丹参酮ⅡA通过抑制PI3K/Akt通路逆转人喉癌细胞顺铂耐药的机制研究

Mechanism of tanshinoneⅡA reversing cisplatin resistance in human laryngeal cancer cells by inhibiting PI3K/Akt pathway

目的研究丹参酮Ⅱ_(A)对人喉癌细胞顺铂耐药的影响作用及机制。方法以不同质量浓度(0、1.0、2.0、4.0、8.0、16.0、32.0 mg/L)顺铂干预对数生长期人喉癌Hep-2细胞和人喉癌顺铂耐药细胞(Hep-2/DDP)48 h,CCK-8法检测细胞增殖抑制率,计算半数抑制浓度(IC_(50))和耐药指数(RI)。以不同质量浓度(0、0.5、1.0、2.0、4.0、8.0、16.0 mg/L)丹参酮Ⅱ_(A)干预对数生长期Hep-2细胞48 h,CCK-8法检测细胞增殖抑制率,计算IC_(50);采用丹参酮Ⅱ_(A)(IC_(50)5.32 mg/L)+不同质量浓度(0、1.0、2.0、4.0、8.0、16.0、32.0 mg/L)顺铂干预对数生长期Hep-2/DDP细胞48 h,CCK-8法检测细胞增殖抑制率,计算2种药物联用的IC_(50)和逆转倍数(RF)。取对数生长期Hep-2/DDP细胞,设置对照组、顺铂组、丹参酮Ⅱ_(A)+顺铂组、丹参酮Ⅱ_(A)+顺铂+胰岛素样生长因子-1(IGF-1)组。各组分别给药干预48 h后,采用CCK-8法、Annexin V-FITC/PI双染法检测细胞增殖抑制率和凋亡率,Western blotting法检测B细胞淋巴瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)、激活型半胱氨酸蛋白酶-3(cleaved Caspase-3)、cleaved Caspase-9、磷脂酰肌醇3激酶(PI3K)、p-PI3K、蛋白激酶B(Akt)蛋白表达情况。结果顺铂对Hep-2细胞的IC_(50)为4.58 mg/L,对Hep-2/DDP细胞的IC_(50)为14.09 mg/L,RI为3.08;丹参酮Ⅱ_(A)对Hep-2细胞的IC_(50)为5.32 mg/L;丹参酮Ⅱ_(A)联合顺铂对Hep-2/DDP细胞的IC_(50)为3.34 mg/L,RF为4.22。与对照组比较,顺铂组Hep-2/DDP细胞增殖抑制率、凋亡率显著升高(P<0.01);与顺铂组比较,丹参酮Ⅱ_(A)+顺铂组Hep-2/DDP细胞增殖抑制率、凋亡率显著升高(P<0.05);与丹参酮Ⅱ_(A)+顺铂组比较,丹参酮Ⅱ_(A)+顺铂+IGF-1组Hep-2/DDP细胞增殖抑制率、凋亡率显著降低(P<0.05)。与对照组相比,顺铂组Hep-2/DDP细胞Bcl-2表达量显著降低,Bax、cleaved Caspase-3、cleaved Caspase-9表达量和Bax/Bcl-2值显著升高(P<0.05)。与顺铂组比较,丹参酮Ⅱ_(A)+顺铂组Hep-2/DDP细胞Bcl-2表达量显著降低,Bax、cleaved Caspase-3、cleaved Caspase-9表达量和Bax/Bcl-2值显著升高(P<0.05)。与丹参酮Ⅱ_(A)+顺铂组比较,丹参酮Ⅱ_(A)+顺铂+IGF-1组Hep-2/DDP细胞Bcl-2表达量显著升高,Bax、cleaved Caspase-3、cleaved Caspase-9表达量和Bax/Bcl-2值显著降低(P<0.05)。与对照组相比,顺铂组Hep-2/DDP细胞p-PI3K、p-Akt表达量及PI3K、Akt磷酸化(pPI3K/PI3K、p-Akt/Akt)水平显著降低(P<0.05)。与顺铂组比较,丹参酮Ⅱ_(A)+顺铂组Hep-2/DDP细胞p-PI3K、p-Akt表达量及PI3K、Akt磷酸化水平显著降低(P<0.05)。与丹参酮Ⅱ_(A)+顺铂组比较,丹参酮Ⅱ_(A)+顺铂+IGF-1组Hep-2/DDP细胞p-PI3K、p-Akt表达量及PI3K、Akt磷酸化水平显著升高(P<0.05)。结论丹参酮Ⅱ_(A)可逆转人喉癌细胞顺铂耐药,其作用机制可能与抑制PI3K/Akt通路活化而促进喉癌细胞凋亡有关。

Objective To study the effect and mechanism of tanshinone Ⅱ_(A) on cisplatin resistance in human laryngeal carcinoma cells.Methods Human laryngeal carcinoma Hep-2 cells and human laryngeal carcinoma cisplatin-resistant cells(Hep-2/DDP)were treated with different concentrations of cisplatin(0,1.0,2.0,4.0,8.0,16.0,32.0 mg/L)for 48 h.The inhibition rate of cell proliferation was detected by CCK-8 method,and the semi-inhibitory concentration(IC_(50))and drug resistance index(RI)were calculated.Hep-2 cells were treated with different concentrations of tanshinoneⅡ_(A)(0,0.5,1.0,2.0,4.0,8.0,16.0 mg/L)in logarithmic growth phase for 48 h.The cell proliferation inhibition rate was detected by CCK-8 method,and the IC_(50) was calculated.Hep-2/DDP cells were treated with tanshinoneⅡ_(A) IC_(50)(5.32 mg/L)+cisplatin at different concentrations(0,1.0,2.0,4.0,8.0,16.0,32.0 mg/L)for 48 h,and the cell proliferation inhibition rate was detected by CCK-8 method.The IC_(50) and reversion fold(RF)of two drug combinations were calculated.Hep-2/DDP cells in logarithmic growth phase were divided into control group,cisplatin group,tanshinoneⅡ_(A)+cisplatin group,and tanshinoneⅡ_(A)+cisplatin+IGF-1 group.The cell proliferation inhibition rate and apoptosis rate were detected by CCK-8 method and Annexin V-FITC/PI double staining method,and the expression of Bcl-2,Bax,Cleaved caspase-3,Cleaved caspase-9,PI3K,p-PI3K,Akt,and p-Akt was detected by Western blotting.Results The IC_(50) of cisplatin on Hep-2 cells was 4.58 mg/L,the IC_(50) of cisplatin on Hep-2/DDP cells was 14.09 mg/L,and the RI was 3.08.The IC_(50) of tanshinoneⅡ_(A) on Hep-2 cells was 5.32 mg/L.The IC_(50) and RF of tanshinoneⅡ_(A) combined with cisplatin were 3.34 mg/L and 4.22,respectively.Compared with the control group,the proliferation inhibition rate and apoptosis rate of Hep-2/DDP cells in cisplatin group were significantly increased(P<0.01).Compared with cisplatin group,the proliferation inhibition rate and apoptosis rate of Hep-2/DDP cells in tanshinoneⅡ_(A)+cisplatin group were significantly increased(P<0.05).Compared with tanshinoneⅡ_(A)+cisplatin group,the proliferation inhibition rate and apoptosis rate of Hep-2/DDP cells in tanshinoneⅡ_(A)+cisplatin+IGF-1 group were significantly decreased(P<0.05).Compared with the control group,the expression level of Bcl-2 in Hep-2/DDP cells in cisplatin group was significantly decreased,while the expression levels of Bax,Cleaved caspase-3,Cleaved caspase-9,and Bax/Bcl-2 ratio were significantly increased(P<0.05).Compared with cisplatin group,the expression level of Bcl-2 in Hep-2/DDP cells in tanshinoneⅡ_(A)+cisplatin group was significantly decreased,and the expression levels of Bax,Cleaved caspase-3,Cleaved caspase-9,and Bax/Bcl-2 ratio were significantly increased(P<0.05).Compared with tanshinoneⅡ_(A)+cisplatin group,the expression level of Bcl-2 in Hep-2/DDP cells in tanshinoneⅡ_(A)+cisplatin+IGF-1 group was significantly increased,and the expression levels of Bax,Cleaved caspase-3,Cleaved caspase-9,and Bax/Bcl-2 ratio were significantly decreased(P<0.05).Compared with the control group,the expressions of p-PI3K and p-Akt and the phosphorylation of PI3K and Akt(p-PI3K/PI3K,p-Akt/Akt)in Hep-2/DDP cells in cisplatin group were significantly decreased(P<0.05).Compared with cisplatin group,the expressions of p-PI3K and p-Akt and the phosphorylation levels of PI3K and Akt in Hep2/DDP cells in tanshinoneⅡ_(A)+cisplatin group were significantly decreased(P<0.05).Compared with tanshinoneⅡ_(A)+cisplatin group,the expressions of p-PI3K and p-Akt and the phosphorylation levels of PI3K and Akt in Hep-2/DDP cells in tanshinoneⅡ_(A)+cisplatin+IGF-1 group were significantly increased(P<0.05).Conclusions TanshinoneⅡ_(A) can reverse the cisplatin resistance of human laryngeal cancer cells,which mechanism may be related to inhibiting the activation of PI3K/Akt pathway and promoting the apoptosis of laryngeal cancer cells.