REDD1 and Related Molecular Mechanism of Zoledronic Acid-treated Human Breast Cancer Cell Lines under Normoxic and Hypoxic Conditions

• Main Authors: Ya-Chun Lan, 藍雅君
• Other Authors: Chin-Wen Chi
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/96953886182507434614

碩士 === 國立陽明大學 === 藥理學研究所 === 100 === Zoledronic acid (ZOL) is a bisphosphonate used to prevent/treat skeletal complications in breast cancer patients with bone metastasis. Previous study has found that ZOL inhibited growth and mTOR expression in breast cancer cells. REDD1 is a regulator of DNA damage response. It not only can inhibit mTOR signaling pathway, but also can be induced under hypoxia and ER stress. In this study, breast cancer cell lines with or without drug resistance were used to investigate the efficacy of ZOL and its related molecular mechanism under normoxic and hypoxic conditions. The results showed that under normoxia the growth of three breast cancer cell lines was decreased obviously after treatment with ZOL. Compared with MCF-7/WT cells, more malignant breast cancer cells including MCF-7/ADR and MDA-MB-231 were more sensitive to ZOL treatment. In contrast to normoxia, ZOL treatment under hypoxia showed stronger growth inhibition effect in breast cancer cell lines. RT-qPCR and Western blot analysis showed that under nomoxia ZOL enhanced the mRNA level and protein expression of REDD1; and the increased level of REDD1 was higher in MCF-7/ADR and MDA-MB-231 cells as compared with that of MCF-7/WT cells. However, under hypoxia the mRNA level of REDD1 was increased, but protein expression of REDD1 was decreased in the three breast cancer cell lines. Moreover, ZOL treatment inhibited mTOR and P70S6K activation. ZOL treatment also induced the protein expression of LC3-an autophagy marker in breast cancer cells. In cell growth assay, co-treatment with ZOL and 3-Methyladenine (3-MA), an autophagy inhibitor, enhanced cell death in MCF-7/ADR and MDA-MB-231 cells. The results showed that autophagy could be a protective function in ZOL-treated breast cancer cells. ZOL treatment of breast cancer cells also resulted in activation of eIF2α. After transfected Ser52A plasmid (a phosphorylation-deficient mutant form of eIF2α) in MDA-MB-231 cells, the cell growth assay result showed that the growth inhibition after ZOL treatment became weaker. Moreover, the increased level of REDD1 protein expression after ZOL treatment was lower and the activated level of P70S6K was also decreased. Therefore, these results together indicated that ZOL-induced cell death was caused by ER stress activated eIF2α to induce REDD1 and inhibit mTOR pathway. These findings may provide important clues for future application in breast cancer therapy.