Objective: To investigate the effects of salinomycin on the proliferation and apoptosis of oral squamous carcinoma cells and to further understand the mechanisms of these effects.
Methods: The human oral squamous carcinoma cell line CAL-27 was cultured in different concentrations of salinomycin and cisplatin. After co-culture with 0, 1, 2, 4, 8, 16 and 32 μmol/L salinomycin or 0, 1.25, 2.5, 5, 10, 20, 40 and 80 μmol/L cisplatin for 24 hours and 48 hours, the proliferation of oral squamous carcinoma cells were detected by cell counting kit-8(CCK-8) assay. After being exposed to 0, 2, 4, 8 μmol/L salinomycin and 0, 5, 10, 20 μmol/L cisplatin for 48 hours, the cell cycle of oral squamous carcinoma cells was detected by flow cytometry assay, and Western blot analysis was performed to analyze the expressions of cysteine-containing aspartate-specific proteases-3(Caspase-3), cysteine-containing aspartate-specific proteases-9(Caspase-9), poly ADP-ribose polymerase (PARP), protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt) protein in oral squamous carcinoma cells.
Results: Both salinomycin and cisplatin significantly inhibited the proliferation of oral squamous cell carcinoma CAL-27 cells in a time- and dose-dependent manner. However, compared with the first-line chemotherapeutic drug cisplatin, salinomycin showed stronger anti-proliferation activity in oral squamous carcinoma cells than cisp-latin (P < 0.001). After being exposed to 8 μmol/L salinomycin, CAL-27 cells exhibited markedly higher proportion in quiescent/ first gap phases (40.40%±1.99% vs. 64.46%±0.90%, P < 0.05), and had a significantly lower proportion in synthesis phases and second gap / mitosis phases (24.32%±2.30% vs. 18.73%±0.61%, P < 0.05; 35.01%±1.24% vs. 16.54%±1.31%, P < 0.05) compared with the dimethyl sulfoxide control group; moreover cisplatin didn't show cell-cycle specific effect on CAL-27. Western blot proved that salinomycin could up-regulate the expressions of Caspase-3 and Caspase-9 protein in oral squamous cell carcinoma CAL-27 cells (P < 0.05). At the same time, the levels of PARP, Akt and p-Akt protein were down-regulated (P < 0.05).
Conclusion: Compared with cisplatin, salinomycin has a better inhibitory effect on the proliferation of oral squamous carcinoma cells and blocks the cell cycle process at the quiescent / first gap phase. At the same time, salinomycin could trigger apoptosis of oral squamous carcinoma cells and the mechanism is associated with the Akt/p-Akt signaling pathway.
目的: 探讨沙利霉素(salinomycin)对口腔鳞癌细胞增殖和凋亡的影响, 并初步探讨沙利霉素对信号通路的影响。
方法: 培养口腔鳞状细胞癌细胞系CAL-27，将1、2、4、8、16、32 μmol/L沙利霉素和1.25、2.5、5、10、20、40、80 μmol/L顺铂与CAL-27细胞共同培养，24 h和48 h后用细胞计数试剂盒-8(cell counting kit-8，CCK-8)法检测沙利霉素和顺铂对CAL-27细胞增殖的影响；0、2、4、8 μmol/L沙利霉素和0、5、10、20 μmol/L顺铂与CAL-27细胞共培养48 h后，通过流式细胞术检测沙利霉素和顺铂对CAL-27细胞周期的影响，蛋白免疫印迹法(Western blot)检测CAL-27细胞中天冬氨酸特异性半胱氨酸蛋白酶-3(cysteine-containing aspartate-specific proteases-3，Caspase-3)、天冬氨酸特异性半胱氨酸蛋白酶-9(cysteine-containing aspartate-specific proteases-9，Caspase-9)、脱氧核糖核酸(deoxyribonucleic acid，DNA)修复酶(poly ADP-ribose polymerase，PARP)、蛋白激酶B(protein kinase B, Akt)和磷酸化蛋白激酶B (phosphorylated protein kinase B，p-Akt)的表达。
结果: CCK-8实验表明沙利霉素和顺铂均能显著抑制口腔鳞状细胞癌CAL-27细胞增殖，且抑制作用呈时间依赖性和药物浓度依赖性，但是相对于临床一线化疗药物顺铂而言，沙利霉素对CAL-27细胞增殖的抑制效果更加显著(P＜0.001)。细胞周期检测表明，与加入二甲基亚砜(dimethyl sulfoxide，DMSO)的对照组相比，8 μmol/L沙利霉素与CAL-27细胞共同培养48 h后，细胞休眠期/DNA合成前期的CAL-27细胞比例明显升高(40.40%±1.99% vs.64.46%±0.90%，P＜0.05)，DNA合成期和DNA合成后期/有丝分裂期的CAL-27细胞比例出现降低(24.32%±2.30% vs.18.73%±0.61%，P＜0.05，35.01%±1.24% vs.16.54%±1.31%，P＜0.05)；顺铂对CAL-27细胞周期没有特异性改变。蛋白免疫印迹法结果显示，沙利霉素在上调CAL-27细胞中Caspase-3和Caspase-9蛋白表达(P＜0.05)的同时下调PARP、Akt和p-Akt蛋白的表达(P＜0.05)。
结论: 相对于顺铂而言，沙利霉素对CAL-27细胞增殖有更强的抑制作用，并且能将口腔鳞状细胞癌CAL-27细胞周期阻滞在细胞休眠期/DNA合成前期，同时能够诱导CAL-27细胞发生凋亡，这一机制可能和Akt/p-Akt信号通路相关。
Keywords: Apoptosis; Cell cycle arrest; Oral squamous cell carcinoma; Salinomycin.