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Downregulation of autophagy by Bcl-2 promotes MCF7 breast cancer cell growth independent of its inhibition of apoptosis.

ABSTRACT: The anti-apoptotic Bcl-2 protein, which confers oncogenic transformation and drug resistance in most human cancers, including breast cancer, has recently been shown to effectively counteract autophagy by directly targeting Beclin1, an essential autophagy mediator and tumor suppressor. However, it remains unknown whether autophagy inhibition contributes to Bcl-2-mediated oncogenesis. Here, by using a loss-of-function mutagenesis study, we show that Bcl-2-mediated antagonism of autophagy has a critical role in enhancing the tumorigenic properties of MCF7 breast cancer cells independent of its anti-apoptosis activity. A Bcl-2 mutant defective in apoptosis inhibition but competent for autophagy suppression promotes MCF7 breast cancer cell growth in vitro and in vivo as efficiently as wild-type Bcl-2. The growth-promoting activity of this Bcl-2 mutant is strongly correlated with its suppression of Beclin1-dependent autophagy, leading to sustained p62 expression and increased DNA damage in xenograft tumors, which may directly contribute to tumorigenesis. Thus, the anti-autophagic property of Bcl-2 is a key feature of Bcl-2-mediated oncogenesis and may in some contexts, serve as an attractive target for breast and other cancer therapies.

SUBMITTER: Oh S

PROVIDER: S-EPMC3017654 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:CNS tumors, including medulloblastoma and pediatric glioblastoma (pGBM) account for the majority of solid pediatric malignancies. There remains an unmet need to identify novel treatment approaches in poor prognosis and relapsed pediatric brain tumors, where therapeutic options are limited. Small-molecule B-cell lymphoma 2 (BCL-2) family inhibitors may enhance tumor cell killing when combined with conventional and targeted chemotherapeutic agents. We investigated the effect of disrupting BCL-2 and B cell lymphoma-extra large (BCL-XL) protein function using ABT-263, ABT-199 and WEHI-539 in medulloblastoma and pGBM cells following treatment with MLN8237, an Aurora kinase inhibitor under investigation as a novel agent for the treatment of malignant brain tumors. Methods:Tumor cell growth and viability were determined by MTT/WST-1 assays and flow cytometry. Effects on cell phenotype, cell cycle progression, and ploidy were determined by live cell imaging and DNA content analysis. Apoptosis was determined by annexin V/propidium iodide staining and time-lapse microscopy and confirmed by measuring caspase-3/7 activity and western blotting and by short interfering RNA (siRNA) knockdown of BCL-2 associated X protein/BCL-2 antagonist killer (BAX/BAK). Results:ABT-263, in combination with MLN8237, reduced mitotic slippage and polyploidy and promoted the elimination of mitotically defective cells via a BAX/BAK-dependent, caspase-mediated apoptotic pathway. The BCL-XL antagonist, WEHI-539, significantly augmented tumor cell killing when used in combination with MLN8237, as well as sensitized resistant brain tumor cells to a novel BAX activator, SMBA1. In addition, siRNA-mediated knockdown of BCL-XL sensitized pGBM and medulloblastoma cells to MLN8237 and mimicked the effect of combination drug treatment. Conclusion:Selective small-molecule inhibitors of BCL-XL may enhance the efficacy of MLN8237 and other targeted chemotherapeutic agents.

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Project description:The purpose of this study was to assess in vitro whether the biological effects of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy are enhanced by inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL in different glioblastoma models. Pre-clinical testing of a microcontroller-based device emitting light of 405 nm wavelength in combination with exposure to 5-ALA (PDT) and the Bcl-2/Bcl-xL inhibitor ABT-263 (navitoclax) was performed in human established and primary cultured glioblastoma cells as well as glioma stem-like cells. We applied cell count analyses to assess cellular proliferation and Annexin V/PI staining to examine pro-apoptotic effects. Western blot analyses and specific knockdown experiments using siRNA were used to examine molecular mechanisms of action. Bcl-2/Bcl-xL inhibition synergistically enhanced apoptosis in combination with PDT. This effect was caspase-dependent. On the molecular level, PDT caused an increased Noxa/Mcl-1 ratio, which was even more pronounced when combined with ABT-263 in a Usp9X-independent manner. Our data showed that Bcl-2/Bcl-xL inhibition increases the response of glioblastoma cells toward photodynamic therapy. This effect can be partly attributed to cytotoxicity and is likely related to a pro-apoptotic shift because of an increased Noxa/Mcl-1 ratio. The results of this study warrant further investigation.

| S-EPMC8393699 | biostudies-literature

Rhazyaminine from Rhazya stricta Inhibits Metastasis and Induces Apoptosis by Downregulating Bcl-2 Gene in MCF7 Cell Line.

Project description:BACKGROUND:The role of alkaloids isolated from Rhazya stricta Decne (Apocynaceae family) (RS) in targeting genes involved in cancer and metastasis remains to be elucidated. OBJECTIVE:Identify and characterize new compounds from RS, which inhibit gene(s) involved in the survival, invasion, self-renewal, and metastatic processes of cancer cells. METHODS:Bioinformatics study was performed using HISAT2, stringtie, and ballgown pipeline to understand expressional differences between a normal epithelial cell line-MCF10A and MCF7. NMR and ATR-FTIR were performed to elucidate the structure of rhazyaminine (R.A), isolated from R stricta. Cell viability assay was performed using 0, 25, and 50 ?g/mL of total extract of R stricta (TERS) and R.A, respectively, for 0, 24, and 48 hours, followed by scratch assay. In addition, total RNA was isolated for RNA- seq analysis of MCF7 cell line treated with R.A followed by qRT-PCR analysis of Bcl-2 gene. RESULTS:Deptor, which is upregulated in MCF7 compared with MCF10A as found in our bioinformatics study was downregulated by R.A. Furthermore, R.A effectively reduced cell viability to around 50% ( P < .05) and restricted cell migration in scratch assay. Thirteen genes, related to metastasis and cancer stem cells, were downregulated by R.A according to RNA- seq analysis. Additionally, qRT-PCR validated the downregulation of Bcl-2 gene in R.A-treated cells by less than 0.5 folds ( P < .05). CONCLUSION:R.A successfully downregulated key genes involved in apoptosis, cell survival, epithelial-mesenchymal transition, cancer stem cell proliferation, and Wnt signal transduction pathway making it an excellent "lead candidate" molecule for in vivo proof-of-concept studies.

| S-EPMC7240879 | biostudies-literature

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