Beclin 1对凋亡和自噬的调控作用

Beclin 1是自噬关键调控蛋白之一,参与自噬体膜形成.近期,大量研究结果指出, Beclin 1是caspase家族蛋白酶的全新底物,可被caspase剪切.剪切后的Beclin 1失去自噬调节功能,转而加剧凋亡进程.因而,Beclin 1对细胞凋亡和自噬起着重要的调控作用. 本文主要对细胞凋亡和自噬的相关性,以及Beclin 1在两通路中的调控作用进行了回顾与总结.在此基础上,进一步讨论了Beclin 1与人类疾病如肿瘤、神经系统退行性疾病的关联.最后,简要介绍了实验室常用于Beclin 1研究的工具.     

Cucurbitacin E Induces Cell Cycle G2/M Phase Arrest and Apoptosis in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a highly aggressive form of breast cancer resistant to many common treatments. In this study, we compared the effects of 12 phytochemical drugs on four cancer cell lines, and noticed that Cucurbitacin E (CuE) significantly inhibited TNBC cell growth by inducing cell cycle G2/M phase arrest and apoptosis. CuE reduced expression of Cyclin D1, Survivin, XIAP, Bcl2, and Mcl-1 in MDA-MB-468 and SW527, and within MDA-MB-468, CuE significantly increased activation of JNK and inhibited activation of AKT and ERK. Collectively, these results suggest that CuE may be a viable compound for developing novel TNBC therapeutics.     

Lactaptin Induces p53-Independent Cell Death Associated with Features of Apoptosis and Autophagy and Delays Growth of Breast Cancer Cells in Mouse Xenografts

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.     

The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.     

Metabotropic Glutamate Receptor-1 Contributes to Progression in Triple Negative Breast Cancer

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.     

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.     

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.     

Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway

Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.     

Monitoring the Antioxidant Mediated Chemosensitization and ARE-Signaling in Triple Negative Breast Cancer Therapy

Chemotherapy often fails due to cellular detoxifying mechanisms, including phase-II enzymes. Activation of Nrf2-Keap1 pathway induces phase-II enzymes expression through ARE-signaling and prevents cancer development. Nrf2-overexpression in cancer cells results in chemo- and/or radioresistance. This necessitates understanding of Nrf2-regulation, and identification of Nrf2 activators/inhibitors sensitizing cancer cells to improve chemotherapy. N-terminal 435-amino acids of Nrf2 are crucial for Keap1 binding during ubiquitination. Identification of a minimum Nrf2-domain required for Keap1 binding without altering endogenous ARE-signaling would be a novel tool to study Nrf2-signaling. Current study developed firefly-luciferase reporter fusion with N-terminal Nrf2-domain of different lengths and examined its response to Nrf2-activators in cells. The results identified FLuc2 fusion with N-terminal 100-aa of Nrf2 is sufficient for measuring Nrf2-activation in cancer cells. We used MDA-MB231 cells expressing this particular construct for studying antioxidant induced Nrf2-activation and chemosensitization in triple-negative breast cancer therapy. While antioxidant EGCG showed chemosensitization of MDA-MB231 cells to cisplatin by activating Nrf2-ARE signaling, PTS, another antioxidant showed chemoprotection. Tumor xenograft study in mouse demonstrates that combinational treatment by cisplatin/EGCG resulted in tumor growth reduction, compared to cisplatin alone treatment. The results of this study highlight the importance of identifying selective combination of antioxidants/chemotherapeutic agents for customized treatment strategy.     

Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells

Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.     

三阴性乳腺癌中环状RNA的表达特点及功能意义

环状RNA是由前体RNA通过反向剪接形成的一类共价闭合环状分子.在过去,环状RNA被认为是DNA转录的"噪音",不参与生物代谢过程.然而,最近研究表明,环状RNA的异常表达可影响包括三阴性乳腺癌在内的多种恶性肿瘤的发生发展.该文综述了环状RNA在肿瘤中的分子机制及其在三阴性乳腺癌细胞增殖、凋亡、迁移、侵袭和药物抗性中的...     

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

Background

Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.

Materials and Methods

PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.

Results

PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.

Conclusions

Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.     

Deguelin Action Involves c-Met and EGFR Signaling Pathways in Triple Negative Breast Cancer Cells

Background

Treatment of breast cancer patients with antiestrogens and aromatase inhibitor(s) or Herceptin have shown significant success in steroid receptor positive or Her-2+ breast cancers respectively. However, choice of treatments for breast cancer patients with negative status for estrogen, progesterone receptors and HER2/neu is limited. As a result, search for appropriate therapy regimen for these triple negative breast cancers (TNBC) has become a major focus of investigations for many laboratories. Recently, Deguelin, a natural product isolated from African plant Mundulea sericea (Leguminossae) has shown both antiproliferative actions in various cancers including breast as well as chemoprenventive activity against carcinogen induced experimental cancers. In this report we evaluated efficacy and mechanism of action of Deguelin in triple negative breast cancer cell lines.

Methods/Findings

In vitro, Deguelin in a dose and time dependent manner inhibited the growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells. Deguelin (2 or 4 mg/kg body weight), when injected intraperitoneally, reduced the in vivo tumor growth of MDA-MB-231 cells transplanted subcutaneously in athymic mice. Moreover it was nontoxic as evident from daily observations on mobility, food and water consumption and comparison of bodyweight and other visceral organ weights with those in control animals at the termination of the study. The western blot analyses and immunostaining studies indicated that the deguelin effects may be mediated through EGFR-PAKT/c-Met p-ERK and NF-κB by down regulating their downstream targets such as p-STAT3, c-Myc, Survivin.

Conclusion/Significance

These results suggest that Deguelin may have a significant therapeutic value for the treatment of TNBC patients.     

A Biomimetic Collagen Derived Peptide Exhibits Anti-Angiogenic Activity in Triple Negative Breast Cancer

We investigated the application of a mimetic 20 amino acid peptide derived from type IV collagen for treatment of breast cancer. We showed that the peptide induced a decrease of proliferation, adhesion, and migration of endothelial and tumor cells in vitro. We also observed an inhibition of triple negative MDA-MB-231 xenograft growth by 75% relative to control when administered intraperitoneally for 27 days at 10 mg/kg. We monitored in vivo the changes in vascular properties throughout the treatment using MRI and found that the vascular volume and permeability surface area product decreased significantly. The treatment also resulted in an increase of caspase-3 activity and in a reduction of microvascular density. The multiple mode of action of this peptide, i.e., anti-angiogenic, and anti-tumorigenic, makes it a viable candidate as a therapeutic agent as a monotherapy or in combination with other compounds.     

长春瑞滨联合顺铂治疗三阴性乳腺癌的临床观察

目的：观察长春瑞滨联合顺铂治疗三阴性乳腺癌临床疗效。方法：选择我院2007年5月～2011年8月期间收治经细胞学或病理学诊断并确诊TNBC患者85例,术后免疫学化疗证实ER、PR、HER-2均呈阴性,随机分组入试,长春瑞滨联合顺铂（NP组）45例,第1、8天予以静脉滴注长春瑞滨25 mg/m2,前3天辅以顺铂25 mg/m2滴注,21天为1个周期,治疗2个周期后观察临床疗效;紫杉醇联合顺铂（TP组）40例,第1天小剂量滴注紫杉醇135～175 mg/m2,,顺铂用量、用法及用药周期同上所示。对比观察病症缓释情况和毒副作用。结果：NP组总有效率（RR）48.89%,控制率（PD）为77.78%,TP组总有效率（RR）为37.50%,控制率（PD）55.56%,两组间有效率及控制率比较差异有统计学意义（P〈0.05）。两组常见毒副反应为骨髓抑制、消化道和胃肠道反应。NP组消化道反应和骨髓抑制微重于TP组,两组间比较有显著差异（P〈0.05）。结论：长春瑞滨联合顺铂（NP）新辅助化疗方案针对三阴性乳腺癌患者耐受性好、毒副反应较小、有效率高、不良反应可逆等优势,可作为临床推广药物。