NBT Affects MCF-7 Proliferation Through The Crosstalk Between Apoptosis And Autophagy In Mitochondria

ObjectiveBreast cancer seriously threaten women’s physical and mental health.Although chemotherapeutics are widely used for the treatment of breast cancer,some shortcomings remain major therapeutic problems such as the offtarget effects.Betulin(BT)is a pentacyclic lupine-type triterpenoid natural product reported to possess antitumor activity in various of cancers.However,its further clinical development was discouraged because of its low bioavailability and poor solubility.Fortunately,we prepared a derivative of BT(NBT)that was modificated by the position 3 of ring A and C-28 as well as introduced NO-releasing moiety.This study mainly explores the mechanism of the NBT treating breast cancer through the crosstalk between apoptosis and autophagy in mitochodria.Methods1.The effect of NBT on viability of human breast carcinomaThe human breast carcinoma MCF-7 cell line,human lung cancer A549 cell line and human cervical carcinoma Hela cell line were treated with different concentration of NBT or BT to verify the cell line,treatment time and concentration by CCK-8 assay.And the cell cycle phase distributione were determined by flow cytometric analysis,the gene expression level of Cyclin D1 was detected by Real-time PCR and the protein expression level were determined by Western Blot.2.The effect of NBT on apoptosis of human breast carcinomaFirstly,we used hoechst 33258 staining and flow cytometric measurement to detect NBT whether induce apoptosis in MCF-7 cells.Then,Caspase-9 activity was carried out using caspase-9 colorimetric assay kit.And we further examed the expression level of PARP by Western Blot.In addition,.mitochondrial membrane potential was detected by flow cytometric measurement and the protein expression level of Bcl-2、Bax、Cyt C were determined by Western Blot to verify the type of apoptosis by NBT.Finally,Western Blot was used to determinate the level of Drpl,Fisl,Mfn2 proteins to reveal the effect of NBT on balance of ffision and fusion in the MCF-7 cells.3.The effect of NBT on autophagy of human breast carcinomaThe ultrastructural changes of MCF-7 cells were observed under a transmission electron microscope.We tested the autophagy-associated proteins expression level such as Beclinl,LC3 and p62 to verify NBT whether induce autophagy in the MCF-7 cells.4.The effect of CQ on NBT-induced apoptosis of human breast carcinomaFirstly,we used flow cytometric measurement determinated the co-treatment of NBT and CQ whether enhanced NBT-induced apoptotic effect in the MCF-7 cells,and Bcl-2,Bax,Cyt C,PARP proteins were detected by Western Blot.Secondly,we used Western Blot to exam the expression level of Drp1,Fisl and Mfn2.Finally,we observed the morphology of autophagy through transmission electron microscope,and Western Blot was carried out to detect the expression level of Beclinl,LC3,p62.5.The effct of NBT on MCF-7 solid tumor in vivoMCF-7 cells(10×106)were injected subcutaneously into the nude mice under the left axillae.The mice were were administrated with normal saline(0.2 mL i.p.),5-Florouracil(22 mg/kg i.p.),BT(100 mg/kg i.p.),NBT(100 mg/kg i.p.)for two weeks when the tumor size reached 8mm3.During the treatment time,its body weight and tumor were examed.And we detected aminotransferase(ALT),aspertate aminotransferase(AST),urea nitrogen(BUN),creatinine(CRE),uric acid(UA)using corresponding kit to test the effects of NBT on nude mice blood biochemical indexes.Moreover,we using HE staining,TUNEL assay to evaluate wether NBT affect the histological morphology and apoptosis of tumor tissues.Results1.NBT inhibited the proliferation of human breast carcinoma and affected its cell cycle distributionThe results showed that the anti-tumor effect of NBT on MCF-7,A549,Hela cells was superior to BT,and MCF-7 cells was the most sensitive to NBT.Therefore,we chose MCF-7 cells as object in our study.The IC50 of NBT for 24 h and 48 h on MCF-7 were 26.53μM and 10.83μM respectively.According to these results,NBT treatment concentrations were identified as 0,6,12,24μM and the time was 24 h.And NBT induced cell cycle arrest in G0/G1 phase by decreasing the gene and protein expression of Cyclin D1.2.NBT induced apoptotic cell death of human breast carcinoma through mitochondrial pathwayThe results showed more cells with brighter nuclei stained,condensation of nuclear chromatin and karyorrhexis,and the numbers of apoptotic cells were enhanced in a dose-dependent manner.At the protein level,the expression level of Bcl-2 was decreased,Bax was increased,Cyt C was accumulated in the cytoplasm,and Caspase-9 and PARP were cleaved,mitochondrial membrane potential loss was increased in MCF-7 cells.In addition,the expressions of Drpl and Fisl were significantly increased,while Mfn2 was decreased by NBT.3.The effect of NBT on autophagy of human breast carcinoma-Treatment with NBT at the dose of 12 μM induced the formation of double-membraned,degrading autophagic vacuoles and swelling,splitting mitochondria in the MCF-7 cells.Moreover,the expression level of Beclin-1 and LC3-Ⅱ were increased,while,p62 were decreased.4.Inhibiting the late stage of autophagy by CQ promoted NBT-induced apoptosis of human breast carcinomaThe results showed that the expressions of Beclinl,LC3-Ⅱ and p62 proteins of the MCF-7 cells treated with NBT along with CQ were increased significantly comparing to NBT.The apoptotic cells were increased and apoptosis associated protein levels such as Bax,cytochrome C in the cytoplasm,Caspase-9,cleaved PARP were enhanced,Bcl-2 level was reduced.In addition,the expression of Drp 1 and Fis 1 were increased,however Mfn 2 was decreased comparing to NBT.5.NBT inhibits the the growth of MCF-7 solid tumorThe experiments in vivo showed that the effect of NBT was similar to 5-Fu that inhibit the proliferation of MCF-7 cells.Compared to the model,there were no significant effct on the body weight,the coefficient of the heart,liver,spleen,kidney and the parameters of AST,ALT,BUN,CRE,UA of nude mice.The HE staining revealed that there exhibited changes of the morphology and necrosis of the cancer cells from the mice treated with NBT.And the numbers of TUNEL-positive cells treated with NBT were more than BT.ConclusionIn this study,we found that NBT possessed a potent antiproliferative activity in MCF-7 cells in vitro and vivo.Mechanistically,NBT induced cell death through the mitochondrial apoptosis pathway.In addition,we found mitochondrial function was disrupted by NBT accompanied with mitochondrial divivion resulting in mitochondrial-dependent factor responsible for its apoptotic potential.In addition,the experiments of MCF-7 cells treted with NBT and CQ combination infer that CQ significantly promote the MCF-7 cell mitochondria to divide,cytochrome C to be released from mitochondria to the cytoplasm resulting in an increasing apoptosis rate by inhibiting the degradation of mitochondrial autophagosome.Therefore,our research revealed NBT was useful and safe in treating breast cancer,specifically by targeting the mitochondrial apoptosis pathway.