system offer enormous potential for cancer treatments. This work devised a promising approach to increase drug loading efficacy and produce sustained release of 5-fluorouracil (5-FU) and Ganoderic acid (GA) as model drugs for breast cancer. Chitosan, aptamer, and carbon quantum dot (CS/Apt/COQ) hydrogels were initially synthesized as a pH-sensitive and biocompatible delivery system. Then, CS/Apt/COQ NPs loaded with 5-FU-GA were made using the W/O/W emulsification method. FT-IR, XRD, DLS, zeta potentiometer, and SEM were used to analyze NP's chemical structure, particle size, and shape. Cell viability was measured using MTT assays in vitro using the MCF-7 cell lines. Real-time PCR measured cell apoptotic gene expression. XRD and FT-IR investigations validated nanocarrier production and revealed their crystalline structure and molecular interactions. DLS showed that nanocarriers include NPs with an average size of 250.6 nm and PDI of 0.057. SEM showed their spherical form, and zeta potential studies showed an average surface charge of +37.8 mV. pH 5.4 had a highly effective and prolonged drug release profile, releasing virtually all 5-FU and GA in 48 h. Entrapment efficiency percentages for 5-FU and GA were 84.7±5.2 and 80.2 %±2.3, respectively. The 5-FU-GA-CS-CQD-Apt group induced the highest cell death, with just 57.9 % of the MCF-7 cells surviving following treatment. 5-FU and GA in CS-CQD-Apt enhanced apoptotic induction by flow cytometry. 5-FU-GA-CS-CQD-Apt also elevated Caspase 9 and downregulated Bcl2. Accordingly, the produced NPs may serve as pH-sensitive nano vehicles for the controlled release of 5-FU and GA in treating breast cancer.     

Synthesis and cytotoxicity evaluation of novel acylated starch nanoparticles

Starch nanoparticles (StNPs) were acylated under ambient conditions to obtain various nanosized derivatives formed stable suspension in water and soluble in organic solvents. The degree of substitution (DS) was determined using ¹H NMR technique. The cytotoxicity potential of the derivatised StNPs was evaluated in mouse embryonic fibroblast (3T3L1) cells and A549 tumor cell line using MTT cell viability assay. Other parameters that determine the oxidative stress viz., reactive oxygen species (ROS) generation, intracellular reduced glutathione (GSH), superoxide generation and acridine orange/ethidium bromide staining were also investigated. The present study led to the conclusion that cytotoxic activity of acylated starch nanoparticles was dependent on their dosage, DS and type of substitution. The non-toxic nature in non-cancerous cells reveals that the nanoparticles (NPs) can be used for cancer therapy and drug delivery. The nanoparticles also offered reasonable binding propensity with CT-DNA.     

Silver nanocrystals sensitize magnetic-nanoparticle-mediated thermo-induced killing of cancer cells

Magnetic nanoparticles (MNPs) can heat up tumor tissues and induce killing of cancer cells under external AC magnetic field. However, magnetic nanoparticles hyperthermia (MNPH) requires high concentration of MNPs that are injected into the tumor in order to obtain clinically needed thermal dose because of the complicated heat transfer in vivo and the limited heat quality of MNPs. To cut down the dose of MNPs and enhance the effect of this Nanotherapy, we prepared silver nanoparticles (AgNPs) with different sizes and investigated the effects of these AgNPs on cancer cells in MNPH treatment. It was found that AgNPs could enhance thermo-sensitivity of glioma cells and this effect was size dependent. AgNPs could induce cell cycles arrested in G(2)/M phase and enhanced the apoptosis rate of cancer cells after hyperthermia. In glioma bearing rats model, MNPH combined with AgNPs could enhance Bax expression in cancer cells. Our results suggested that AgNPs could be a potential thermo-sensitizer and could be further developed for the design of Ag nanostructure-based thermal seeds for MNPH therapy.     

Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells

We investigated the impact of bisphenol A (BPA) on murine ovarian granulosa cells. Ovarian granulosa cells were cultured with 100 fM to 100 microM BPA for 24 h to 72 h. BPA decreased granulosa cell viability in a dose- and time-dependent manner. The lowest concentration that induced a significant decrease was 100 pM (89.2 +/- 4.0% of the control). TUNEL analysis demonstrated that treatment with BPA increased apoptosis of granulosa cells in a dose- and time-dependent manner. In addition, flow cytometry analyses revealed that treatment with BPA resulted in G2-to-M arrest, which was most prominent at 48 h. BPA increased the expression of Bax and concomitantly decreased the expression of Bcl2 at both protein and mRNA levels of granulosa cells. These findings suggest that low, presumably environmentally relevant doses of BPA, decrease the viability of granulosa cells by inducing apoptosis and G2-to-M arrest. Up-regulation of Bax and down-regulation of Bcl2 were suggested to be involved in this apoptotic effect.     

热疗联合奥沙利铂对SW480细胞增殖和凋亡的影响

目的:观察奥沙利铂联合热疗对人结肠癌细胞SW480增殖及凋亡的影响,确定联合用药的效果,为临床方案提供参考。方法:采用MTT(四唑盐)法检测热疗、奥沙利铂及联合用药对细胞增殖的影响;瑞士吉姆萨染色法观察细胞形态;流式细胞仪检测细胞凋亡和周期;Western blot检测Bax、Bcl-2以及Caspase8蛋白表达量变化;q PCR检测Bax、Bcl-2以及Caspase8 m RNA的积累。结果:热疗联合奥沙利铂可以显著抑制细胞增殖,与对照组相比,热疗组、化疗组、联合组细胞凋亡率分别为16.2%、20.5%和36.1%,具有显著性差异(P0.01);细胞学形态中,热疗组细胞发生皱缩,化疗组细胞膜破裂;化疗将细胞阻滞在G2/M期,热疗和联合组将细胞阻滞S期;Western blot和qPCR显示Bax/Bcl-2比值上升,Caspase8表达量增加,联合组三种蛋白的表达量均与对照组具有显著性差异(P0.01)。结论:热疗联合奥沙利铂可以显著促进细胞凋亡,提高治疗效果,为结肠癌的治疗提供参考。     

Potential of bacterial culture media in biofabrication of metal nanoparticles and the therapeutic potential of the as-synthesized nanoparticles in conjunction with artemisinin against MDA-MB-231 breast cancer cells

In the recent past, various groups have proposed diverse biocompatible methods for the synthesis of metal nanoparticles (NPs). Besides culture biomass, culture supernatants (CS) are increasingly being explored for the synthesis of NPs; however, with the ever-increasing exploration of various CS in the biofabrication of NPs, it is equally important to explore the potential of various culture media (CMs) in the synthesis of metal NPs. Considering these aspects, in the present investigation, we explore the possible applicability of various CMs in the biofabrication of metal NPs. The synthesis of NPs was primarily followed by UV/VIS spectroscopy, and, thereafter, the NPs were characterized by various physiochemical techniques, including EM, EDX, FT_IR, X-ray diffraction, and DLS measurements, and finally, their anticancer potentialities were investigated against breast cancer. In addition, the NPs were examined in conjunction with artemisinin for therapeutic benefits against aggressive and highly metastatic MDA-MB-231 breast cancer cells. Cumulatively, the results of the present study collated the potentials of various bacterial CMs in the biofabrication of metal NPs and ascertained the efficacy of the as-synthesized silver nanoparticles, especially the combinatorial entity as intriguing breast cancer therapeutics. The data of the present study plausibly assist in advancing the therapeutic applicability of the combinatorial amalgam against aggressive and highly metastatic MDA-MB-231 breast cancer cells.     

Anti-KDEL-coated nanoparticles: A promising tumor targeting approach for ovarian cancer?

The purpose of this study was to target ovarian cancer cells by coupling paclitaxel (Tx)-loaded nanoparticles (NPs-Tx) to antibodies against KDEL sequence, able to recognize GRP94 and GRP78 that are located at cell surface in cancer cells whereas they are in the endoplasmic reticulum in healthy cells. Tx-loaded poly (dl-lactic acid) nanoparticles coated with anti-KDEL antibodies (NPs-Tx-KDEL) were successfully prepared and characterized. Interaction between tumor cells and NPs-Tx or NPs-Tx-KDEL was observed by microscopy with fluorescently labeled NPs and the efficacy of the different formulations was compared by a viability assay.     

Involvement of NF-κB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A

The plant Artocarpus obtusus is a tropical plant that belongs to the family Moraceae. In the present study a xanthone compound Pyranocycloartobiloxanthone A (PA) was isolated from this plant and the apoptosis mechanism was investigated. PA induced cytotoxicity was observed using MTT assay. High content screening (HCS) was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Reactive oxygen species formation was investigated on treated cells by using fluorescent analysis. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition mRNA levels of Bax and Bcl2 were also checked using RT-PCR. Caspase 3/7, 8 and 9 were measured for their induction while treatment. The involvement of NF-κB was analyzed using HCS assay. The results showed that PA possesses the characteristics of selectively inducing cell death of tumor cells as no inhibition was observed in non-tumorigenic cells even at 30μg/ml. Treatment of MCF7 cells with PA induced apoptosis with cell death-transducing signals, that regulate the MMP by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of cytochrome c triggered the activation of caspases-9, then activates downstream executioner caspase-3/7 and consequently cleaved specific substrates leading to apoptotic changes. This form of apoptosis was found closely associated with the extrinsic pathway caspase (caspase-8) and inhibition of translocation of NF-κB from cytoplasm to nucleus. The results demonstrated that PA induced apoptosis of MCF7 cells through NF-κB and Bcl2/Bax signaling pathways with the involvement of caspases.     

Synergistic effect of retinoic acid and vitamin D analog EB1089-induced apoptosis of hepatocellular cancer cells

Previous report showed that leukemia cells’ differentiation could be induced by retinoic acid (RA), and prostate cancer cells’ proliferation could be inhibited by Vitamin D or its analog. This study aimed to examine whether RA and vitamin D analog EB1089 have synergistic effect on hepatocellular cancer cells’ apoptosis. The hepatocellular cancer cell lines’ viability was determined by MTT method after treating by RA and EB1089 alone or in combination, cell cycle of SSMC-7721 cell analyzed by FACS, mitochondrial membrane potential of SSMC-7721 under different treatments were detected using MitoTracker Red CMXRos. TUNEL analysis was also used for cell apoptosis detection. Real time-PCR and Western Blot assay were used to detect the expression of Bcl-2 and Bax. Moreover, hepatocellular cancer model was developed by subcutaneously (S.C.) challenging H22 cells to nude mice. In the combination group (10 μmol/L RA, 10 nmol/L EB1089), the viability of hepatocellular cancer cells decreased significantly compared with drugs used alone (P < 0.05). From the TUNEL analysis, SSMC-7721 cells have a higher apoptotic ratio in the combined drug group than in the groups for which the drugs were used separately. In a hepatocellular cancer model, the tumor weight of H22 tumor bearing mice was more reduced in the combined drug treated group when compared to the groups for which the drugs were used alone (P < 0.05), in addition, significantly prolonged survival was observed. Combination of RA and EB1089 exert synergistic growth inhibition and apoptosis induction on hepatocellular cancers cells.     

Combination of AT-101/cisplatin overcomes chemoresistance by inducing apoptosis and modulating epigenetics in human ovarian cancer cells

We investigated the effects of AT-101/cisplatin combination treatment on the expression levels of apoptotic proteins and epigenetic events such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme activities in OVCAR-3 and MDAH-2774 ovarian cancer cells. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were performed. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. DNMT and HDAC activities were evaluated by ELISA assay and mRNA levels of DNMT1 and HDAC1 genes were quantified by qRT-PCR. Combination of AT-101/cisplatin resulted in strong synergistic cytotoxicity and apoptosis in human ovarian cancer cells. Combination treatment reduced some pivotal anti-apoptotic proteins such as Bcl-2, HIF-1A, cIAP-1, XIAP in OVCAR-3 cells, whereas p21, Bcl-2, cIAP-1, HSP27, Clusterin and XIAP in MDAH-2774 cells. Among the pro-apoptotic proteins, Bad, Bax, Fas, phospho-p53 (S46), Cleaved caspase-3, SMAC/Diablo, TNFR1 and Cytochrome c were induced in OVCAR-3 cells, whereas, Bax, TRAILR2, FADD, p27, phospho-p53 (S46), Cleaved caspase-3, Cytochrome c, SMAC/Diablo and TNFR1 were induced in MDAH-2774 cells. Combination treatment also inhibited both DNMT and HDAC activities and also mRNA levels in both ovarian cancer cells. AT-101 exhibits great potential in sensitization of human ovarian cancer cells to cisplatin treatment in vitro, suggesting that the combination of AT-101 with cisplatin may hold great promise for development as a novel chemotherapeutic approach to overcome platinum-resistance in human ovarian cancer.