Apoptosis and Autophagy in Breast Cancer Cells following Exemestane Treatment

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G(0)/G1 or in G(2)/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.     

Role of Autophagy in Breast Cancer

Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1^+/- mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer-promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.

Addendum to:

Autophagy Mitigates Metabolic Stress and Genome Damage in Mammary Tumorigenesis

V. Karantza-Wadsworth, S. Patel, O. Kravchuk, G. Chen, R. Mathew, S. Jin and E. White

Genes Dev 2007; 21:1621-35     

Icaritin Synergistically Enhances the Radiosensitivity of 4T1 Breast Cancer Cells

Icaritin (ICT) is a hydrolytic form of icariin isolated from plants of the genus Epimedium. This study was to investigate the radiosensitization effect of icaritin and its possible underlying mechanism using murine 4T1 breast cancer cells. The combination of Icaritin at 3 µM or 6 µM with 6 or 8 Gy of ionizing radiation (IR) in the clonogenic assay yielded an ER (enhancement ratio) of 1.18 or 1.28, CI (combination index) of 0.38 or 0.19 and DRI (dose reducing index) of 2.51 or 5.07, respectively. These strongly suggest that Icaritin exerted a synergistic killing (?) effect with radiation on the tumor cells. This effect might relate with bioactivities of ICT: 1) exert an anti-proliferative effect in a dose- and time-dependent manner, which is different from IR killing effect but likely work together with the IR effect; 2) suppress the IR-induced activation of two survival paths, ERK1/2 and AKT; 3) induce the G2/M blockage, enhancing IR killing effect; and 4) synergize with IR to enhance cell apoptosis. In addition, ICT suppressed angiogenesis in chick embryo chorioallantoic membrane (CAM) assay. Taken together, ICT is a new radiosensitizer and can enhance anti-cancer effect of IR or other therapies.     

RNA干涉介导的Plk1沉默对乳腺癌细胞恶性生物表型的影响

目的：研究乳腺癌细胞中丝／苏氨酸蛋白激酶Plk1基因表达下调后对其恶性生物表型的影响。方法：利用pSitencer4．1-CMVneo质粒,分别构建针对Plk1基因的RNA干涉载体（pSilencer4．1-shPlk1）,利用脂质体Lipofectamine2000转染MCF-7细胞,G418筛选稳定的转染细胞系。半定量RT—PCR和Western blot分别检测Plk1基因mRNA和蛋白表达,MTT和克隆形成试验检测细胞增殖活性的变化,流式细胞仪分析细胞周期和凋亡的变化,最后分析MCF-7细胞对紫杉类药物（紫杉醇和多西他赛）化疗敏感性的变化。结果：成功筛选了稳定转染细胞系（MCF-7／shPlk1和MCF-7／shcontro1）。同MCF-7／shPlk1细胞相比,MCF-7／shPtkl细胞中Plk1基因mRNA和蛋白表达水平分别下调65．8％和74．4％（P〈0．05）。同MCF-7／shcontrol,MCF-7tshPlk1细胞增殖速度显著抑制,到第5天时抑制率达到44．9±3．2％（P〈0．05）。同时,MCF-7／shPlk1细胞的克隆形成能力显著降低（P〈0．01）流式细胞仪技术分析细胞周期结果表明：MCF-7／shPlk1细胞的G2／M期细胞比例显著增加了21．1±4．1％,而S期细胞比例则显著降低了（18．5±3．1％;P〈0．05）。流式细胞仪技术分析细胞凋亡结果表明：MCF-7／shPlk1细胞的凋亡率约显著增加了13．1±213％（P〈0．05）,同时还发现：MCF-7／shPlk1细胞中激活的caspase-3蛋白显著增加,Bcl-2蛋白显著降低,而Bax蛋白则显著增加。结论：RNA干涉载体能特异性下调乳腺癌细胞中Plk1基因的表达,从而抑制乳腺癌细胞的增殖和体外克隆形成能力,同时诱导乳腺癌细胞的G2／M期阻滞和细胞凋亡率显著增加。因此,靶向Plk1基因的生物治疗有望成为未来临床乳腺癌的一个重要的辅助治疗策略.     

Induction of Apoptosis and Autophagy Using Ectopic DSCR1 Expression in Breast Cancer Cells

Down syndrome critical region 1 gene (DSCR1) is an anti-angiogenesis gene that inhibits the growth of tumor cells. In this study, the role of autophagy and apoptosis in DSCR1-induced cytotoxicity were investigated in MDA-MB-468 breast cancer cells. Lentivirus vector harboring DSCR1 (LV-DSCR1⁺) was constructed in HEK 293 cells and the optimal dosage of lentivirus vector for infection was determined by the MTT assay. After infection of cells using LV-DSCR1⁺, acridine orange and ethidium bromide staining was performed to investigation of apoptosis and autophagy. Expression of DSCR1 and marker genes for angiogenesis (VEGF), apoptosis (Bax and Bcl2) and autophagy (LC3 and Beclin) were determined by Real time PCR. The cellular morphological changes related to apoptosis and autophagy was happened after 48 hours of viral infection. Fragmented bright orange nucleuses and vacuoles were observed due to the cell apoptosis and autophagy after acridine orange and ethidium bromide staining. Upregulation of Bax, Lc3, DSCR1 and Beclin1 and downregulation of Bcl2 and VEGF was detected due to treatment with LV-DSCR1⁺. These results demonstrated that LV-DSCR1⁺ can induce apoptosis and autophagy, therefore suggesting that it may serves as an efficient tool to breast cancer treatment.     

RNA 干涉介导的Plk1 沉默对乳腺癌细胞恶性生物表型的影响

目的:研究乳腺癌细胞中丝/苏氨酸蛋白激酶Plk1基因表达下调后对其恶性生物表型的影响。方法:利用pSilencer4.1-CMVneo质粒,分别构建针对Plk1基因的RNA干涉载体(pSilencer4.1-shPlk1),利用脂质体Lipofectamine2000转染MCF-7细胞,G418筛选稳定的转染细胞系。半定量RT-PCR和Western blot分别检测Plk1基因mRNA和蛋白表达,MTT和克隆形成试验检测细胞增殖活性的变化,流式细胞仪分析细胞周期和凋亡的变化,最后分析MCF-7细胞对紫杉类药物(紫杉醇和多西他赛)化疗敏感性的变化。结果:成功筛选了稳定转染细胞系(MCF-7/shPlk1和MCF-7/shcontrol)。同MCF-7/shPlk1细胞相比,MCF-7/shPlk1细胞中Plk1基因mRNA和蛋白表达水平分别下调65.8%和74.4%(P<0.05)。同MCF-7/shcontrol,MCF-7/shPlk1细胞增殖速度显著抑制,到第5天时抑制率达到44.9±3.2%(P<0.05)。同时,MCF-7/shPlk1细胞的克隆形成能力显著降低(P<0.01)。流式细胞仪技术分析细胞周期结果表明:MCF-7/shPlk1细胞的G2/M期细胞比例显著增加了21.1±4.1%,而S期细胞比例则显著降低了(18.5±3.1%;P<0.05)。流式细胞仪技术分析细胞凋亡结果表明:MCF-7/shPlk1细胞的凋亡率约显著增加了13.1±2.3%(P<0.05)。同时还发现:MCF-7/shPlk1细胞中激活的caspase-3蛋白显著增加,Bcl-2蛋白显著降低,而Bax蛋白则显著增加。结论:RNA干涉载体能特异性下调乳腺癌细胞中Plk1基因的表达,从而抑制乳腺癌细胞的增殖和体外克隆形成能力,同时诱导乳腺癌细胞的G2/M期阻滞和细胞凋亡率显著增加。因此,靶向Plk1基因的生物治疗有望成为未来临床乳腺癌的一个重要的辅助治疗策略。     

BID-Deficient Breast Cancer MCF-7 Cells as a Model for the Study of Autophagy in Cancer Therapy

The BH3-only death factors share just the short BH3 domain with the other Bcl-2 family subclasses. With the exception of BID, which might also bind to BAX, they are thought to act by binding to and neutralizing Bcl-2 like survival factors.Camptothecin (CPT)-induced apoptosis in breast cancer MCF-7 cells is associated with activation of cathepsin B and aggregation of BAX and BID on mitochondria. BID knock down protects cancer cells against apoptosis and induces autophagy, manifested with increased expression of Beclin1 and MAP1LC3. The compensatory increase in the concentration of Hrk (another member of the BH3-only protein family) and its co-localization with BCL-2 on organelles in BID(-) breast cancer cells has also been observed. Nonetheless, Hrk is not able to substitute for BID in triggering apoptosis. Its role in autophagy induction is also doubtful, since MAP1LC3 expression was equally high in BID(-)Hrk(-) and BID(-)Hrk(+) breast cancer cells exposed to CPT. We conclude that BID can serve as a molecular switch between apoptosis and autophagy. BID(+) and BID(-) breast cancer MCF-7 cells could be considered to be a useful model for the study of the molecular interdependences between apoptosis and autophagy and the role of both processes in cancer therapy.

Addenda to:

Cathepsins and BID are Involved in the Molecular Switch between Apoptosis and Autophagy in Breast Cancer MCF-7 Cells Exposed to Camptothecin

M. Lamparska-Przybysz, B. Gajkowska and T. Motyl

J Physiol Pharmacol 2005; 56:159-79     

Arctigenin Enhances Chemosensitivity to Cisplatin in Human Nonsmall Lung Cancer H460 Cells through Downregulation of Survivin Expression

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin‐mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin‐treated non‐small‐cell lung cancer (NSCLC) H460 cells. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and annexin‐V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose‐dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase‐3 and poly(ADP‐ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin‐induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell‐cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina‐tion with chemotherapeutic agents for NSLC.     

抑制c-FLIP基因促进TRAIL诱导的乳腺癌细胞凋亡(英文)

目的:探讨抑制c-FLIP的表达对TRAIL诱导乳腺癌细胞MCF-7凋亡的影响。方法:重组腺病毒Ad-c-FLIP-siRNA和Ad-sTRAIL单独及联合感染对TRAIL耐药的乳腺癌细胞MCF-7,应用实时荧光定量聚合酶链反应(Real-time PCR)检测病毒感染后各组细胞内c-FLIP和TRAIL的mRNA表达变化;MTT法和结晶紫染色法检测MCF-7细胞活性,Hoechst 33258荧光染色检测各组细胞的凋亡情况。结果:与阴性对照组比较,c-FLIP-siRNA组和c-FLIP-siRNA+TRAIL组c-FLIP的mRNA相对表达量分别是(0.32±0.16)和(0.39±0.48)倍;TRAIL组和c-FLIP-siRNA+TRAIL组TRAIL的mRNA相对表达量分别是(96.21±1.54)和(87.33±1.66)倍;TRAIL组、c-FLIP-siRNA组及c-FLIP-siRNA+TRAIL组的抑制率(%)分别为(60.27±1.25)、(11.34±1.74)及(74.91±2.12)。对比阴性对照组的凋亡率(3.12±1.54),TRAIL组(12.79±2.46)和c-FLIP-siRNA+TRAIL组(25.50±3.17)组的凋亡率明显增高(P0.05),c-FLIP-siRNA组(6.85±2.82)的凋亡率变化不明显,差异无统计学意义(P0.05)。结论:siRNA抑制c-FLIP基因的表达能显著促进TRAIL对乳腺癌细胞MCF-7凋亡的诱导作用。     

抑制c-FLIP基因促进TRAIL诱导的乳腺癌细胞凋亡

目的：探讨抑制c-FLIP的表达对TRAIL诱导乳腺癌细胞MCF-7凋亡的影响。方法：重组腺病毒Ad-c-FLIP-siRNA和Ad-sTRAIL单独及联合感染对TRAIL耐药的乳腺癌细胞MCF-7,应用实时荧光定量聚合酶链反应（Real-time PCR）检测病毒感染后各组细胞内c-FLIP和TRAIL的mRNA表达变化;MTT法和结晶紫染色法检测MCF-7细胞活性,Hoechst 33258荧光染色检测各组细胞的凋亡情况。结果：与阴性对照组比较,c-FLIP-siRNA组和c-FLIP-siRNA＋TRAIL组c-FLIP的mRNA相对表达量分别是（0.32±0.16）和（0.39±0.48）倍;TRAIL组和c-FLIP-siRNA＋TRAIL组TRAIL的mRNA相对表达量分别是（96.21±1.54）和（87.33±1.66）倍;TRAIL组、c-FLIP-siRNA组及c-FLIP-siRNA＋TRAIL组的抑制率（%）分别为（60.27±1.25）、（11.34±1.74）及（74.91±2.12）。对比阴性对照组的凋亡率（3.12±1.54）,TRAIL组（12.79±2.46）和c-FLIP-siRNA＋TRAIL组（25.50±3.17）组的凋亡率明显增高（P〈0.05）,c-FLIP-siRNA组（6.85±2.82）的凋亡率变化不明显,差异无统计学意义（P〉0.05）。结论：siRNA抑制c-FLIP基因的表达能显著促进TRAIL对乳腺癌细胞MCF-7凋亡的诱导作用。     

HDAC Inhibition Induces Increased Choline Uptake and Elevated Phosphocholine Levels in MCF7 Breast Cancer Cells

Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using ¹³C MRS to monitor [1,2-¹³C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by ³¹P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA₂) isoforms, resulting in increased PLA₂ activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using ¹H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment.     

Lin28 Induces Epithelial-to-Mesenchymal Transition and Stemness via Downregulation of Let-7a in Breast Cancer Cells

The RNA-binding protein Lin28 is known to promote malignancy by inhibiting the biogenesis of let-7, which functions as a tumor suppressor. However, the role of the Lin28/let-7 axis in the epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer has not been clearly expatiated. In our previous study, we demonstrated that let-7 regulates self-renewal and tumorigenicity of breast cancer stem cells. In the present study, we demonstrated that Lin28 was highly expressed in mesenchymal (M) type cells (MDA-MB-231 and SK-3rd), but it was barely detectable in epithelial (E) type cells (MCF-7 and BT-474). Lin28 remarkably induced the EMT, increased a higher mammosphere formation rate and ALDH activity and subsequently promoted colony formation, as well as adhesion and migration in breast cancer cells. Furthermore, we demonstrated that Lin28 induced EMT in breast cancer cells via downregulation of let-7a. Strikingly, Lin28 overexpression was found in breast cancers that had undergone metastasis and was strongly predictive of poor prognoses in breast cancers. Given that Lin28 induced the EMT via let-7a and promoted breast cancer metastasis, Lin28 may be a therapeutic target for the eradication of breast cancer metastasis.     

长链非编码RNA RP11-214F16.8 促进乳腺癌细胞增殖

长链非编码RNAs (long non-coding RNAs, lncRNAs)在乳腺癌发生发展中的作用备受瞩目。本研究通过大数据分析发现,lncRNA RP11-214F16.8在乳腺癌组织中的表达量显著高于正常组织,且其表达量与乳腺癌患者预后负相关。因此,本文采用qRT-PCR技术,在收集的临床样本中验证RP11-214F16.8的表达,证实其在乳腺癌组织中相对表达量为5.65±0.72,其在癌旁组织中表达量为2.63±0.35,且RP11-214F16.8的表达量与乳腺癌瘤体大小和临床TNM分期相关。此外发现,RP11-214F16.8在乳腺癌细胞中的表达量高于正常乳腺上皮细胞。在乳腺癌MCF-7细胞中,过表达RP11-214F16.8后,MCF-7细胞的增殖能力显著增强。而在MDA-MB-231细胞中,敲低RP11-214F16.8的表达,获得相反的结果。进一步研究发现,RP11-214F16.8可上调细胞周期蛋白 D3、CDK4的表达,而下调p18蛋白表达量,进而加速细胞增殖。总之,RP11-214F16.8在乳腺癌中高表达,且促进乳腺癌细胞增殖,进而推动乳腺癌进程。这一研究发现,将为乳腺癌发生发展的网络机制研究提供新的理论依据。     

LncRNA ITGA9-AS1抑制乳腺癌细胞增殖并增强其对顺铂的敏感性

长链非编码RNAs (long non-coding RNAs, lncRNAs) 是一类长度大于200 nt,无蛋白质编码功能的RNAs。近年来,lncRNAs在肿瘤发生发展中的作用备受关注。LncRNAs芯片分析结合后期实时荧光定量PCR验证发现,ITGA9-AS1在MCF-7细胞中的表达量显著高于耐药细胞MCF-7/5Fu,且其在乳腺癌细胞中的表达量显著低于正常乳腺上皮细胞。生物信息学预测,ITGA9 AS1无蛋白质编码功能。在乳腺癌细胞T47D中过表达ITGA9-AS1,可显著抑制该细胞的增殖和克隆形成能力,增加该细胞对化疗药物顺铂（cisplatin, cDDP）的敏感性。相反,在乳腺上皮细胞MCF-10A中敲低ITGA9-AS1的表达,能够明显增加该细胞的增殖能力和克隆形成能力,同时降低该细胞对cDDP的敏感性。总之,lncRNA ITGA9-AS1可抑制乳腺癌细胞增殖,增强乳腺癌细胞对化疗药物的敏感性。