Resistin Confers Resistance To Doxorubicin-induced Apoptosis In Human Breast Cancer Cells Through Autophagy Induction

Aim:Clear evidence has linked obesity to a high risk of incidence as well as poor clinical outcome of breast cancer.It has been proven that changes in the levels of adipokines caused by obesity are associated with the initiation and progression of breast cancer.Resistin is a novel adipokine that is upregulated in breast cancer patients and promotes breast cancer cell growth,invasion,and migration.The aim of the study was to investigate whether resistin affected the efficacy of doxorubicin(Dox),one of the most effective anthracycline chemotherapeutic agents in the treatment of breast cancer.Methods:1.Human breast cancer cell lines MCF-7 and MDA-MB-231 were treated with Dox and/or resistin.Annexin V binding assay was used to evaluate apoptosis of the treated cells.Western blot analysis was used for determining the expression levels of Cytochrome c,cleaved-Caspase-9 and cleaved-PARP to further confirm apoptotic level.2.Human breast cancer cell lines MCF-7 and MDA-MB-231 were cultured with or without resistin.Immunofluorescence staining was used to demonstrate LC3 expression to reflect autophagy.Western blot analysis was used to test the expression levels of autophagy-related proteins(BECN1,SQSTM1,LC3B-I/II,LAMP1).And quantitative analysis of LC3B-II/LC3B-I ratio was used to further confirm the autophagy level.3.Human breast cancer cell lines MCF-7 and MDA-MB-231 were pretreated with autophagy inhibitor 3-methyladenine(3-MA)and then cultured in the media containing Dox and/or resistin.Flow cytometry and Western Blot were used to test the apoptosis.Then,the cells were infected by lentivirus containing Atg5-sh RNA toknockdown the expression of Atg5.Annexin V binding assay was used to test apoptosis.4.Human breast cancer cell lines MCF-7 and MDA-MB-231 were treated with or without resistin.Western Blot analysis was used to test the expression levels of components of AMPK/m TOR and JNK signaling pathways.Western Blot analysis for LC3B-I/LC3B-II and annexin V binding assay for apoptosis were used when cells were treated with Compound C and SP600125,which are inhibitors of AMPK/m TOR and JNK signaling pathways respectively.Results:First,we demonstrated that Dox effectively induced the apoptosis of both MCF-7and MDA-MB-231 cells.And we found that the addition of resistin significantly decreased Dox-induced apoptosis of breast cancer cells in a dose-dependent and time-dependent manner.As expected,MCF-7 and MDA-MB-231 cells treated with Dox alone had significantly higher levels of cytochrome c,cleaved caspase-9,and cleaved PARP than untreated cells,while addition of resistin significantly decreased the levels of these proteins in the presence of Dox.Next,we detected the accumulation of LC3,a hallmark of mammalian autophagy,by immunofluorescence.Addition of resistin resulted in a remarkable increase in LC3 dots in MCF-7 and MDA-MB-231 cells.It was also confirmed by Western Blot results showing that addition of resistin dramatically increased the expression of LC3B-Ⅱ,BECN1,LAMP1,and the ratio of LC3B-Ⅱ to LC3B-Ⅰ,and decreased the expression of SQSTM1 in a dose-dependent manner.These results suggest that resistin activates autophagy in breast cancer cells.To further confirm that resistin-induced resistance to Dox was mediated through activated autophagy,we used 3-MA(a specific autophagy inhibitor)and a lentiviral vector containing sh RNA of Atg5 inhibit autophagy.Annexin V binding assay and Western blot analysis both confirmed that inhibition of autophagy in breast cancer cells abrogated the protective effect of resistin in Dox-treated cells.Western blot analyses showed that addition of resistin significantly upregulated the expression of p-AMPK and downregulated the expression of p-m TOR and p-ULK1 Ser757 in a dose-dependent manner.Resistin was also found to upregulate the expression of p-JNK and p-Bcl-2 in a dose dependent manner.Meanwhile,autophagy induced by resistin was abrogated by inhibitors of the two pathways,Compound Cand SP600125.These data indicate that activation of AMPK/m TOR/ULK1 and JNK signalings are responsible for pro-survival autophagy induced by resistin.Conclusions:(1)Resistin protects human breast cancer cell lines MCF-7 and MDA-MB-231 against Dox-induced apoptosis;(2)Resistin activates autophagy in human breast cancer cell lines MCF-7 and MDA-MB-231;(3)Autophagy induced by resistin confers Dox resistance in breast cancer cell lines MCF-7 and MDA-MB-231;(4)Resistin induces autophagy via AMPK/m TOR and JNK signaling.Our data firstly propose that adipokine resistin is able to attenuate Dox-induced apoptosis through autophagy induction.This finding suggests that upregulated levels of resistin in breast cancer patients could facilitate the chemoresistance of tumor cells.Thus,our study reveals a new role of resistin in breast cancer,and provides a potential molecular target to overcome chemoresistance in the treatment of breast cancer.