左旋含羞草碱对人咽鳞状细胞癌FaDu细胞凋亡及DNA损伤相关蛋白表达的影响

The effects of L-mimosine on apoptosis and DNA damage-related protein expression in human pharyngeal squamous cell carcinoma FaDu cells

目的:探讨左旋含羞草碱对人咽鳞状细胞癌FaDu细胞凋亡及DNA损伤相关蛋白表达的影响。方法:观察左旋含羞草碱作用不同时间后FaDu细胞的生长状况;以流式细胞仪检测左旋含羞草碱与枸橼酸铁铵对FaDu细胞凋亡的影响;CCK-8法检测左旋含羞草碱与枸橼酸铁铵作用后FaDu细胞的增殖活性;Western blot法检测左旋含羞草碱作用于FaDu细胞后p-Histone-H2AX、p-ATR、p-ATM、p-mTOR等DNA损伤相关蛋白的表达。结果:不同浓度的左旋含羞草碱作用于咽鳞状细胞癌FaDu细胞不同时间后细胞的生长受到抑制;100μmol/L枸橼酸铁铵对FaDu细胞的凋亡具有抑制作用,缺铁或铁过量对肿瘤细胞凋亡具有促进作用;与对照组比,左旋含羞草碱组(200μmol/L)FaDu细胞凋亡最显著,左旋含羞草碱200μmol/L+枸橼酸铁铵50μmol/L组和左旋含羞草碱200μmol/L+枸橼酸铁铵100μmol/L组FaDu细胞凋亡差异无统计学意义,枸橼酸铁铵组(50、100μmol/L)FaDu细胞凋亡明显减低;对FaDu细胞增殖活性影响由强到弱依次为左旋含羞草碱组(200μmol/L)、左旋含羞草碱200μmol/L+枸橼酸铁铵50μmol/L组、左旋含羞草碱200μmol/L+枸橼酸铁铵100μmol/L组,与对照组比,差异有统计学意义(P<0.05);随着左旋含羞草碱浓度的增加,Fa Du细胞p-ATR蛋白的表达降低,p-Histone-H2AX、p-ATM、p-m TOR等蛋白的表达增强。结论:左旋含羞草碱可能通过干扰FaDu细胞铁代谢,调控AKT/mTOR等信号通路,引起细胞DNA双链损伤,导致肿瘤细胞凋亡。

Objective:To explore the effect of L-mimosine on FaDu cells apoptosis and DNA damage related protein expression in human pharyngeal squamous cell carcinoma.Methods:The growth of FaDu cells after treating with L-mimosine at different time was observed by an inverted microscope.Flow cytometry was used to detect the apoptosis effects after different concentrations of L-mimosine and ammonium ferric citrate treating FaDu cells.Cell growth proliferation activity was examined by CCK-8 method after different concentrations of Lmimosine and ammonium ferric citrate processing FaDu cells.The expressions of p-Histone-H2 AX,p-ATR,pATM and p-mTOR proteins related to DNA damage in FaDu cells were detected by western-blot after different concentrations of L-mimosine treating FaDu cells.Results:Different concentrations of L-mimosine inhibited the growth of FaDu cells at different times.Ferric ammonium citrate of 100μmol/L could inhibit the apoptosis of FaDu cells,while deficient or excessive iron could promote the apoptosis of tumor cells.Compared with the control group,the apoptosis rate of FaDu cells was found between L-mimosine group(200μmol/L)was the most significant.No significant difference was found between L-mimosine(200μmol/L)+ammonium ferric citrate(50μmol/L)group and L-mimosine(200μmol/L)+ammonium ferric citrate(100μmol/L)group.Meanwhile the apoptosis rate of FaDu cells in the ferric ammonium citrate group(50,100μmol/L)was significantly reduced.The effect on the proliferation activity of FaDu cells,ranging from strong to weak,were L-mimosine(200μmol/L)group,L-mimosine(200μmol/L)+ammonium ferric citrate(50μmol/L)group and L-mimosine(200μmol/L)+ammonium ferric citrate(100μmol/L)group.Compared with the control group,the difference was significant(P<0.05).With the increase of L-mimosine concentration,the expression of p-ATR protein decreased,while p-Histone-H2 AX,p-ATM,p-mTOR in FaDu cells increased.Conclusion:L-mimosine cause DNA double strand damage and lead to tumor cell apoptosis via interference with iron metabolism of FaDu cells to regulate Akt/mTOR and other signaling pathways.