Induction of the death-promoting gene bax-α sensitizes cultured breast-cancer cells to drug-induced apoptosis

Resistance to apoptosis plays an important role in malignancies that are refractory to chemotherapy treatment. Recently we have shown that the expression of bax-α, a death-promoting member of the bcl-2 family, is down-regulated in breast cancer and have provided evidence that low bax expression might contribute to the pathogenesis of breast cancer. In this study we were able to demonstrate the role of this gene in chemotherapy-induced apoptosis. We transfected bax-α into the breast-cancer cell lines R30C and MCF-7 under the control of an inducible tetracycline-dependent expression system. Induction of bax-α expression did not affect viability by itself but strongly increased chemosensitivity to epirubicin. We were able to demonstrate that this sensitization is due to apoptosis. These data might explain the recently published observation that reduced expression of bax is associated with poor response rates to chemotherapy in breast cancer. © 1996 Wiley-Liss, Inc.     

紫杉醇脂质体对乳腺癌MCF-7细胞生长抑制作用的机制

目的 分析紫杉醇脂质体对乳腺癌MCF-7细胞的生长抑制作用,观察药物作用的时间浓度关系并探究其作用机制.方法 应用不同剂量紫杉醇脂质体对MCF-7细胞进行处理,通过MTT法检测细胞存活率,倒置显微镜观察细胞形态,流式细胞分析技术检测细胞凋亡情况及其对细胞周期的影响,Western blot法检测bcl-2蛋白表达情况.结果 紫杉醇脂质体对MCF-7细胞有明显的剂量时间依赖效应.紫杉醇与紫杉醇脂质体均可将MCF-7细胞阻滞在G2/M期,且随药物浓度增加,凋亡细胞所占比例逐渐增加.通过Western blot法检测发现,紫杉醇脂质体作用后MCF-7细胞bcl-2蛋白表达明显下降,bax蛋白表达明显上调,bax/bcl-2比例明显上调.结论 与紫杉醇相比,紫杉醇以脂质体对人乳腺癌MCF 7细胞具有同样的抑制作用,其促凋亡机制之一为调控bcl-2及bax蛋白表达.     

Low doses of alpha particle irradiation modify the expression of genes regulating apoptosis in human MCF-7 breast cancer cells

The possibility of modifying apoptosis-related genes in tumor cells is an interesting line of research that calls for multidisciplinary experimentation to describe its characteristics and the conditions required. In vitro low doses of alpha particle irradiation due to radon have an antiproliferative effect on the growth of MCF-7 cells and increase the sensibility of cancer cells to taxol, a chemotherapeutic agent that induces cellular apoptosis. The present study examines the in vitro effects of low doses of alpha particle irradiation from the gas radon on the expression of some bcl-2 family apoptosis-related genes. The analyzed genes were bax, bcl-2 and bcl-x, with known responses to genotoxic stress (bcl-2) or ionising radiation (bax and bcl-x) in MCF-7 human breast cancer cells. The results obtained indicate that the cell line studied expresses the mentioned genes and they demonstrate that irradiation with low radon doses of MCF-7 cells induces underexpression of both bax and bcl-2 genes. Interestingly, the mRNA levels of the full-length bcl-x gene (bcl-xL) were overexpressed after irradiation, and we found significant mRNA levels of an alternative mRNA splicing form of the same gene (bcl-xS), which enhances the apoptotic sensitivity of the cell. The increased sensitivity to apoptosis resulting from bcl-xS overexpression is important because it might improve the efficacy of chemotherapeutic agents used to treat cancers which act through induction of apoptosis. The finding that low radiation doses of alpha particles from the gas radon modulate the expression of apoptosis-related genes suggests a therapeutic utility for this naturally occurring agent.     

Tamoxifen-induced apoptosis in breast cancer cells relates to down-regulation of bcl-2, but not bax and bcl-X(L), without alteration of p53 protein levels.

Tamoxifen (TAM) has been shown to induce apoptosis in breast cancer cells. bcl-2 family genes, which can interact with each other, have been shown to interfere with apoptosis after various stimuli. In this study, we investigated the effects of TAM on bcl-2 family gene products bcl-2, bax, and bcl-X(L) and on p53 levels in estrogen receptor-positive MCF-7 breast cancer cells. We found that TAM induced time- and concentration-dependent down-regulation of bcl-2 at both the mRNA and protein level. Down-regulation of bcl-2 correlated with TAM-induced apoptosis. In addition, estradiol treatment significantly increased bcl-2 protein expression and blocked the reduction of bcl-2 by TAM. TAM did not, however, affect bax, bcl-X(L), or p53 expression at the mRNA or protein level. Our results demonstrate that TAM can induce apoptosis in a time- and dose-dependent manner by modulating bcl-2 levels in breast cancer cells, and down-regulation of bcl-2 induced by TAM was not accompanied by alterations in p53 levels.     

miR-620通过靶向ING4调控乳腺癌MCF-7细胞放射敏感性

目的：探讨miR-620对乳腺癌MCF-7细胞放射敏感性的影响及其机制。方法：收集2017年3月至2018年3月在海南省儋州市人民医院手术切除的21例乳腺癌患者的癌及癌旁组织标本,以及乳腺癌细胞MCF-7、BCaP-37和乳腺上皮细胞HBL-100,采用qPCR法检测癌组织和细胞中miR-620和生长抑制因子4(ING4)mRNA的表达。利用脂质体转染技术,分别将miR-620抑制剂（anti-miR-620）和抑制剂阴性对照（anti-miR-NC）、anti-miR-620和ING4小干扰RNA(si-ING4)、anti-miR-620和小干扰RNA阴性对照序列（si-NC）转染至MCF-7细胞,经放射处理后（依次记为IR+anti-miR-620组、IR+anti-miR-NC组、IR+antimiR-620+si-ING4组、IR+anti-miR-620+si-NC组）,利用克隆形成实验、MTT法和FCM分别检测细胞放射敏感性、细胞增殖活力、细胞周期分布和凋亡率。双荧光素酶报告基因实验和WB法验证miR-620和ING4的靶向关系。结果：与癌旁组织和HBL-100细胞比较,...     

Differential sensitivity of MCF-7 and LCC2 cells, to multiple growth inhibitory agents: possible relation to high bcl-2/bax ratio?

Comparison of LCC2, the E(2)-independent, tamoxifen-resistant subline of the MCF-7 human breast cancer cell line with its parent line, disclosed that it is more resistant to growth inhibition and apoptosis induction by a variety of agents acting by diverse mechanisms. Thus, LCC2 cells can serve as a useful in-vitro model for the study of the molecular mechanisms of this resistance. It was found that bcl-2 protein and mRNA were elevated and that bax protein and mRNA were reduced in LCC2 compared with MCF-7 cells. Incubation of both lines in the presence of bcl-2 antisense caused growth inhibition and reduced bcl-2 protein levels only in MCF-7 cells, suggesting the involvement of bcl-2 in the regulation of normal growth of breast cancer cells. Increased bcl-2 expression in breast cancer cells may correlate with their resistance to growth inhibitory agents. Bcl-2 is a useful target for enhancing the effects of growth inhibitory agents.     

Curcumin improves MMC-based chemotherapy by simultaneously sensitising cancer cells to MMC and reducing MMC-associated side-effects

Mitomycin C (MMC) potently suppresses tumour growth. However, its use is limited by its severe toxicity to the kidney and bone marrow. The purpose of this study is to investigate whether the chemoprevention agent curcumin can reduce MMC-associated side-effects and improve MMC efficacy in a breast cancer xenograft model. We first determined the effectiveness of combined MMC and curcumin to inhibit in vitro cell growth and to regress in vivo tumour outgrowth. We then investigated the mechanisms associated with MMC/curcumin-induced cell death by examining the effect of MMC/curcumin treatment on apoptosis, the activation of caspase-3, 8 and 9 and the expression of bcl-2 and bax. We also evaluated the ability of curcumin to alleviate MMC-associated side-effects by comparing the levels of creatinine/blood urea nitrogen (Cr/BUN) and glutamic oxalacetic transaminase/glutamic pyruvic transaminase (GPT/GOT) in serum between animals receiving MMC alone and MMC/curcumin. Curcumin significantly sensitised MCF-7 and MDA-MB-231 cells to MMC-induced cell death and improved MMC’s ability to regress MCF-7 xenograft. MMC and curcumin together synergistically enhanced apoptosis in MCF-7 cells and the apoptosis most likely resulted from both the activation of caspases and modulation of bcl-2/bax expression. Most importantly, the inclusion of curcumin in MMC treatment decreased MMC-caused severe side-effects evidenced by significant improvement in the kidney function. Enhancing the tumoricidal effect of MMC, curcumin greatly reduces MMC-associated severe side-effects. Therefore, the combination treatment of MMC and curcumin may be of significant therapeutic benefit in treating breast cancer.     

Expression of the bcl-2 gene family in normal and malignant breast tissue: Low bax-α expression in tumor cells correlates with resistance towards apoptosis

We have studied the expression of the apoptosis-regulating genes bcl-2, bcl-x, bax and APO-1/fas (CD95) in human breast cancer. The expression pattern of these genes in human breast-cancer tissues and breast-cancer-derived cell lines was compared to that seen in normal breast epithelium and breast epithelial cell lines. No difference with regard to bcl-2 and bcl-x_L expression was observed between normal breast epithelium and tumor tissue or breast cancer and non-malignant epithelial cell lines. In contrast, bax-α, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal cell lines and breast tissue, whereas only weak or no expression could be detected in cancer-cell lines and malignant tissue. In contrast to malignant cell lines, which express low levels of bax-α, non-malignant epithelial cell lines displaying high amounts of bax-α were highly sensitive to induction of programmed cell death by both serum starvation and APO-1/fas (CD95) triggering. We therefore propose that dysregulation of apoptosis contributes to the pathogenesis of breast cancer, at least in part, due to an imbalance between anti-apoptosis genes (such as bcl-2/bcl-x) and apoptosis-promoting genes (bax).     

Induction of apoptosis by <Emphasis Type="Italic">p53</Emphasis>, <Emphasis Type="Italic">bax</Emphasis>, <Emphasis Type="Italic">bcl-2</Emphasis>, and <Emphasis Type="Italic">p21</Emphasis> expressed in colorectal cancer

Background We investigated the influence of genes on the apoptosis of colorectal tumor cells, based on DNA and mRNA kinetics.Methods In 30 colorectal cancer patients, we examined the mRNA expression of p53, bax, bcl-2, and p21 ^WAF1 , and we also investigated the development of tumor cell apoptosis, using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method.Results TUNEL-positive cells showed a positive correlation with bax (P = 0.010) and a negative correlation with p21 (P = 0.04). We also investigated the relationship between p53 point mutation, p21 immunostaining degree, and apoptosis, based on DNA ladder expression. A remarkable correlation (P = 0.0090) was found between p21 and apoptosis.Conclusions The present study findings suggest that tumor cell apoptosis is (1) strongly inhibited by p21, (2) induced by bax, and (3) influenced by bcl-2, which, presumably, inhibits tumor cell apoptosis.     

乳腺癌耐药细胞中c-fos抗凋亡作用的研究

背景与目的：乳腺癌是女性最常见的恶性肿瘤之一,且患者死亡率居高不下,其中乳腺癌耐药是导致临床化疗失败的主要原因。该课题组采用已建立的乳腺癌耐药细胞模型MCF-7/ADR及其敏感株MCF-7,旨在探讨c-fos在乳腺癌耐药细胞中的详细作用机制。方法：MTT检测上述细胞对多柔比星的敏感性,并通过实时定量PCR(real-time PCR,RT-PCR)检测上述细胞中mdr-1及c-fos的mRNA表达情况;3 μmol多柔比星分别处理MCF-7细胞12、24、36 h后采用RT-PCR检测细胞中c-fos mRNA的表达,以监测化疗药物处理过程中乳腺癌细胞c-fos的表达变化过程;构建相应载体得到了c-fos稳定干扰细胞株及其对照细胞株：MCF-7/ADR/si-fos-8B、MCF-7/ADR/si-fos-3D和MCF-7/ADR/siNC,采用MTT检测干扰c-fos后对5-FU及顺铂的敏感性变化;流式细胞术检测上述药物及γ射线照射处理后细胞凋亡的情况;罗丹明123检测乳腺癌耐药细胞的外排能力;RT-PCR及蛋白质印迹法(Western blot)分别检测c-fos干扰后,凋亡相关基因bax、bcl-2、puma、p53及mdr-1等的表达。结果：与敏感细胞MCF-7相比,MCF-7/ADR细胞中c-fos及mdr-1的表达显著升高,且对多柔比星的耐药倍数为敏感株的近40倍;在3 μmol多柔比星处理MCF-7之后c-fos的表达逐渐升高,结果显示,c-fos在乳腺癌的耐药表型形成早期即开始发挥作用;干扰c-fos后,细胞对药物(5-FU、顺铂)的敏感性增高,且经药物处理及γ射线照射后稳定干扰细胞株的凋亡率显著升高,说明干扰c-fos后乳腺癌耐药细胞在受到外界刺激后,更易于发生凋亡;RT-PCR及Western blot检测结果显示,干扰c-fos后,bax、puma、p53的表达明显升高,而bcl-2及mdr-1的表达显著降低。结论：在乳腺癌耐药细胞MCF-7/ADR中发现,c-fos呈异常高表达,其可能通过调节乳腺癌细胞凋亡信号通路蛋白的变化抑制乳腺癌细胞的凋亡,促进了乳腺癌耐药表型的形成。由此,c-fos可作为克服乳腺癌耐药治疗的一个潜在靶点用于今后的药物开发。     

Apoptosis-mediated inhibition of human breast cancer cell proliferation by lemon citrus extract

Dietary phytochemicals have a variety of antitumor properties. In the present study, the antitumor activity of methanolic extract of lemon fruit (lemon extract; LE) (LE) on the MCF-7 breast cancer cell line was investigated in vitro. Apoptotic cell death was analyzed using the TUNEL assay. In addition, the apoptosis mediated by LE extract in the MCF-7 cells was associated with the increased expression of the tumor suppressor p53 and caspase-3. Additionally, the expression of a pro-apoptotic gene, bax, was increased, and the expression of an anti-apoptotic gene, bcl-2, was decreased by LE extract treatment, resulting in a shift in the Bax:Bcl-2 ratio to one that favored apoptosis. The expression of a major apoptotic gene, caspase-3, was increased by LE extract treatment. In light of the above results, we concluded that LE extract can induce the apoptosis of MCF-7 breast cancer cells via Bax-related caspase-3 activation. This study provides experimental data that are relevant to the possible future clinical use of LE to treat breast cancer.     

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的：研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法：采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果：甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达（68.40±4.87）%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论：甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。     

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的:研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法:采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果:甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达(68.40±4.87)%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论:甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。     

Daidzein induces MCF-7 breast cancer cell apoptosis via the mitochondrial pathway

BackgroundIn order to study the anticancer effects and cellular apoptosis pathways induced by daidzein.Materials and methodsWe used the human MCF-7 breast cancer cell line as a model and examined the apoptosis by Hoechst–propidium iodide staining fluorescence imaging and flow cytometry.ResultsOur data indicated that daidzein induces antiproliferative effects in a concentration- and time-dependent manner. We demonstrated that daidzein-induced apoptosis in MCF-7 cells was initiated by the generation of reactive oxygen species (ROS). Furthermore, we showed that this daidzein-induced ROS generation was accompanied by disruption of mitochondrial transmembrane potential, down-regulation of bcl-2, and up-regulation of bax, which led to the release of cytochrome C from the mitochondria into the cytosol, which, in turn, resulted in the activation of caspase-9 and caspase-7, and ultimately in cell death. The induction of the mitochondrial caspase-dependent pathway was confirmed by pretreatment with pan-caspase inhibitor z-VAD-fmk and antioxidant N-acetyl-L-cysteine.ConclusionAccordingly, daidzein could induce breast cancer cell apoptosis through the mitochondrial caspase-dependent cell death pathway.     

Gold nanoparticles induce apoptosis in MCF-7 human breast cancer cells

Background: Gold nanoparticles have recently been investigated with respect to biocompatibility accordingto their interactions with cells. The purpose of this study was to examine cytotoxicity and apoptosis induction bywell-characterized gold nanoparticles in human breast epithelial MCF-7 cells. Methods: Apoptosis was assessedby TUNEL, cytotoxicity by MTT assay and caspase 3, 9, p53, Bax and Bcl expression by real-time PCR assays.Results: Gold nanoparticles at up to 200 μg/mL for 24 hours exerted concentration-dependent cytotoxicity andsignificant upregulation of mRNA expression of p53, bax, caspase-3 & caspase-9, whereas expression of antiapoptoticbcl-2 was down-regulated. Conclusion: To the best of our knowledge this is the first report showingthat gold nanoparticles induce apoptosis in MCF-7cells via p53, bax/bcl-2 and caspase pathways.     

lncRNA H19靶向miR-106a-5p调控乳腺癌细胞放射敏感性的分子机制

目的:研究lncRNA H19对乳腺癌细胞MCF-7增殖、凋亡及放射敏感性的影响,并探讨其机制。方法:运用qRT-PCR检测MCF-7细胞中H19、miR-106a-5p的表达;将si-NC组（转染si-con）、si-H19组（转染si-H19）、si-H19+anti-miR-NC组（si-H19和anti-miR-NC共转染）、si-H19+anti-miR-106a-5p组（si-H19和anti-miR-106a-5p共转染）,均用脂质体转染至MCF-7细胞,再用4 Gy放射照射;MTT法检测各组细胞的增殖情况;流式细胞术检测各组细胞的凋亡情况;双荧光素酶报告基因检测实验检测各组细胞的荧光活性。结果:与对照组相比,放射照射组（IR）MCF-7细胞中H19显著降低,miR-106a-5p显著升高（P＜0.05）;敲减H19、过表达miR-106a-5p均可抑制MCF-7细胞增殖并促进凋亡,增强细胞的放射敏感性;H19靶向miR-106a-5p。抑制miR-106a-5p可逆转敲减H19对MCF-7细胞放射敏感性的增强作用。结论:敲减H19可抑制乳腺癌细胞增殖,促进凋亡,增强放射治疗的敏感性,其机制可能与靶向miR-106a-5p有关,可为提高乳腺癌的放射敏感性提供新靶点。     

Tyrphostin AG 1024 modulates radiosensitivity in human breast cancer cells.

Insulin-like growth factor-1 (IGF-1) plays an important growth-promoting effect by activating the PI3K/Akt signalling pathway, inhibiting apoptotic pathways and mediating mitogenic actions. Tyrphostin AG 1024, one selective inhibitor of IGF-1R, was used to evaluate effects on proliferation, radiosensitivity, and radiation-induced cell apoptosis in a human breast cancer cell line MCF-7. Exposure to Tyrphostin AG 1024 inhibited proliferation and induced apoptosis in a time-dependent manner, and the degree of growth inhibition for IC20 plus irradiation (4 Gy) was up to 50% compared to the control. Examination of Tyrphostin AG 1024 effects on radiation response demonstrated a marked enhancement in radiosensitivity and amplification of radiation-induced apoptosis. Western blot analysis indicated that Tyrphostin AG 1024-induced apoptosis was associated with a downregulation of expression of phospho-Akt1, increased expression of Bax, p53 and p21, and a decreased expression of bcl-2 expression, especially when combined with irradiation. To our knowledge, this is the first report showing that an IGF-1 inhibitor was able to markedly increase the response of tumour cells to ionizing radiation. These results suggest that Tyrphostin AG 1024 could be used as a potential therapeutic agent in combination with irradiation.     

多西紫杉醇诱导人乳腺癌MCF-7细胞凋亡及相关基因表达的影响

目的探讨多西紫杉醇(docetaxcel)诱导体外培养人乳腺癌MCF-7细胞凋亡的作用及凋亡的分子机制。方法通过MTT比色法分析多西紫杉醇对人乳腺癌MCF-7细胞增殖的影响;应用光镜、电镜观察细胞形态学的改变;Annexin-V/PI双染流式细胞术检测肿瘤细胞周期动力学和凋亡诱导率的影响;免疫组化方法检测凋亡相关基因bax、bc1-2的表达变化。结果多西紫杉醇可明显抑制人乳腺癌MCF-7细胞的增殖,并可导致其形态学的改变;随着加药时间的增长,G2/M期细胞增多,G2/M期细胞和S期细胞的比值也随剂量呈递增关系;加药(IC50)48h后早期凋亡细胞明显增多,同时可上调bax和下调bc1-2的蛋白表达量。结论多西紫杉醇可明显抑制MCF-7肿瘤细胞的生长,诱导细胞凋亡。其分子机制可能与激活bax和抑制bc1-2等凋亡调控基因有关。