| Objective: Given that the expression of androgen receptor(AR)was found in about 70-90% of breast cancer and approximately 30% of triple negative breast cancer(TNBC),AR is promising to become a new potential therapeutic target for breast cancer.It has been reported that anti-androgen therapy can improve the prognosis of AR+TNBC patients,but its molecular mechanism is still unknown.The aim of this study is to evaluate the biological functions of a new artificial androgen receptor(AR)antagonist HC-1102 in TNBC,and to explore its potential molecular mechanism in TNBC.Methods: AR+ TNBC BT549 cells were selected as cell model.And the optimal concentration of AR antagonist HC-1102 and bicalutamide(BICA)were determined by cell counting kit-8(CCK-8)assay,respectively.Then the selected cells were treated with HC-1102 and BICA alone respectively,or treated with combination of the two medicines.Besides the AR expression in TNBC BT549 cells was tested by WB,the cell viability was detected using CCK8 assay,the apoptotic rate and cell cycle distribution were analyzed using Flow Cytometry,and the migration and invasion capabilities were evaluated using Transwell assay.Furthermore,the potential molecular mechanism was explored by RNAsequencing and bioinformatics in BT549 cells treated with HC-1102,and was verified by western blot(WB).In addition,the mouse orthotopic triple negative breast cancer model was established in BALB/c nude mouse using TNBC BT549 cell line.The tumor formation and tumor size of the mouse were assessed on the21 th day after the establishment of the model.On day 22,the AR antagonist of HC-1102(20 mg/Kg),BICA(20 mg/Kg)alone respectively,or combination of both were injected intraperitoneal for 4 weeks.Finally,the mammary tissues of mice,stimulated by HC-1102 and BICA alone respectively or combination of two compounds,were taken and were prepared in paraffin sections.The morphological feature of the tumor tissue was observed by HE staining,cell apoptotic ratio was performed by TUNNEL method and the potential molecular mechanism in vivo regulated by HC-1102 was identified by WB.Results: The optimal concentration of HC-1102(100μmol/L)and BICA(150μmol/L)was determined in TNBC BT549 cells,respectively.HC-1102 and BICA both inhibited the BT549 cell proliferation in a concentration-dependent manner(P<0.05).BT549 cells without treatment of HC-1102 and BICA were used as the control group(CON group),while BT549 cells were stimulated with100μmol/L HC-1102 and 150μmol/L BICA alone respectively,or in combination,as HC-1102 group,BICA group and HC-1102/BICA group.Compared with the control group(0.72 ± 0.01),HC-1102(0.26 ± 0.01,P<0.01)and BICA(0.33 ±0.01,P<0.05)both reduced the AR expression in relevant groups,and the combination of HC-1102 and BICA depressed significantly the AR repression(0.19 ± 0.01,P<0.01).Then,At the different times of medicines treatment(24h,48 h and 72h),HC-1102 inhibited the cell viability by 32.08 ± 2.10%,52.18 ±0.65% and 80.58 ± 0.66%(P<0.01),BICA restrained cell viability by 17.77 ±2.43%,37.98 ± 1.66% and 70.01 ± 1.12%(P<0.05),and combination of HC-1102 and BICA decreased cell viability by 51.98 ± 1.16%,72.34 ± 0.14% and86.44 ± 0.54%(P<0.01).HC-1102 and BICA both inhibited BT549 cell proliferation in a time-dependent manner.Compared with the control group(the early apoptosis percentage: 7.07 ± 0.11%,the percentage of G1/S phase:46.34 ±1.68%),HC-1102 and BICA significantly enhanced apoptosis percentage:(9.98± 0.10% vs.8.51 ± 0.19%,P<0.01)and G1/S phase percentage(60.02 ± 1.18%vs.50.33 ± 1.68%,P<0.05),while combination of HC-1102 and BICA obviously increased the apoptotic percentage(10.96 ± 1.03%,P<0.001)and G1/S phase percentage(58.51 ± 1.39%,P<0.001).Compared with the control group(number of motility cells: 180.52 ± 8.51;number of invaded cells: 92.81 ± 6.33),HC-1102 and BICA inhibited BT549 cell migration and invasion capabilities(number of motility cells: 91.54 ± 6.52 vs.101.00 ± 4.00,P<0.01,number of invaded cells:57.75 ± 4.25 vs.59.5 ± 3.5,P<0.001),and combination of HC-1102 and BICA repressed obviously cell migration and invasion capabilities(number of motility cells: 71.08 ± 6.40,P<0.001,number of invasion cells: 49.55 ± 2.57,P<0.01).RNA-sequencing and bioinformatics analysis data showed that HC-1102 regulated BT549 cells proliferation,migration and invasion,apoptosis and cell cycle via the PI3K/AKT/mTOR and MAPK signaling pathways.WB data further supported that HC-1102 could inhibit the expressions of Akt,S6,ERK1/2 and its phosphorylated product,p-mTOR,MEK1/2,and RSK2 protein(P<0.05),while it could not affect the expressions of mTOR and p-MEK1/ 2 protein in BT549cells(P>0.05).Compared with the control group,HC-1102 and BICA reduced the tumor size(P<0.05),while the combination of HC-1102 and BICA decreased obviously the size of TNBC in vivo(P<0.01)in constructed mouse orthotopic triple negative breast cancer model.Compared with the control group(the apoptotic percentage: 0.55 ± 0.06%),HC-1102 and BICA both induced the apoptosis separately(5.33 ± 0.94% vs.5.82 ± 0.24%,P<0.001),while the combination of HC-1102 and BICA could increase obviously the apoptotic percentage in vivo(19.98 ± 1.21%,P<0.001).WB results demonstrated that HC-1102 could inhibit the expressions of Akt,S6,ERK1/2 and its phosphorylated product,p-mTOR,MEK1/2,and RSK2 protein(P<0.01),while it could not affect the expressions of mTOR and p-MEK1/ 2 protein in orthotopic TNBC tissues(P>0.05),implying HC-1102 could play an important role in antitumor effects via the PI3K/AKT/mTOR and MAPK signaling pathways in vivo.Conclusion: The artificially synthesized AR antagonist HC-1102 significantly inhibited the proliferation,motility and invasion capabilities,induced cell apoptosis and arrested the cell cycle distrition at G1/S phase in TNBC BT549 cells and the mouse orthotopic triple negative breast cancer.And HC-1102 could play an important role in anti-tumor effects via the PI3K/AKT/mTOR and MAPK signaling pathways in vitro and in vivo. |
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