Anti-breast Cancer Effect Of Nigericin Based On PERK/CHOP Signaling Pathway

Breast cancer is one of the major diseases that threaten womens health,and the morbidity and mortality rates are increasing year by year.Despite the great progress in the research of endocrine drugs and targeted drugs for breast cancer in recent years,the existing drugs are not effective for certain molecular types of breast cancer,and the development of new effective drugs is still of great significance.Nigericin?Nigericinsodium salt,NSS?is an antibiotic derived from streptomyces hygroscopicus,an ionophore that promotes K⁺/H⁺exchange across the mitochondrial membrane.NSS has a variety of biological activities,such as antibacterial,antiviral,antimalarial,coccidiosis,etc.In recent years,its research on tumor stem cells,colon cancer,nasopharyngeal cancer and other antitumor effects has made some progress,but related to its role The research on the molecular mechanism of the method is less and needs further study.Most anti-tumor drugs work by inducing apoptosis.There is a complex interaction between apoptosis and endoplasmic reticulum stress and autophagy.In the endoplasmic reticulum stress,the PERK-mediated pathway is the most important.It is activated by oligomerization and autophosphorylation to phosphorylate its downstream factor,eukaryotic translation initiation factor 2??eIF2??subunit,to maintain the intracellular Environmental balance.When the stimulus continues to extend or increase,endoplasmic reticulum stress can up-regulate the expression of caspase family and induce apoptosis.It has been observed in previous studies that NSS can induce apoptosis,which is consistent with the reports.However,the effect of NSS on the endoplasmic reticulum stress pathway and its relationship with the induction of apoptosis are not clear.This study includes the following four parts:Part IStudy on the anti-breast cancer effect of NSS in vitroMethods:4T1 mouse breast cancer cells,MCF-7,MDA-MB-231 human breast cancer cells as well as L929 mouse fibroblasts,human umbilical vein endothelial cells?HUVEC?were respectively treated with NSS with final concentrations of 1.25?mol/L,2.50?mol/L,5.00?mol/L,10.00?mol/L,20.00?mol/Lfor 24h and 48h.The proliferation of these cells was determined by MTT method.Results:The proliferation of 4T1,MCF-7and MDA-MB-231 cells treated with 2.50?mol/L NSS for 24hcan be inhibited to a certain extent;with the action time extended to 48 hours,the inhibitory effect of NSS on the proliferation of the three types of breast cancer cells was significantly increased in a dose-dependent manner;the 48h IC₅₀ of 4T1,MCF-7 and MDA-MB-231 wererespectively?1.47±0.01??mol/L,?1.74±0.19??mol/L and?2.21±0.15??mol/L.When L929 and HUVEC cells were treated by NSS for 24h,the cell proliferation was inhibited to a certain extent;with the prolongation of the action time,the inhibitory effect of NSS on their proliferation was also significantly increased and was dose-dependent.The IC₅₀ of L929 and HUVEC at 48h were?7.44±1.1??mol/L,?3.43±1.1??mol/L.Part? The effect of NSS on cell cycle and apoptosis of breast cancerMethods:Using flow cytometry,the effect of NSS on the cell cycle of 4T1,MCF-7 and MDA-MB-231 was detected by PI single staining method;the proportion of apoptotic cells in each group was detected by Annexin-V and PI double staining method;The caspase inhibitor Z-VAD-FMKwas used to reversely verify the apoptosis on breast cancer cellsinduced by NSS;and the effects of NSS on the expression of apoptosis-related proteins Bax,Bcl-2,caspase3,activated caspase3,caspase9,activatedcaspase9 of breast cancer cells were detected by immunoblotting.Results:?1?Thecell cycle process phases of 4T1,MCF-7 and MDA-MB-231treated with NSS has been changed,and the percentage of G2/M significantly increased;?2?When 4T1,MCF-7 and MDA-MB-231 cells were treated with NSS at a final concentration of 2?mol/L for 48 h,the apoptosis rates were respectively 90.11%±0.16%,94.41%±2.77%and 67.38%±3.61%,compared with the control group,the difference is significant;?3?After Z-VAD-FMK treatment with a final concentration of 25.00?mol/L,the growth inhibition of thesebreast cancer cells induced by NSS was significantly decreased,that is,the survival rate of the cells increased;?4?After treatment with NSS at a final concentration of 0.75?mol/L compared with the control group,the expression ofBax,activated caspase3,and activated caspase9 protein in thesbreast cancer cells showed a gradual increase trend with the extension of the NSS action time.But Bcl-2 showed a gradual downward trend,and caspase3 and caspase9 showed no significant changes.Part? The effect of NSS on ER stress and autophagein breast cancer cells Methods:4T1,MCF-7 and MDA-MB-231 were treated with a final concertration of 0.75?mol/L NSS,and the expression of P-PERK,PERK,P-eIF2,eIF2,Chop,LC3was detected by immunoblotting at 6h,12h and 24h.Results:?1?The expression ratios of P-PERK/PERK,P-eIF2/eIF2 protein,and Chop protein in 4T1 and MCF-7 cells showed a gradual increase after 6 hours of action,difference was significant compared with the control group;the expression ratios of P-PERK/PERK,P-eIF2/eIF2 protein,and Chop protein in MDA-MB-231 cells showed a gradual increase after 12 hours,difference was significant compared with the control group.?2?After the breast cancer cells treated with NSS at a final concertration of0.75?mol/Lfor 6h,12h and 24h,the expression of Bax/BcL-2 and LC3significantly increased with the extension of the NSS action time.Part IV Anti-tumor effect of NSS on 4T1 tumor-bearing Balb/C miceMethods:The mouse models of 4T1 breast cancer tumor-bearing mice were established and randomly divided into 4 groups,that is:the model control group?saline?,DOX group?1.5mg/kg?,NSS group?4mg/kg,2mg/kg?;tail vein injection,once every other day,three times in a row.The general state of mice,the tumor inhibition rate,and the toxic effects to the major organs such as heart,liver,spleen,kidneyin each groupwere observed.Results:At the fourth day of the last injection of NSS,the tumors were taken out and the tumor weight and tumor inhibition rate of each group was compared.The tumor weight of the groups of 4mg/kg NSS,2mg/kg NSS and DOX was significantly reduced compared with the model group;tumor inhibition rate of the groups of 4mg/kg NSS,2mg/kg NSS,significantly increased compared with the DOX group.During the experiment period,the mice in each group moved normally,without mental or other abnormal performance,and no mice died.Histological evaluation of the heart,liver,spleen,kidney of mice in each group was performed,and no pathological changes such as inflammation,necrosis,and edema were observed in the model group,positive control group and NSS groups.Conclusion:1.NSS can significantly inhibit the proliferation of breast cancer cells,induce apoptosis and arrest the cell cycle in G2/M phase;2.NSS down-regulates the expression of Bcl-2 and up-regulates the expression of Bax protein,activates the caspase family,and induce apoptosis of breast cancer cells through mitochondrial pathway;3.NSS up-regulats the expression of P-PERK,P-eIF2,Chopand LC3?/LC3?proteins,and induces endoplasmic reticulum stress and autophage;4.NSS also showed a certain anti-tumor effect in 4T1 tumor-bearing Balb/C mice.