Study On Mechanism Of Ionizing Radiation Inhibiting The Proliferation In ER~+ Breast Cancer Cells By Reducing The Level Of Estrogen Synthesis

Nowadays,breast cancer is the cancer with the highest incidence among female malignant tumors.According to the clinical and pathological characteristics,breast cancer is usually classified into estrogen receptor positive(ER⁺)breast cancer,human epidermal growth factor receptor 2 positive and triple negative breast cancer.Of which,ER⁺breast cancer accounts for more than two-thirds of total breast cancer.In clinic,surgery,chemotherapy,endocrine therapy and radiotherapy are commonly used to therapy breast cancer.Endocrine therapy,such as tamoxifen,is the first drug to therapy ER⁺breast cancer.However,more than one-thirds of patients have developed drug resistance after tamoxifen treatment for 5 years,which in turn leads to more serious tumor recurrence or metastasis.Radiotherapy is a commonly auxiliary therapy for treatment of breast cancer.In our previous studies,we found that the cell proliferation was inhibited as well as the production of endogenous estradiol(E2)was significantly reduced at different doses of ¹²C⁶⁺and X ray irradiation in MCF7 cells.Regrettably,mechanism of the decreased E2 induced by ionizing radiation is still unclear.E2 often combines with estrogen receptor?(ER?)forming dimers to improve the proliferation of breast cancer cells.At the same time,CYP19A that located in the endoplasmic reticulum is a key enzyme for the synthesis of endogenous E2 and has a positive regulatory effect on the development of breast cancer.The endoplasmic reticulum is the place where proteins are synthesized.Under normal conditions,immunoglobulin heavy chain binding protein(Bip)binds to the endoplasmic reticulum chaperones protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1),respectively.When endoplasmic reticulum is compelled,Bip will separate from these chaperones,binds the misfolded proteins and activates the unfolded protein response to maintain the homeostasis of endoplasmic reticulum.Numerous researches have reported,ionizing radiation can also cause endoplasmic reticulum stress and activate unfolded protein response.Therefore,we put forward a hypothesis that“Ionizing radiation(X ray)affects the expression of CYP19A by resulting in endoplasmic reticulum stress,which reduce the synthesis level of E2 and inhibit the proliferation of ER⁺breast cancer cells.”In order to verify the hypothesis,we chose human ER⁺breast cancer cell lines MCF7 and T47D cells as our materials.Under normal culture,E2 conditioned medium culture conditions which simulated an environment in vivo,and in tamoxifen-resistant cells,experimental methods including cell proliferation,colony formation,real-time quantitative PCR,western blot,immunohistochemistry and immunofluorescence were used to explore the expression of CYP19A and the mechanism of inhibiting the cell proliferation caused by ionizing radiation,respectively.Additionally,we also used immunohistochemistry to explore the mechanism of ionizing radiation inhibiting tumor growth in Balb/c nude mice.We obtained some results through above experiments and summarized as follows:Firstly,under normal culture or E2 conditioned medium culture conditions,ionizing radiation suppressed the proliferation of ER⁺breast cancer cells by reducing the synthesis of E2,down-regulating the expression levels of ER?,its regulated protein SGK3 and phosphorylation of ER?at serine 118(s118);Second,under normal culture or E2 conditioned medium culture conditions,ionizing radiation not only caused endoplasmic reticulum stress by elevating the protein expression levels of Bip,CNX and CRT in ER⁺breast cancer cells,but also led to endoplasmic reticulum autophagy by up-regulating the expression levels of LC3B?/?and Beclin1 as well as down-regulating the expression level of P62through activating the IRE1/XBP1 signaling pathway,which decreased the level of CYP19A;Third,ionizing radiation reduced the synthesis level of E2,which attenuated the expression of CYP19A through endoplasmic reticulum phagy,and inhibited the proliferation of tamoxifen-resistant cells MCF7/TAM and T47D/TAM cells.Fourth,ionizing radiation could inhibit the proliferation of ER⁺breast cancer cells by up-regulating the expression of XBP1s and down-regulating the expression levels of CYP19A,ER?and Ki67 in vivo.From the above results,we drew a conclusion as follows:Ionizing radiation induced endoplasmic reticulum stress in ER⁺breast cancer cells,and inhibited the synthesis of E2 via degrading CYP19A through endoplasmic reticulum phagy mediated by IRE1/XBP1 pathway;at the same time,it down-regulated the expression levels of ER?,its regulatory protein SGK3 and phosphorylation of ER?at serine 118(s118);in summary,above results explained the mechanism of ionizing radiation inhibiting the proliferation of ER⁺breast cancer cells.