Research On The Anti-proliferative Effect Of Shikonin On Breast Cancer Cells And Its ER Mediated Mechanism

ObjectiveBreast Cancer is one of the most common malignant femal tumors in the world at present,which has a great influence on the life of the patients.In recent years,the morbidity as well as mortality of breast cancer is increasing.The pathogenic factors of breast cancer are complex and varied,including genetic factors,biological factors,reproductive factors,living factors,environmental factors and psychological factors.Unquestionably,the high expression of estrogen is an important factor in breast cancer.Estrogen receptor?ER?plays a key role when estrogen works in the body.ER is a kind of steroid substance,which can be divided into different subtypes.Nuclear receptors?nERs?are classic estrogen receptors,including ER alpha and ER beta,which mediate the slow genomic effect of estrogen.In recent years,the research has confirmed that the new membrane receptor?mER?G protein coupled estrogen receptor?GPER?can also play an important role in the occurrence and proliferation of breast cancer,cervical cancer,endometrial cancer,ovarian cancer and other related tumours.Owing to its no need for simultaneous expression of any nuclear receptor,GPER has become a research hotspot recently.Many studies have shown that Shikonin?SK?,as a major component of Chinese herbal medicine zicao,shows anti-tumor,anti-inflammatory,antioxidation,and wound healing,especially the anti-tumor effect.There are many studies on breast cancer and cervical cancer.The mechanism of action of Shikonin includes the possible inhibition of phosphorus by PI3K/Akt signaling pathway.Finally it will induce apoptotic cell membrane surface exposure calcium reticulin,and regulating Bcl-2 family proteins,etc.This experiment choose two kind of breast cancer cells MCF-7?ER alpha +/ER beta-/GPER+?and breast cancer SK-BR-3?ER alpha-/ER beta-/GPER+?.The effect of SK on the proliferation,cell cycle and apoptosis of breast cancer cells were investigated.And the effect of SK on nER/mER and their downstream target genes were investigated.Ultimately,we discussed the research on the anti-proliferative effect of Shikonin on breast cancer cells and its ER mediated mechanismMethods&Results1 Effects of shikonin on proliferation and apoptosis of breast cancer cellsThe proliferation of MCF-7 and SK-BR-3 cells was detected by MTT cell proliferation test,and the effect of proliferation cycle of MCF-7 and SK-BR-3 cells was detected by flow cytometry,and the effect of apoptosis of MCF-7 and SK-BR-3 cells was detected by Annexin V FITC/PI double staining.Results:Compared with the blank control group,the shikonin group of 1×10^-6?1×10^-7?1×10^-8mol/Lhad an inhibitory effect on MCF-7 and SK-BR-3 cells with statistical significance?P<0.05?.The rate of 1×10-mol/L E2 group was higher than that in the blank group?P<0.01?.After flow cytometry,MCF-7 cells were blocked at G0/G1,cells were increased in S phase,resulting cell cycle delayed.But the cycle of SK-BR-3 cells was not significantly changed.Annexin V FITC/PI double staining showed that the apoptosis rate of MCF-7 and SK-BR-3 cells increased by shikonin.2 The effect of shikonin on the expression of estrogen receptor?nERs?and membrane receptor?mER?in breast cancer cells.The expression of three proteinsERa,ER(3 and GPER were tested by Western blot immunoblotting in MCF-7 cells and SK-BR-3 cells.Besides,the expression level of ERa and GPER were detected by shikonin.The influence of SK among the E2 was detected,and finally the localization of ER and GPER protein in MCF-7 and SK-BR-3 cells were detected by immunohistochemistry.Results:ERa,ER? and GPER were positive in MCF-7 cells,while in SK-BR-3 cells,ERa and ER? were negative but GPER showed positive expression.In MCF-7 cells,the expression of ERa and GPER could be reduced in all groups influenced by shikonin.In SK-BR-3 cells,the concentration group of 1×10^-6mol/L shikonin had inhibitory effect on GPER protein,suggesting that shikonin could inhibit MCF-7 and SK-BR-3 cells through the ER pathway.Compared with the blank control group,the expression of ER alpha and GPER increased in the MCF-7 cells after adding E2,and the expression of ERa and GPER decreased after 1×10^-6mol/L SK alone.The expression of ERa and GPER decreased after the coaction of E2 and SK,suggesting that SK still inhibited proliferation of cells in the human estrogen environment.The results of cellular immunohistochemistry showed that in MCF-7 cells,the positive expression of ERa was located in the nucleus.Compared with the blank control group,the expression of E2 group was significantly increased,showing a deeper brown color.The expression of SK was light brown located in the cell membrane.And the expression in the E2 group increased and was dark brown,however the SK were less expressed in each group with light brown.3 The effect of shikonin on the expression of downstreamprotein of GPER signaling pathway EGFR/p-ERKin breast cancer cells.The effects of SK on the expression of EGFR and p-ERK expressionwere studied with Western blot under G1,G15 and E2.And the targeting mechanism of inhibition and cell proliferation was discussed.The localization and expression of EGFR and p-ERKin MCF-7 and SK-BR-3 cells were detected by immunohistochemistry.Results:In MCF-7 cells,the expression of EGFR and p-ERK were significantly reduced in each group of SK obviously.In SK-BR-3 cells,SK could reduce the expression of EGFR and p-ERKsuggesting that SK could inhibit the expression of EGFR and p-ERK protein in breast cancer cells.After the combination influence of SK and G1,the expression of EGFR and p-ERK decreased in MCF-7 cells,and the expression of p-ERK in SK-BR-3 cells was inhibited.The binding of GPER agonists to SK further inhibited the expression of EGFR and p-ERK.After the coaction of SK and E2,the expression of EGFR and p-ERK in MCF-7 and SK-BR-3 cells decreased significantly compared with the blank control group,suggesting that SK had inhibitory effect on EGFR and p-ERK protein in estrogen environment.The results of cellular immunohistochemistry showed that in MCF-7 cells,the expression of EGFR protein was located in the cell membrane.Compared with the blank control group,the expression of E2 group increased significantly and were darker brown.The expression of SK in each group were less,and the expression of p-ERK protein was in the cytoplasm,finally the expression of E2 group increased and was dark brown.The expression of SK in each group were light brown.4 The effect of shikonin on the expression of cyclin D1 protein in ER positive breast cancer cells.Taking MCF-7 cells as the research object,Western blot method was used to observe the effect of SK and E2 on the expression of cyclin D1 in the downstream gene of ER?,and to explore the cyclin D1 targeting mechanism of SK affecting the MCF-7 cell cycle.The localization and expression of cyclin D1 in MCF-7 cells was detected by immunohistochemistry.Results:After treatment with SK,the expression level of cyclin D1 decreased compared with the blank control group,of whichl ×10^-6mol/L SK had the strongest inhibitory effect on cyclin D1 expression.Compared with the blank control group,the protein expression of cyclin D1 was improved after the E2 intervention,and the protein expression of cyclin D1 decreased significantly after the action of 10^-6mol/L SK alone,and the combined action of E2 and SK inhibited the expression of cyclin D1 protein.The immunohistochemical results showed that the expression of cyclin D1 protein was in cytoplasm and nucleus in MCF-7 cells.Compared with the blank control group,the expression of E2 group increased,and the expression of SK was less in each group.Conclusions1 The cell proliferation and apoptosis experiments showed that SK could inhibit the proliferation of MCF-7 and SK-BR-3 cells,inducing apoptosis of two cells and blocking the cycle of MCF-7 cells in G0/G1 stage.2 The mechanism of SK inhibiting the proliferation of MCF-7 and SK-BR-3 cells is ER targeted.After the action of SK on these two cells,the expression of the nuclear receptor ER alpha and GPER protein in the ER pathway was reduced,and the inhibitory effect was not affected by the estrogen environment.3 SK inhibits proliferation of breast cancer cells through membrane receptor signaling pathway,and induces cell apoptosis mediated by GPER/EGFR/p-ERK signaling pathway.4 SK affects the increment cycle of ER alpha+ breast cancer MCF-7 cells through the nuclear receptor signaling pathway and delays the cell proliferation process,which may be mediated by the cyclin D1 protein.