| Background Breast cancer(Breast cancer,Bca)is the most common malignant tumor in women and one of the three most common cancers in the world.Based on the different expression of hormone receptors,breast cancer is divided into progesterone receptor(PR)positive,estrogen receptor(ER)positive,human epidermal growth factor receptor(HER2)positive and triple negative breast cancer.70% of breast cancers are classified as estrogen receptor α(Estrogen Receptor α,ERα)positive.ERα is also one of the most effective targets for the treatment of breast cancer,which is related to the prognosis of breast cancer.At present,drug resistance has appeared in some patients with endocrine therapy targeting ERα for breast cancer.Therefore,it is urgent to find a new compound targeting ERα to treat breast cancer.Rotundine(also known as L-Tetrahydropalmatine,L-THP)is a natural tetrahydropalmatine isoquinoline alkaloids isolated from the genera Stephania and Corydalis.Clinically,rotundine has been used as a traditional Chinese medicine which treat cardiovascular disease and many pain for 40 years.However its effect on ER-positive breast cancer cell lines has not been reported..We found that rotundine inhibits the proliferation of ERα positive breast cancer cells by inducing cell cycle arrest rather than apoptosis.In addition,rotundine enhances the sensitivity of ERα positive breast cancer cells to tamoxifen and fluvestrant.In addition,rotundine promotes ERα ubiquitination degradation,and ERα overexpression rescue the rotundine-mediated growth inhibition in breast cancer cells.overall,our study suggests that rotundine inhibits breast cancer cell growth by promoting ERα degradation,which providing a potential therapeutic option for the treatment of ERα positive breast cancer.Methods 1.Cell viability assay: According to the experimental design,cell viability was carried out with MTS reagent.2.Clone formation experiment: after treated the cells according to the experiment,the cells were cultured in a six-well plate for about two weeks(until colonies were formed),the colonies were fixed and then stained with crystal violet,and each treatment group was photographed with a digital camera and counted.3.Ed U cell proliferation assay: according to the instructions of Ed U cell proliferation detection kit,the treated cells were stained,the cells were photographed with a fluorescence microscope and the percentage of cells of Ed U positive cells /total cells was counted.4.Flow cytometry:(1)Cell cycle assay: after the cells were treated according to the experimental design,the cells were fixed with 70% ethanol.The next day,the cells were stained with the cell cycle kit,and the cells of each period were analyzed by flow cytometry.(2)Apoptosis assay: After cells were treated,Annexin V-FITC and PI were used to stain the cells,and flow cytometry was used to analyze the apoptotic cells at each stage.5.Western blot assay: After the cells were treated according to the experimental design,the protein lysates of each group were collected,and the expression of target protein was analyzed by Western blot assay.6.Immunofluorescence experiment: After cells were treated according to the experimental design,the fluorescence intensity and localization of target proteins in each group were observed through the immunofluorescence experiment.7.Double luciferase detection experiment: The plasmid with the target reporter gene was transferred into the cells,then treated the cells.The transcriptional activity of the target gene was detected with the enzyme labeled instrument in accordance with the instructions of the double luciferase kit.8.Molecular docking simulation experiment: Auto Dock software was used to conduct docking between Rotundine and the target molecule,analyzed the docking situation of the two.9.Real-time quantitative fluorescent polymerase chain reaction(RT-q PCR): After the cells were treated according to the experimental design,RNA was extracted with RNAiso Plus,and c DNA was obtained through reverse transcription.Then SYBR Green Dye was used for quantitative PCR,and the expression of target genes was analyzed by obtaining the amplification products.Results 1.Rotundine inhibits the growth of ERα positive breast cancer cells ERα positive breast cancer cells T47 D,MCF-7 were treated with rotundine at different concentrations(0-200 μM)for 24,48,72 H.MTS assay was used to detect the cell viability,we found that rotundine significantly inhibited the growth of ERα positive breast cancer cell line in a concentration-dependent and time-dependent manner.In addition,the results of colony formation assay showed that rotundine had a long-term inhibitory effect on ERα positive breast cancer cells.The results of Ed U staining experiment also support this view.2.Anti-proliferation effect of rotundine depends on cell cycle arrest on ERα positive breast cancer cells Flow cytometry was used to detect cell cycle and apoptosis.we found that the number of cells in G0 / G1 phase upregulated after the treatment of rotundine,which confirmed that rotundine blocked the transition of ERα positive breast cancer cells from G1 phase to S phase.In addition,we detected the expression of cell cycle related proteins by Western blotting,the expression of p27 was up-regulated and the expression of CDK4,Cyclin D1,Rb and p-Rb was down-regulated.However,the results of apoptosis detected by flow cytometry showed that there was no significant apoptosis in MCF-7 cells treated with rotundine,and the results of apoptosis-related proteins PARP and Bcl-2 detected by Western blotting also supported this view.3.Rotundine induces the downregulation of ERα expression Western blotting showed that after treatment with rotundine,the expression of ERα protein was down-regulated in MCF-7 cells and T47 D,but the expression of mRNA was not affected.In addition,as ERα is a nuclear transcription factor,estrogen(E2)bind to ERα,activate its transcriptional activity and promote its degradation.the expression of ERα protein decreased more significantly when E2 was added.Immunofluorescence showed that rotundine could significantly reduce the abundance of ERα,but there was no nuclear translocation of ERα.4.Rotundine down-regulates expression of ERα by promoting its degradation.The results of RT-q PCR showed that although rotundine could not induce a significant change in the level of ERα mRNA,the downstream gene PS2,Cyclin D1 of ERα were regulated by rotundine;In addition,double luciferase reporter gene assay showed that rotundine significantly inhibited the transcriptional activity of ERα.CHX(cycloacetimide)was used to inhibit protein synthesis,and rotundine accelerated the decrease of ERα protein under the treatment of CHX.Furthermore,Co-IP showed that rotundine increased the levels of polyubiquitin and K48 ubiquitin of ERα,which confirmed that rotundine degraded ERα through ubiquitin proteasome pathway.5.Rotundine interacts with ERα The results of molecular docking simulation experiments showed that rotundine directly target ERα.6.Overexpression of ERα attenuates partial growth inhibition mediated by rotundine The plasmid of human ERα was transferred into T47 D and MCF-7 cells,we found that overexpression of ERα attenuated the cell cycle arrest induced by rotundine.In addition,we evaluated the expression of Rb and P-Rb by Western blotting,the results showed that the overexpression of ERα reversed part of the downregulation induced by rotundine.7.Rotundine enhances the sensitivity of breast cancer cells to tamoxifen.The MTS results showed that the combination of rotundine and tamoxifen enhanced the anti-proliferation effect,the results was also confirmed by the clone formation assay.Western blotting assay for ERα and its related target proteins Cyclin D1 showed that the expressions of the two proteins were down-regulated more significantly in combination of rotundine and tamoxifen than that in the single treatment group.In addition,when we detected apoptosis by flow cytometry,although rotundine did not induce apoptosis in ERα-positive breast cancer cells,apoptosis increased when rotundine was used in combination with tamoxifen.The expressions of Bcl-2 and PARP detected by western blotting assay also support this view.8.Rotundine and fulvestrant synergistically inhibit the growth of breast cancer cells The results of MTS showed that rotundine enhanced the anti-proliferation ability of fulvestrant in ERα positive breast cancer cells,which was also confirmed by clone formation assay and Ed U proliferation assay.In addition,the results of Western blotting also showed that the expression of ERα and its target protein Cyclin D1 decreased more significantly in combination than when they were treated alone.Conclusion Rotundine inhibits the growth of ERα-positive breast cancer cells by targeting ERα proteasome-mediated degradation. |
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