Study On The Mechanism Of ER - Mediated Tanshinone â…¡A In Anti - Gynecological

ObjectiveAs an essential hormone, estrogen is widely distributed inside human body. Estrogen plays regulative functions in various target points including female reproductive systems, nervous system, cardiovascular system and skeletal system. Estrogen plays an important role in development and prognosis of breast cancer, ovarian cancer, cervical cancer, osteoporosis, postmenopausal syndrome and other related diseases. The function of estrogen mainly depends on the key targets of estrogen:estrogen receptors. ER includs two subtypes:ER alpha and ER beta. ER located in the cytoplasm or nucleus with the role of transcription factors. Estrogen combined with ERto form dimers. Then estrogencombines with estrogen receptor components (estrogen response element, ERE) to stimulate the target gene transcription which regulates estrogen promote cell proliferation and differentiation.It can maintain estrogen’s normal physiological function.The classical pathwaybelongs to genotype slow effect.A large number of studies have found that estrogen’s effects on target cells, in addition to genotype slow effect,there arenon-genotype rapid effect.In recent years, many researches have found a membranous-G protein coupled receptor of estrogen receptor (GPER). Estrogen combines with GPER to trans-active epidermal growth factor receptor (EGFR) and regulate the activities of extracellular regulate kinase (ERK) and mitogen activated protein kinase (MAPK).Then their combination activates the corresponding signal transduction pathways and causes non-genotype rapid effect to participate in the pathophysiology of the regulation of the body.Phytoestrogens have similar structures with endogenous estrogen. Thus phytoestrogens could also bind with estrogen receptors and play estrogen or anti-estrogen effects inside human body.Now it is known that Salvia has estrogenic effects. Sal via is the dry root and rhizome of Salvia miltiorrhiza, a Labiatae plant with the effects of promoting blood circulation, regulating menstruation,removing blood stasis, analgesia, cooling the blood and eliminating stasis. Modern pharmacological studies have shown that Salvia miltiorrhiza in organisms can be involved in many kinds of important life activities. For example, it can protect cardiovascular system, enhance immune function and show the effects of anti-bacterial, anti-inflammatory, anti-oxidation, analgesia, anti-aging and anti-tumor in target organs. Tanshinone ⅡA is one of the main component and pharmacological basis of TCM Salvia miltiorrhiza. In the following study, we will concentrate on the target points and pharmacological pathways of tanshinone ⅡA in treating gynecological diseases and provide important experimental basis for the clinical application of traditional Chinese medicine.This thesis is mainly study the inhibitory effects and ER-mediated pathway of tanshinone ⅡA to cervical cancer siha cells, breast cancer T47D cells and endometrial carcinoma Ishikawa cells proliferation.MethodsThe effects of different doses of tanshinone ⅡA on proliferation were measured by MTT assay and flow cytometry analysis. The effects of tanshinone ⅡA on expression levels of estrogenic effects of proteins were measured by Western blot.Results1. The influence of tanshinone IIA on proliferation of cervical cancer siha cells, breast cancer T47D cells and endometrial carcinoma Ishikawa cells.Cervical cancer siha cells, breast cancer T47D cells and endometrial carcinoma Ishikawa cells for the study of the carrier, tanshinoneⅡA as the research objects,MTT test was used to evaluate cell proliferation.1.1 The effects of different concentration of tanshinone ⅡA on proliferation of siha cells, T47D cells and Ishikawa cells.Results:tanshinone HA showed a significant effect on inhibiting siha, T47D and Ishikawa cells proliferation rate at 48h. 1.2 Influence of ER antagonist, ERaantagonist, ERβantagonist, ERa agonist and ERβagonist on the effect of tanshinone IIA on siha and T47D cell proliferation.Results:(1)tanshinone ⅡA significantly inhibits siha cells proliferation and such effect could be enhanced by ERa antagonist MPP and attenuated by ERβ antagonist PHTPP.(2) tanshinone IIA significantly inhibits T47D cells proliferation and such effect could be enhanced by ERa and ERβ antagonist ICI182780.1.3 The influence of the same concentration of tanshinone ⅡA on proliferation of cervical cancer siha cells, breast cancer T47D cells and endometrial carcinoma Ishikawa cells.Results:The same concentration of tanshinone ⅡA showed a significant effect on inhibiting siha, T47D and Ishikawa cell proliferation rate at 48h.Compared with two other kinds of cells,cervical cancer siha cells are more sensitive to tanshinone ⅡA andbreast cancer T47D cells is relatively strong tolerance to tanshinone ⅡA drug.2.The effects of tanshinone IIA on cell cycle distribution of cervical cancer siha cells and breast cancer T47D cells.The flow cytometry analysis was used to detect the change of cell cycle distribution of siha and T47D cells induced by tanshinone IIA.Results.We selected the 48h group to perform the cell cycle test. Estrogen could significantly increase the proliferation index of siha cells and T47D cells. But tanshinone IIA could significantly decrease the proliferation index of sihacells and T47D cells.The main function is a reduction of the proportion of cells in Sphase and a significant increase of the proportion of cells in G0/G1 phase.3.The effect of tanshinone ⅡA on ER and ER related protein expression in siha cells, T47D cells and Ishikawa cells.3.1P-ERK and cyclin D expression levels influenced by tanshinone IIA in siha cells were evaluated by Western blot.Results:Tanshinone ⅡA could significantly reduce the protein expressions levels of p-ERK and cyclinD in a dose-dependent manner.3.2 GPER、p-ERK and cyclin D expression levels influenced by tanshinone IIA in T47D cells were evaluated by Western blot.Results:Tanshinone ⅡA could significantly reduce the protein expressions level of GPER,p-ERK and cyclinDin a dose-dependent manner.3.3Cyclin D expression levels influenced by tanshinone ⅡA in Ishikawa cells were evaluated by Western blot.Results:Tanshinone ⅡA could significantly reduce the protein expressions level of cyclin D in a dose-dependent manner.4.Using gene silencing technology to build GPER negative cells model and the effects of different concentration of tanshinone IIA on proliferation of GPER SiRNA T47D cells.4.1 Western blot analysis to evaluate the expression of GPER in T47D cells and GPER gene silence T47D cells.Results:Compared with the normal group,the protein expressions levels of GPER in GPER SiRNAtransfection group was obviously reduced.4.2 The effects of different concentration of tanshinone HA on proliferation of GPER SiRNA T47D cells.Results:Estrogen showed an effect of increasing T47D cells proliferation rate, and such effect was significantly decreased after transfection of GPER SiRNA. Tanshinone IIA showed a significant effect on inhibiting siha cells and T47D cells proliferation rate and its effect was significantly enhanced after GPERSiRNA transfection.5. The effects of different concentrations of tanshinone IIA on endometrial cancer cell’s apoptosis.The morphological changes of human endometrial carcinoma Ishikawa cells treated by Tanshinone IIAwas observed by Hoechst33342 staining.Results:Hoechst33342 staining showed that the negative control group in the Ishikawa cells even chromatin, nuclear form of rules, drug-treated cells showed compact fluorescent stain, cell morphology showed apoptosis phenomenon.Conclusion1. Tanshinone IIA significantly inhibits siha cells proliferation, decrease the proliferation index of siha and reduce the protein expressions level of p-ERK and cyclin D.2. Tanshinone IIA significantly inhibitsT47D cells proliferation, decrease the proliferation index of siha and reduce the protein expressions level of GPER, p-ERK and cyclin D.3. Tanshinone IIA significantly inhibitslshikawa cells proliferation, induced apoptosis of Ishikawaand reduce the protein expressions level of cyclin D.