Dihydrotanshinone Ⅰ Induces Apoptosis Through STAT3 In Esophageal Squamous Cell Carcinoma Cells

Part Ⅰ Dihydrotanshinone I exerts proliferation inhibition and apoptosis induction in esophageal squamous cell carcinoma cellsObjective:To investigate the role of dihydrotanshinone I(DHTS)in esophageal squamous cell carcinoma cells and the underlying mechanism.Methods:The human ESCC cell lines KYSE30,Eca109,KYSE450,and KYSE510 and human normal esophageal epithelial cell line Het-1A were treated with DHTS of different concentrations.Cell proliferation was detected using CCK-8 assay,cell cycle was analyzed using flow cytometry,apoptosis was detected using Annexin V-PE/7-AAD double staining and Hoechst 33258 staining,proteins associated with cell cycle and the intrinsic mitochondrial apoptotic pathway were detected using western blot,these proteins include CyclinA2,CyclinD1,CyclinE1P21,Bax,Bcl2,cleaved caspase3,cleaved caspase7,cleaved caspase9,Cyt C and Survivin.Results:Proliferation in ESCC cells was inhibited by DHTS dose-dependently and time-dependently,while DHTS showed no obvious proliferation inhibition in normal esophageal epithelial cells.DHTS induced apoptosis in ESCC cells in a dose-dependent manner but showed no obvious apoptosis induction in normal esophageal epithelial cells.DHTS arrested cell cycle at G0/1 phase through the regulation of CyclinA2,CyclinD1,CyclinE1 and P21.Meanwhile,DHTS activated the intrinsic mitochondrial apoptotic pathway and induced apoptosis in ESCC cells.Conclusion:DHTS inhibited proliferation and induced apoptosis in ESCC cells while showed no obvious proliferation inhibition and apoptosis induction in normal esphageal epithelial cells.Part Ⅱ Dihydrotanshinone I induces apoptosis through STAT3 in esophageal squamous cell carcinoma cellsObjective:To investigate the role of signal transducer and activator of transcription 3(STAT3)in apoptosis induced by DHTS.Methods:Treated KYSE30 and Eca109 cells with DHTS and detected the levels of STAT3 and pSTAT3 using western blot.Constructed STAT3-knockdown and STAT3-overexpressed ESCC cells and treated the cells with DHTS.Detected apoptosis of the cells using Annexin-PE/7-AAD double staining and Hoechst 33258 staining.Detected the levels of proteins associated with the intrinsic mitochondrial apoptotic pathway using western blot.Results:DHTS inhibited the expression of pSTAT3 dose-dependently.The expression of pSTAT3 stayed in line with STAT3 in STAT3-knockdown and STAT3-overexpressed ESCC cells.After STAT3 knockdown,DHTS treatment induced more significant apoptosis,the expression of cleaved caspase3,cleaved caspase7,cleaved caspase9,Cyt C and Bax increased,while the expression of Bc12 and survivin decreased,STAT3 knockdown promoted the apoptosis induced by DHTS.After STAT3 overexpression,the levels of pro-apoptotic proteins decreased while the levels of anti-apoptotic proteins increased compared with negative control cells treated by DHTS,STAT3 overexpression blocked the apoptosis induced by DHTS.Conclusion:DHTS inhibited the phosphorylation of STAT3 and thus activated the intrinsic mitochondrial apoptotic pathway,DHTS induces apoptosis in ESCC cells.Part Ⅲ DHTS inhibited proliferation in ESCC cells in vivoObjective:To investigate the role of DHTS in ESCC cells in vivo.Methods:Cultured ESCC cells KYSE30 and implanted the cells into the right flank in the dorsal region of nude mice to generate a xenograft tumor model in vivo.When the longest diameter of the tumor was about 5 mm,treated the mice with DHTS.Measured the longest diameter and shortest diameter of the tumor.Weighed the body weight of the mice.Tumor sizes were measured using a Vernier caliper every other day.Tumor volume(TV)was calculated using the following formula:TV(mm3)=0.5xd2 xD;d is the shortest diameter while D is the longest diameter.All mice were weighed every other day.After treatment for 9 times,tumor specimens were collected and weighed.Xenograft tumors were fixed in 10%neutral buffered formalin,then embedded in paraffin,and used to prepare 4 μm-thick sections.TUNEL assay was used to detect apoptosis with an in situ apoptosis detection kit.Levels of Ki-67 were detected using immunohistochemistry.Results:The transplanted tumors grew rapidly in the control group but were suppressed in DHTS treatment groups in a dose-dependent manner.The weight of tumors in the DHTS treatment groups also decreased in a dose-dependent manner,while the body weight of mice in the four groups showed no difference during the treatment of DHTS.TUNEL assay of the tumors revealed more brown spots(which represent DNA fragments)in the DHTS treatment groups,indicating that cell apoptosis in the tumor increased significantly with the increase of DHTS dose.Immunohistochemistry revealed that levels of Ki-67 in the tumor decreased significantly with the increase of DHTS dose.Conclusion:DHTS inhibited tumor growth and induced apoptosis in ESCC in vivo.DHTS exerts anti-tumor effects in vivo.