| Background and Objective: Breast cancer has been being the most common women cancer,and the first leading cause of cancer death in women.Despite the development of therapeutic methods including surgery,chemotherapy,radiotherapy and targeted therapy,the risk of it augments with age.Therefore,the development of novel therapeutic strategies is critical for these patients.Recent researches have paid more attention on sesquiterpene lactone which demonstrates a series of biology activities such as anti-cancer,anti-inflammation,anti-bacteria and antioxidation.Parthenolide is a sesquiterpene lactone found in the medicinal plants,feverfew.It has been used for the treatment of fever,headache and arthritis for many years.Parthenolide has an anticancer activity against pancreatic cancer,prostate cancer,colorectal cancer,melanoma,and cholangiocarcinoma.Similarly,researches indicated that parthenolide can inhibit breast cancer,but the detailed mechanisms need to be further elucidated.Autophagy has been named second programmed cell death pathway,which is different from apoptosis.The process through which unnecessary or dysfunctional cellular components are removed via the union of lysosomes and autophagosomes to create autolysosomes.Autophagy levels are usually lower under normal conditions compared with starvation or nutrition-deficiency conditions.It is significant to sustain cellular homeostasis.It can be notably inhibited by knocking down ATGs,which leads to the damaged proteins and cell organelles,such as the mitochondria,not being removed,thus creating a toxic environment that can affect the survival of normal cells.Autophagy plays a significant role in all stages of cancer development.Recently,it was reported that autophagy is a ?double-edged sword‘.On one hand,it can decrease sensitivity to anticancer treatments,eliminate organelle damage,DNA fragmentation,and preserve the integrity of the cells.On the other side,excessive autophagy can lead to autophagic death.Based on above all information,it is extremely critical to detect whether parthenolide can induce autophagy or not and the mechanism of it induced autophagy.Parthenolide could induce autophagy and apoptosis in breast cancer cells.Based on some researches,autophagy induced by anti-cancer drugs may exert a protective effect.The mechanism of autophagy and apoptosis induced by parthenolide needs to be examined,which is significant to breast cancer therapy.Method: In this paper,CCK-8 assay,colony formation assay were used to evaluate the effect of parthenolide in breast cancer.Autophagy was measured through immunofluorescence and the formation of autophagosomes.Flow cytometry analysis was used to measure apoptosis.The western blot analysis was used to examine the mechanism of autophagy induced by parthenolide on the expression of PI3 K,AKT,phosphorylation of AKT,mTOR,ATG13 and ATG14.Moreover,we also confirmed apoptosis and autophagy via Western Blot,RT-qPCR was used to explore the expression of ATG13 and ATG14.Result:1.Parthenolide was found to exert an effect on the cell viability of human MCF-7 and MDA-MB-231 cells,which could be examined by CCK-8 assay.Compared with the control group,other groups treated with different concentrations of parthenolide exhibited reduced survival rates of the cells.The rates of cell viability were 89.1,80.1,62.5,47.3 and 36.6% following treatment of MCF-7 cells with parthenolide concentrations of 2,4,6,8,and 10 ?M,respectively,for 24 h.Compared with MCF-7 cells,the cell viability rates of MDA-MB-231 cells treated with parthenolide at the same concentrations for 24 h were 96.2,90.1,81.3,65.9,and 50.9%.To further confirm the effect of parthenolide on breast cancer cells,colony-formation assays were performed.Parthenolide clearly inhibited the colony-formation abilities of MCF-7 and MDA-MB-231 cells in a dose-dependent manner.2.The present study further assessed whether parthenolide could induce apoptosis in breast cancer.An annexinV/PI apoptosis assay was performed using flow cytometry.The percentages of apoptotic MCF-7 cells were 12.66 and 21.79% following treatment with 4 and 8 ?M parthenolide,respectively.Similarly,the percentages in MDA-MB-231 cells were found to be 12.24 and 17.62% under the same experimental conditions.Finally,western blot analysis was performed to confirm the apoptosis results.A high expression level of caspase-3,and a low expression level of Bcl-2 were observed in MCF-7 and MDA-MB-231 cells.3.MDC staining,which reveals the formation of autophagic vacuoles,appearing as dot-like structures in the cytoplasm compared with control cells,demonstrated that the cells with parthenolide treatment exhibited several dot-like structures.Autophagy induction by parthenolide was further confirmed by microscopy after staining the breast cancer cells with Beclin 1.Immunofluorescence experiments revealed red staining in MCF-7 and MDA-MB-231 cells following 24 h treatment with parthenolide at concentrations of 4 and 8 ?M,and 5 and 10 ?M,respectively.Furthermore,the western blotting results also showed higher expression levels of Beclin 1 and lower expression levels of P62/SQSTM1 upon parthenolide treatment compared with the control groups.4.The PI3K/AKT/mTOR signaling pathway has a critical role in regulating apoptosis and autophagy.Consequently,the effect of parthenolide on the expression levels of mTOR,AKT,p-AKT,PTEN and PI3 K were examined by western blot analysis,which revealed that the expression levels of the proteins were decreased.Conclusion:1.Parthenolide suppresses the growth of human breast cancer cells2.Induction of apoptosis by parthenolide in human breast cancer cells3.Parthenolide induces autophagy of breast cancer sells4.Parthenolide can inhibit PI3K/AKT/mTOR signal pathway... |
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