川楝素调节Shh/Gli1信号通路对舌鳞状细胞癌细胞增殖、凋亡及裸鼠移植瘤生长影响的研究OA

Objective To investigate the effects of toosendanin regulating the sonic hedgehog/glioma-associated oncogene homolog 1(Shh/Gli1)signaling pathway on the proliferation and apoptosis of tongue squamous cell carcinoma(TSCC)cells and the transplanted tumor growth in nude mice.Methods In cell experiments,human TSCC cell lines CAL-27 and Tca8113 were randomly assigned into the cell control group(without drug treatment),the cell low-dose experimental group,the cell medium-dose experimental group and the cell high-dose experimental group.The cells in experiment groups were intervened by 5,10,and 15 nmol·L-1 toosendanin for 48 hours.The 5-Ethynyl-2′-deoxyuridine(Edu)staining,plate clone formation assay,Hoechst 33342/propidine iodide(PI)double staining,flow cytometry assay were used to measure cell proliferation and apoptosis,and immunofluorescence staining was used to measure cell proliferation and apoptosis-related proteins.The protein relative expression levels of the Shh/Gli1 signaling pathway in TSCC cells of each group was detected by Western blot.In animal experiments,the TSCC xenograft tumor model was constructed by subcutaneous inoculation of CAL-27 and Tca8113 cells.The successfully modeled animals were divided into animal control group,animal low,medium and high-dose experimental groups,with 6 animals in each group.The low,medium,and high-dose groups were given 2.5,5 and 10 mg·kg-1 toosendanin,respectively,via intraperitoneal injection once daily for 4 weeks.The control group was given an equivalent volume of sterile normal saline on the same schedule.The growth of transplanted tumors in nude mice was evaluated by measuring the volume and mass of transplanted tumors.In the rescue experiment:human TSCC cell lines CAL-27 and Tca8113 were randomly assigned into experimental group(intervened with 15 nmol·L-1 toosendanin),SAG group(intervened with a combination of 15 nmol·L-1 toosendanin and 0.6 μmol·L-1 SAG),pcDNA group(treated with 15 nmol·L-1 toosendanin+transfected empty plasmid)and toosendanin+pcDNA-Shh group(treated with 15 nmol·L-1 toosendanin+transfected Shh overexpression plasmid).After intervention with toosendanin,Shh signaling pathway agonist SAG and plasmid,the cell proliferation index and apoptosis rate were detected by the same method.Results Cell experiment:In CAL-27 cells,the proliferation indices of the cell low,medium,high dose experimental groups and the cell control group were(60.45±3.83)％,(43.03±3.45)％,(24.19±2.27)％and(79.87±4.61)％,respectively;the apoptosis rates were(24.75±2.19)％,(45.96±1.87)％,(67.12±3.65)％and(2.98±0.74)％,respectively;the relative expression levels Shh protein were 0.79±0.06,0.45±0.05,0.11±0.02 and 1.15±0.08,respectively;the relative expression levels of Gli1 protein were 0.71±0.04,0.39±0.05,0.08±0.01 and 1.03±0.06,respectively;In Tca8113 cells,the proliferation indices were(61.76±3.82)％,(42.17±4.06)％,(25.31±2.85)％and(81.03±5.14)％,respectively;the apoptosis rates were(23.21±1.93)％,(43.62±2.54)％,(65.31±3.73)％and(2.42±0.76)％,respectively;the relative expression levels of Shh protein were 0.82±0.08,0.48±0.06,0.15±0.03 and 1.19±0.10,respectively;the relative expression levels of Gli1 protein were 0.74±0.05,0.41±0.03,0.09±0.02 and 1.08±0.07,respectively;,compared the cell low,medium,and high-dose experimental groups with control group compared among the cell low,medium and high-dose experimental groups,the differences were all statistically significant(all P＜0.05).Animal experiments:In TSCC nude mice constructed with CAL-27,the tumor volumes of the animal low,medium and high dose experimental groups and the control group were(374.65±22.83),(260.94±27.31),(161.29±17.65)and(479.86±31.20)mm3,respectively;In TSCC nude mice constructed with Tca8113,the transplant tumor volumes were(386.57±27.60),(265.83±24.35),(174.35±15.96)and(491.18±32.24)mm3,respectively.,The transplant tumor volumes of compared the animal low,medium,and high dose experimental groups with the control group,compared among animal low,medium,and high dose experimental groups,the above indicators were all statistically significant(all P＜0.05).Rescue experiment:In CAL-27 cells,the proliferation indices of the experimental group,SAG group,pcDNA group and pcDNA Shh group were(25.83±3.20)％,(73.16±5.14)％,(26.92±4.31)％and(75.24±6.05)％,respectively;the apoptosis rates were(65.84±4.17)％,(4.83±1.28)％,(64.52±3.15)％and(4.14±1.31)％,respectively;In Tca8113 cells,the proliferation indices were(26.42±3.64)％,(76.13±5.25)％,(27.56±4.36)％and(78.05±6.17)％,respectively;the apoptosis rates were(63.91±3.62)％,(4.53±1.25)％,(64.02±2.98)％and(4.07±1.19)％,respectively;,compared the SAG group and pcDNA Shh group with the experimental group,the above indicators were all significantly different(all P＜0.05).Conclusion Toosendanin can induce apoptosis of TSCC cells,inhibit their proliferation and the transplanted tumor growth in nude mice,possibly by blocking the Shh/Gli1 signaling pathway.