Chloroquine Selectively Enhances The Inhibitory Effect Of Isorhamnetin On Triple-negative Breast Cancer Cells

BackgroundBreast cancer is the most common malignancy and is a leading cause of cancer-related deaths in women worldwide.Among its different subtypes,triple-negative breast cancer（TNBC）has been a major concern due to its high invasiveness,high recurrence rate and poor prognosis.Therefore,developing new drugs for the treatment of TNBC is urgently required.Natural products have always been a new source of anticancer drugs,and finding new anti-breast cancer drugs from natural products is still an important strategy in the treatment of TNBC patients.Isorhamnetin（IH）is a flavonoid compound isolated from the fruit of the Hippophae fhamnoides L.In the present study,we found that IH selectively inhibits the proliferation of TNBC cells,and inhibition of autophagy/mitophagy by chloroquine（CQ）selectively enhances IH-induced mitochondrial fission and apoptosis in TNBC cells but not in estrogen-dependent breast cancer cells.These findings suggest that IH could potentially be further developed as a novel chemotherapeutic agent,and a combination of IH with classic autophagy/mitophagy inhibitor（CQ）could represent a novel therapeutic strategy for treatment of TNBC.PurposeTo explore the mechanism of CQ/IH combination in inhibiting cell profileration in TNBC,and to develop the combination of CQ/IH as a novel therapeutic strategy in the treatment of TNBC.MethodsTNBC cells and xenografted mice were treated with a combination of chloroquine（CQ）and isorhamnetin（IH）.Apoptosis and ROS were determined by flow cytometry.Mitochondrial fission and mitophagy were determined by confocal microscopy.The expression of related proteins was determined by western blot.The expression of related proteins in the tumor was detected by immunohistochemistry assay.Results1.IH induces autophagy in TNBC cellsWestern blot analysis showed that treatment of triple negative breast cancer MDA-MB-231 and BT549 cells with IH resulted in a dose-and time-dependent decrease in levels of p62and increase in levels of LC3-II,Beclin1 and ATG5.Next,cells transfected with a tandem reporter construct（tfLC3）were treated with IH followed by assessment of EGFP-LC3 and mRFP-LC3 puncta colocalization.We found that exposure of cells to IH caused pronounced formation of LC3 puncta that displayed only red fluorescence intensity,which was similar to that in cells treated with rapamycin（a typical autophagy inducer）.These data suggest that IH act as an autophagy inducer in TNBC cells.2.Chloroquine dramatically potentiates isorhamnetin-mediated inhibition of cell proliferation and induction of apoptosis in triple negative breast cancer cellsCompared with CQ or IH treatment alone,exposure to a subtoxic concentration of CQ（20?M）significantly decreased the cell viability in both triple-negative breast cancer MDA-MB-231 and BT549 cells treated with a nontoxic concentration of IH.In contrast,CQ in combination with IH exerted little effects on cell viability toward MCF-7（an estrogen-dependent）cells and normal breast epithelial MCF-10A cells.The median dose-effect analysis of cell viability in cells exposed to CQ and IH for 48 h at fixed ratios yielded CI values consistently less than 1.0 in MDA-MB-231 and BT549 cells but greater than 1.0 in MCF-7 cells,suggest that CQ interacts synergistically with IH selectively inhibits cell proliferation in TNBC cells.Flow cytometry analysis showed that the combined treatment with CQ/IH resulted in a pronounced increase in apoptosis in MDA-MB-231 and BT549 cells.Western blot analysis showed that the combined treatment with CQ/IH resulted in activation of caspases-3,release of cytochrome c into the cytosolic fraction,and the translocation of Bax from the cytosol to the mitochondria.3.Excessive accumulation of mitophagosomes contributes to mitochondrial injury and apoptosis mediated by combination of CQ/IHBy using western blot analysis,we found that combined exposure of cells to CQ/IH resulted in excessive accumulation of LC3B-II in mitochondria.Knockdown of ATG5 markedly reduced combination-mediated LC3B-II accumulation in mitochondria,and abrogated combination-induced apoptosis.4.The combination of CQ/IH induces mitochondrial fission through phosphorylation of CaMKⅡ（Thr286）and Drp1（Ser616）and their mitochondrial translocationWestern blot analysis showed that combined treatment of TNBC cells with CQ/IH resulted in increases in levels of phospho-Drp1（Ser 616）and phospho-CaMKII（Thr286）,but had no effect on phosphorylation of Drp1 at Ser 637.Combination of CQ/IH also led to mitochondrial translocation of Drp1 and CaMKII.Similarly,immunofluorescence analysis showed CaMKII and Drp1 signal at the mitochondria of cells treated with a combination of CQ/IH.To further investigate the role of CaMKII phosphorylation at Thr286 in mitochondrial fission and apoptosis induced by the combination of CQ/IH,We generated a mutant of CaMKII^T286A to occlude Thr286 phosphorylation or a mutant of CaMKII^T286D to mimic Thr286 phosphorylation.Overexpression of CaMKII^T286A blocked phosphorylation of CaMKII（Thr286）/Drp1（S616）and mitochondrial translocation of CaMKII/Drp1 in cells treated with a combination of CQ/IH,whereas overexpression of CaMKII^T286D promoted phosphorylation of CaMKII（Thr286）/Drp1（S616）and mitochondrial translocation of CaMKII/Drp1 in cells treated with either CQ or IH alone or combination.Furthermore,CaMKII^T286A attenuated combination-mediatedmitochondrial fission and apoptosis,whereas CaMKII^T286D promoted these events mediated by either CQ or IH alone or combination.5.Combined treatment with CQ/IH induces generation of reactive oxygen species（ROS）By using flow cytometry analysis,we found that combined exposure of cells to CQ/IH resulted in significant increases in generation of ROS.To explore further the role of individual ROS on combination-mediated mitochondrial fission and apoptosis,we employed three antioxidants TBAP（a cell permeable SOD mimetic）,catalase,and sodium formate,which primarily act on O2^·-,H2O2,and OH·,respectively.We found that pretreatment with TBAP,an O₂^·-scavenger,essentially abrogated combination-mediated ROS generation in both MDA-MB-231 and BT549 cells.Addition of TBAP essentially abrogated combination-mediated phosphorylation of CaMKII（T286）/Drp1（S616）,and mitochondrial translocation of CaMKII/Drp1.Furthermore,addition of TBAP markedly abrogated combination-induced mitochondrial fission and apoptosis.6.Inhibition of mitophagy enhances the inhibitory effect of IH on tumor growth in a TNBC xenograft mouse model in vivoTo determine whether our in vitro findings that inhibition of autophagy by CQ can sensitize to IH-induced cell death could be replicated in vivo,we next examined the effect of CQ on the inhibitory efficacy of IH in vivo using a TNBC xenograft mouse model.We found that exposure of mice to CQ/IH significantly increased the median survival of the mice.Furthermore,combination of CQ/IH caused greater inhibition of tumor growth.Immunohistochemistryanalyses showed that combine treatment with CQ/IH led to significant increase in apoptosis,cleaved caspase 3 and the interaction of CaMKII and Drp1 in tumor sections of mice.ConclusionsIn this study,we found that inhibition of autophagy/mitophagy selectively potentiates IH-induced mitochondrial fission and apoptosis in TNBC cells.In this model,the CQ/IH combination induces production of ROS,particularly the O₂^·-free radical.This,in turn,promotes the phosphorylation of CaMKII/Drp1 and their mitochondrial translocation,this culminated in mitochondrial fission,caspase activation,and apoptosis.Our findings suggest that IH has the potential for further development as a novel chemotherapeutic agent,and that a combination of IH with classic autophagy/mitophagy inhibitor could represent a novel therapeutic strategy for the treatment of TNBC.