Macroautophagy inhibition sensitizes tamoxifen-resistant breast cancer cells and enhances mitochondrial depolarization

Macroautophagy (autophagy), a process for lysosomal degradation of organelles and long-lived proteins, has been linked to various pathologies including cancer and to the cellular response to anticancer therapies. In the human estrogen receptor positive MCF7 breast adenocarcinoma cell line, treatment with the endocrine therapeutic tamoxifen was shown previously to induce cell cycle arrest, cell death, and autophagy. To investigate specifically the role of autophagy in tamoxifen treated breast cancer cell lines, we used a siRNA approach, targeting three different autophagy genes, Atg5, Beclin-1, and Atg7. We found that knockdown of autophagy, in combination with tamoxifen in MCF7 cells, results in decreased cell viability concomitant with increased mitochondrial-mediated apoptosis. The combination of autophagy knockdown and tamoxifen treatment similarly resulted in reduced cell viability in the breast cancer cell lines, estrogen receptor positive T-47D and tamoxifen-resistant MCF7-HER2. Together, these results indicate that autophagy has a primary pro-survival role following tamoxifen treatment, and suggest that autophagy knockdown may be useful in a combination therapy setting to sensitize breast cancer cells, including tamoxifen-resistant breast cancer cells, to tamoxifen therapy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of action of the anti-neoplastic drug lonidamine on drug-sensitive and drug-resistant human breast cancer cells:31 P and 13C nuclear magnetic resonance studies

Lonidamine (LND) is a relatively new anti-cancer drug,and several clinical trials have indicated that it may be effectivein combinations with other therapeutic modalities. LND isclassified within the metabolic inhibitor agents. Multidrugresistance (MDR) phenomenon is often associated with increasedenergy requirements, and enhanced glycolysis rate. These studieswere performed to delineate the mechanism of action of LNDon MDR human breast cancer cells, and to investigate whetherLND as a single agent, or in combination with anotheranti-metabolism drug, 2-deoxyglucose (2-DG), may be usefulagainst MDR tumors. The effects of LND on intact perfuseddrug-sensitive (WT) and 33-fold resistant to Adriamycin(Adr) MCF-7 cells, embedded in alginate micro capsules, were continuouslymonitored by ³¹P and ¹³C nuclearmagnetic resonance (NMR) spectroscopy. ³¹PNMR studies showed that LND induced intracellular acidificationand depletion of NTP in both WT and Adr cells. However, pH and NTPlevels decreased less in the Adr cells than in the WT cells(p < 0.05 for both parameters). ¹³CNMR demonstrated that LND inhibited lactate transport,and lactate signals were elevated in both cell lines. However, theintracellular lactate levels increased to a greater extentin the WT than in the Adr cells (p < 0.05).There were major differences in the effects of LND onmetabolism between sensitive and resistant cells.While LND enhanced glucose uptake in the WT cells, and itsadministration was followed by continuous increase oflactate signal, both processes were not affected by LNDin the Adr cells. 2-DG is a glucose analogue that inhibitsboth cellular uptake and utilization of glucose, leading to cell starvation. Combined treatment with LND and2-DG yielded at best additive, but not synergistic,cellular toxicity, and the metabolic effects of LNDwere attenuated by 2-DG. These results showed that the principalmechanism of action of LND is inhibition of lactate transportleading to intracellular lactate accumulation and acidificationin both WT and Adr cells. The Adr cells were only 2-fold resistantto LND (compared to the WT cells), and since cellular uptakeof alkaloid chemotherapy is improved in acidic environment,LND may have a role in the treatment protocols of MDR tumors,especially when given as the initial means for inductionof intracellular acidification.     

奈达铂联合顺铂对人食管癌细胞株（Eca-109）抑制作用机制的研究

目的研究奈达铂（NDP）联合顺铂（DDP）对人食管癌细胞株Eca-109的增殖及凋亡的影响,并探讨其机制。方法采用MTT法检测NDP联合DDP对Eca-109细胞的增殖抑制率,中效原理法判断联合用药的相互作用;采用RT-PCR和蛋白印迹法分析Ki-67及Bax表达的变化。结果NDP、DDP均明显抑制Eca-109细胞的生长,且呈剂量依赖性;两者联合使用低浓度时呈拮抗效应,高浓度时呈协同效应;IC50浓度的NDP、DDP和1/2IC50（NDP）＋1/2IC50（DDP）对Eca-109细胞的抑制率分别为（41.9±4.1）%、（47.4±2.9）%和（52.5±0.9）%;与单独用药组比较,联合用药组Ki-67基因（蛋白）表达显著降低,而Bax基因（蛋白）表达显著增高。结论1/2IC50浓度NDP和DDP联合应用与两药IC50浓度单独应用相比,对Eca-109细胞增殖的抑制作用和诱导凋亡能力均有明显增强。     

miR-221增强乳腺癌细胞对多西他赛的耐药性

目的:探究miR-221在人乳腺癌细胞T47D和MCF-7多西他赛(docetaxel)耐药性中的作用及机制。方法:qPCR检测多西他赛处理乳腺癌细胞T47D和MCF-7不同时间点,细胞中miR-221含量的变化;qPCR和Western blot检测miR-221 mimics的转染效率。用阴性对照（NC）或miR-221 mimics转染细胞,不同浓度的多西他赛刺激细胞72小时后,MTT法检测细胞对多西他赛的耐药性;PI和Annexin V双染法检测miR-221 过表达对T47D和MCF-7细胞凋亡的影响;qPCR和Western blot检测靶蛋白p27的表达。结果:在T47D和MCD-7中多西他赛刺激明显促进miR-221的表达量升高;miR-221在细胞内可以发挥生物学效应,降低靶蛋白p27的表达;且过表达miR-221明显增强T47D和MCF-7对多西他赛的耐药性,降低其凋亡率。结论:miR-221过表达可明显增强T47D和MCF-7细胞对多西他赛的耐药性。     

诺美孕酮逆转乳腺癌耐药细胞株多药耐药性的研究

目的 研究诺美孕酮（NOM）对乳腺癌耐药细胞株（MCF7／ADR）多药耐药性（MDR）的影响,探讨其调节机制。方法 应用MTT法,研究NOM对MCF7／ADR药物敏感性的影响。采用半定量逆转录聚合酶联反应（RT－PCR）和免疫细胞化学染色,分析耐药基因MDR1、谷胱甘肽S转移酶Pi(GSTπ）、拓扑异构酶Ⅱα（Topo Ⅱα)和多药耐药相关蛋白（MRP）表达的变化。通过流式细胞技术观察NOM对MCF7／ADR细胞内药物积累和细胞周期的影响。结果 NOM对MCF7／ADR的MDR具有明显的逆转作用,在20,10和5μmol/L浓度时的逆转倍数分别为21,12和8倍,逆转强度明显高于前身化合物甲地孕酮,而与维拉帕米相当。5μmol/L NOM处理MCF7／ADR后,MDRI的mRNA表达水平明显降低,呈现时间依赖性变化;P糖蛋白（P－gp)和GSTπ蛋白的变化符合相应mRNA表达的变化;MRP和Topo Ⅱα基因表达未见明显变化。用NOM 20,10和5μmol/L分别处理2h后,ADR在细胞内的积累分别增加到2．7倍、2．3倍和1．5倍。同时,NOM可明显加强ADR对MCF7／ADR细胞在G2M期的阻滞作用。结论 NOM具有较强的逆转MCF7／ADR细胞MDR的作用,其逆转机制为多种途径,包括时间依赖性下调MDRI和GSTπ基因的表达,增加细胞内药物积累,加强ADR对MCF7／ADR在G2M期的阻滞作用等。     

Enhancement of cisplatin activity by lonidamine in human ovarian cancer cells.

The ability of lonidamine, an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxicity of cisplatin was investigated in human ovarian-cancer cell lines sensitive (A2780) or with experimentally induced resistance (A2780/cp8) to the alkylating agent. A 24-hr post-incubation with 300 microM lonidamine significantly potentiated the activity of a 1-hr cisplatin treatment in both cell lines. In particular, the cisplatin IC50 value was reduced 4-fold in the sensitive line and 5-fold in the resistant line. Flow cytometric analysis showed that, in the resistant cell line, lonidamine alone did not affect cell kinetics, but when given after cisplatin it was able to transform the temporary G2 + M cell accumulation induced by the alkylating agent to a persistent block in S/G2 + M. In the A2780/cp8 cell line, lonidamine was also able to significantly enhance the accumulation of cisplatin-induced DNA interstrand cross-links. Our results suggest that lonidamine can positively modulate the anti-tumor activity of cisplatin in ovarian cancer cells and also indicate that the drug is potentially useful in combination therapy including the alkylating agent for ovarian cancer patients.     

Modulation of resistance to cisplatin by amphotericin B and aphidicolin in human larynx carcinoma cells

The aim of this study was to examine whether resistance to cisplatin [cis-diamminedichloroplatinum (II)] (CDDP) could be overcome by amphotericin B, cyclosporin A and aphidicolin in two sublines of human larynx carcinoma HEp2 cells. The sensitivity of parental and cisplatin-resistant CA3 and CK2 cells to amphotericin B, cyclosporin A and aphidicolin, and also the effects of these drugs (given in maximal nontoxic concentrations) on cisplatin sensitivity were determined by clonogenic survival assay. CA3 ad CK2 cells were sensitive to amphotericin B, and resistant to cyclosporin A and aphidicolin, compared with their parental cells. Amphotericin B increased cisplatin toxicity 2-fold in CA3 cells and 2.7-fold in CK2 cells, while it had no effect in parental HEp2 cells. Cyclosporin A did not influence the sensitivity of examined cells to cisplatin. The sensitizing effect of aphidicolin was more obvious in cisplatin-resistant cells. Cisplatin toxicity was increased by aphidicolin: 1.5-fold in HEp2 cells, 2-fold in CA3 cells, and 1.9-fold in CK2 cells. Therefore, the resistance to cisplatin in human larynx carcinoma CA3 and CK2 cells can be partially reversed by amphotericin B and aphidicolin.     

赫赛汀与泰索帝序贯联合应用对乳腺癌细胞抑制作用的研究

目的探讨赫赛汀与泰索帝联合作用于乳腺癌时,抑制作用最强的序贯方式;赫赛汀对于泰索帝作用的影响及其机制。方法利用MTT法及流式细胞技术测定单纯赫赛汀组、单纯化疗组以及不同序贯方式赫赛汀结合化疗组对人乳腺癌细胞(MDA-MB543)的抑制率及细胞周期的影响。结果赫赛汀作用18h后应用泰索帝,两药联合作用对乳腺癌细胞的抑制率最强(53.12%),优于单纯化疗组(41.55%)及其他序贯方式作用组,特别是化疗后应用赫赛汀组(P<0.01);此时泰索帝抑制率最高为(46.15%)。此种序贯方式与其他序贯方式相比可更有效地将细胞阻滞在G0/G1和G2/M期。结论赫赛汀应用18h后应用泰索帝,可最大程度增强泰索帝的抑制作用,优于单药及其他序贯方式,且此时两药联合对于肿瘤细胞的抑制作用最强。较其它序贯方式可更有效地将细胞阻滞在G0/G1和G2/M期,影响细胞的生长及凋亡,而发挥协同作用。     

Cold‐inducible RNA‐binding protein contributes to human antigen R and cyclin E1 deregulation in breast cancer

The cell cycle regulator cyclin E1 is aberrantly expressed in a variety of human cancers. In breast cancer, elevated cyclin E1 correlates with poor outcome, as do high cytoplasmic levels of the stress‐induced RNA‐binding protein human antigen R (HuR). We showed previously that increased cytoplasmic HuR elevates cyclin E1 in MCF‐7 breast cancer cells by stabilizing its mRNA. We show here that cold‐inducible RNA‐binding protein (CIRP) co‐regulates cyclin E1 with HuR in breast cancer cells. CIRP had been shown to interact with HuR in Xenopus laevis oocytes and to be decreased in endometrial cancer. To investigate if human CIRP and HuR co‐regulate cyclin E1, HuR and CIRP levels were altered in MCF‐7 cells and effects on cyclin E1 assessed. Altering HuR expression resulted in a reciprocal change in CIRP expression, while altering CIRP expression resulted in corresponding changes in HuR and cyclin E1 expression. CIRP and HuR co‐precipitated in the presence of RNA and CIRP enhanced HuR binding to the cyclin E1 mRNA and increased cyclin E1 mRNA stability. CIRP co‐localized with HuR predominantly in the nucleus, but also in discrete cytoplasmic foci identified as stress granules (SGs). CIRP overexpression increased the number of HuR‐containing SGs, while its knockdown decreased them. Our results suggest that CIRP positively regulates HuR, ultimately resulting in increased protein synthesis of at least one of its targets. © 2009 Wiley‐Liss, Inc.     

干扰B7-H4表达通过下调E2F家族相关转录因子抑制乳腺癌细胞的增殖

目的：探讨干扰B7-H4表达对乳腺癌细胞增殖、凋亡、周期以及相关下游分子表达的影响。方法：利用脂质体转染技术分别将特异性靶向B7-H4的siRNA(siB7-H4)及其阴性对照（siNC）转染至对数生长期的乳腺癌T47D和MCF-7细胞,分别命名为T47D-siB7-H4、T47D-siNC、MCF-7-siB7-H4和MCF-7-siNC组。用qPCR法和WB法验证siRNA干扰效果及其对细胞周期分子cyclin D1表达的影响,CCK-8法和FCM分别检测干扰B7-H4表达对T47D和MCF-7细胞增殖、周期和凋亡的影响,qPCR法检测B7-H4干扰对E2F家族相关转录因子表达的影响。结果：成功构建干扰B7-H4表达的乳腺癌T47D和MCF-7细胞。与T47D-siNC和MCF-7-siNC组相比,T47D-siB7-H4和MCF-7-siB7-H4组细胞中B7-H4 mRNA和蛋白表达水平均显著降低、细胞增殖能力显著降低（均P<0.01）,并伴有G1/S期细胞周期阻滞以及cyclin D1表达下调（均P<0.01）,但细胞凋亡率差异无统计学意义（均P>0.05）...     

健择联合顺铂治疗晚期非小细胞肺癌疗效观察

目的:评价健择(GEM)联合顺铂(DDP)治疗晚期非小细胞肺癌(NSCLC)的疗效和毒副反应。方法:收集NSCLC患者38例给予GP方案化疗,GEM1000mg/m2iv d1,8;DDP80mg/m2iv d1,28天为一周期,应用2周期后评价疗效。结果:全组有效率46.9%,无CR病例,鳞癌和腺癌有效率分别为44.0%和46.1%,初治、复治有效率分别为46.9%、33.3%,Ⅲ、Ⅳ期有效率为47.6%、41.2%。毒副反应主要为白细胞下降68.4%,其次为消化道症状为42.1%。结论:GP方案治疗NSCLC疗效较好,毒副反应轻。     

奥曲肽对人肺癌细胞株A549化疗增效作用的体外研究

目的：探讨生长抑素类似物奥曲肽能否提高化疗药物对于人肺癌细胞株A549敏感性。方法：以体外培养的人肺癌细胞株A549为靶细胞,运用四氮唑盐法（MTT法）观察奥曲肽单独作用及与化疗药物顺铂共同作用对于A549生长的影响,并以流式细胞仪分析其对细胞周期的影响。结果：MTT法结果显示奥曲肽抑制A549的生长,浓度0.098mg/L-25mg/L范围内,呈剂量依赖性,与顺铂共同作用后,能够增强顺铂对于A549的敏感性。流式细胞仪测定结果显示：奥曲肽作用后,G0/G1期细胞比例增加,G2/M期细胞比例减少,二者共同作用后,此趋势更加明显。结论：奥曲肽能够抑制体外培养的人肺癌细胞株A549增殖,且与顺铂有协同作用,其机制可能是发生G0/G1期阻滞。     

Protection of platinum-DNA adduct formation and reversal of cisplatin resistance by anti-MRP2 hammerhead ribozymes in human cancer cells

Resistance to platinum-containing antineoplastic drugs is the major limitation in their clinical use. To elucidate the role of the ABC transporter MRP2 in platinum drug resistance, its expression was analyzed in human cisplatin-resistant cell lines: the ovarian carcinoma line A2780RCIS, the adrenocortical carcinoma line D43/86RCIS and the melanoma line MeWoCIS1. All these cells showed overexpression of MRP2. For reversal of platinum resistance, 2 anti-MRP2 hammerhead ribozymes were introduced into A2780RCIS cells. Both ribozymes showed gene-silencing activities and reversed the drug-resistant phenotype. Moreover, formation of platinum-induced intrastrand cross-links was measured in DNA. The level of DNA platination corresponded inversely to the level of MRP2 expression and was accompanied by increased caspase-3-dependent apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity was not altered; the decrease in platinum-DNA adduct formation was rather a reflection of the protecting activity of MRP2. In conclusion, functional inhibition of MRP2 might be a promising strategy in the reversal of resistance to platinum-based anticancer drugs. This was reflected by the specific inhibition of MRP2 by ribozyme technology, indicating that this gene therapeutic approach may be applicable as a specific means to overcome platinum resistance in human neoplasms.     

长春瑞滨联合顺铂治疗晚期非小细胞肺癌疗效观察

目的评价长春瑞滨(NVB)联合顺铂(DDP)治疗晚期非小细胞肺癌(NSCLC)的疗效和毒副反应。方法收集晚期NSCLC患者46例给予NVB DDP方案化疗，NVB25mg／m^2iv d1、8，DDP75mg／m^2iv d1，21天为1周期，应用2周期后评价疗效。结果全组有效率为47．8％，无CR病例，其中初、复治有效率分别为51．5％、38．4％，Ⅲa、Ⅲb、Ⅳ期有效率分别为45．5％、52．3％、42．8％，腺癌和鳞癌有效率分别为57．1％、40．0％。毒副反应主要为白细胞下降(95．6％)、恶心呕吐(39．1％)，其次为静脉炎，发生率19．6％。结论NVB DDP方案治疗NSCLC疗效较好，毒副反应可以耐受。     

多西他赛联合顺铂治疗转移性乳腺癌的临床观察

目的:观察多西他赛联合顺铂治疗转移性乳腺癌的疗效及不良反应.方法:选择我科2005年6月-2008年6月收治的31例转移性乳腺癌患者,给予多西他赛联合顺铂(TP方案)化疗,21d为1个周期,2周期后评价疗效.有效者给予6周期的化疗.结果:31例患者有效率51.6%,其中CR2例,PR14例.中位疾病进展时间8.6个月,中位生存时间19.2个月.主要不良反应为骨髓抑制及消化道反应和脱发.其中Ⅲ度以上白细胞减少的发生率26.0%.结论:多西他赛联合顺铂治疗转移性乳腺癌的疗效确切,不良反应可以耐受.     

多西他赛联合顺铂治疗转移性乳腺癌的临床观察

目的：观察多西他赛联合顺铂治疗转移性乳腺癌的疗效及不良反应。方法：选择我科2005年6月-2008年6月收治的31例转移性乳腺癌患者,给予多西他赛联合顺铂（TP方案）化疗,21d为1个周期,2周期后评价疗效。有效者给予6周期的化疗。结果：31例患者有效率51．6％,其中012例,PR14例。中位疾病进展时间8．6个月,中位生存时间19．2个月。主要不良反应为骨髓抑制及消化道反应和脱发。其中Ⅲ度以上白细胞减少的发生率26．0％。结论：多西他赛联合顺铂治疗转移性乳腺癌的疗效确切,不良反应可以耐受。     

褪黑素通过促凋亡协同增强顺铂对人喉癌细胞增殖的抑制作用

目的:研究褪黑素(melatonin,MT)对人喉癌细胞增殖与凋亡的影响及MT增强人喉癌细胞对顺铂(cisplatin,DDP)治疗的敏感性.方法:采用不同质量浓度MT和DDP单独或联合处理Hep-2细胞;通过CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡和细胞周期,采用两药相互作用指数(co-efficient of drug interaction,CDI)评估MT是否影响Hep-2细胞对DDP的敏感性.结果:CCK-8检测结果显示,单用MT或DDP可浓度依赖性抑制Hep-2细胞的增殖,MT可协同增强DDP对Hep-2细胞的增殖抑制作用(CDI＜1).流式细胞术检测细胞凋亡和细胞周期结果显示,MT可促进Hep-2细胞凋亡以及增加亚G1期细胞比例(P＜0.01),MT可协同DDP促进Hep-2细胞凋亡[0.5 mmol/L MT联合20μg/ml DDP组的细胞凋亡率显著高于20 μg/ml DDP组,(40.9±3.0)％vs(11.0±0.9)％,P＜0.01]以及亚G1期细胞比例[0.5 mmol/L MT联合20 μg/mlDDP组的亚G1期细胞比例显著高于20 μg/ml DDP组,(73.0±2.4)％vs(40.4±3.0)％,P＜0.01].加入Caspase抑制剂Z-VAD-fmk可逆转MT和/或DDP对Hep-2细胞的增殖抑制作用和凋亡诱导作用(均P＜0.01).结论:MT能以Caspase依赖的方式诱导人喉癌细胞Hep-2的凋亡,从而协同增强DDP对细胞的增殖抑制作用.     

42℃温热增强顺铂对人肺癌细胞PLA-801 D毒性的实验研究

目的探讨42℃温热对化疗药顺铂(DDP)增敏的规律及其机制。方法应用四氮唑蓝(MTT)快速比色法测定42℃热疗组、单纯化疗组以及以不同序贯方式结合的热化疗组对人肺癌细胞(PLA-801D)的生长抑制率;流式细胞仪测定各组细胞周期的变化。结果42℃单独热疗和单独药物均对肺癌细胞有抑制和杀伤作用;42℃热疗与药物以不同序贯方式联合,抑制作用均强于单纯化疗组和单纯热疗组(P<0.05),尤以热化疗同时作用组效果最佳(P<0.001);细胞周期分析发现42℃热疗降低了S期细胞所占比例;单独顺铂使细胞周期阻滞于S期;与单纯化疗组相比,先化疗后热疗组、先热疗后化疗组和热化疗同时组的S期细胞所占比例减少,G2/M期细胞增多,其中热化疗同时组增减的幅度最大。结论42℃温热可以明显增强化疗药顺铂的毒性,结合方式上以热化疗同时进行最佳,其作用机制可能与干扰细胞周期有关。     

吉西他滨联合顺铂二线治疗晚期乳腺癌的临床观察

目的:探讨吉西他滨联合顺铂二线治疗晚期乳腺癌的疗效和不良反应。方法:选择蒽环类和(或)紫杉类化疗后的转移性乳腺癌患者30例,采用吉西他滨1000mg/m2,静脉滴注,第1、8天;顺铂25 mg/m2,静脉滴注,第1-3天,21d为1周期,至少2周期后评价疗效。结果:CR2例(6.7%),PR12例(40.0%),总有效率为46.7%。中位生存期12.8个月,中位TTP 5.6个月。主要不良反应为骨髓抑制及胃肠道反应。结论:吉西他滨联合顺铂二线治疗晚期乳腺癌的近期疗效较好,患者耐受性好,值得临床推广。