Effect of Pristimerin on Proliferation of Oral Squamous Cell Carcinoma CAL-27 by Regulating Autophagy
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摘要:
目的 探讨扁塑藤素对人口腔鳞状细胞癌细胞CAL-27增殖能力的影响及其相关作用机制。 方法 CCK8检测不同浓度的扁塑藤素处理CAL-27细胞后的增殖生物活性，计算不同作用时间段所对应的药物浓度水平（IC50）；细胞集落形成实验检测CAL-27细胞克隆形成能力；蛋白质印迹法检测细胞增殖指标PCNA以及相关自噬通量BECLIN1、LC3B-II、LC3B-I蛋白表达水平；扁塑藤素联合自噬激动剂雷帕霉素（RAPA）处理CAL-27细胞，蛋白质印迹法检测PCNA以及相关自噬通量BECLIN1、LC3B-II、LC3B-I蛋白表达水平。 结果 较于空白组，各浓度梯度的扁塑藤素可抑制CAL-27细胞活力（P < 0.05），具有浓度-时间依耐性；相较于空白组，经扁塑藤素处理后细胞中PCNA、BECLIN1、LC3B-II/LC3B-I蛋白表达水平都出现明显上调（P < 0.05）；扁塑藤素联合自噬激活剂雷帕霉素（RAPA）处理CAL-27细胞相较于仅使用扁塑藤素组细胞活性有所增强（P < 0.05），PCNA、BECLIN1、LC3B-II/LC3B-I蛋白的表达水平均出现明显上调（P < 0.05）。 结论 扁塑藤素在体外能够比较显著抑制口腔鳞状癌系CAL-27细胞的增殖，这可能与其下调自噬相关基因BECLIN1、LC3B-II、LC3B-I的表达相关。 Abstract:Objective To investigate the effect of pristimerin on the proliferation ability of human oral squamous cell carcinoma cells CAL-27 and its related mechanisms. Methods CCK-8 assay was performed to determine the proliferative bioactivity of CAL-27 cells treated with different concentrations of pristimerin, and the IC50 was calculated for different time periods. Colony formation assay was used to detect the cloning ability of CAL-27 cells. Western blotting was used to detect the protein expression levels of the proliferation marker PCNA and the autophagy flux markers BECLIN1, LC3B-II, and LC3B-I. CAL-27 cells were treated with pristimerin in combination with the autophagy inducer rapamycin （RAPA）, and Western blotting was used to detect the protein expression levels of PCNA and the autophagy flux markers BECLIN1, LC3B-II, and LC3B-I. Results Compared with the blank group, the pristimerin at various concentration gradients can inhibit the viability of CAL-27 cells （P < 0.05）, and it exhibits concentration-time dependence. Compared with the blank group, the expression levels of PCNA, BECLIN1 and LC3B-II/LC3B-I proteins in cells were significantly upregulated after treatment with the pristimerin （P < 0.05）. Compared with the group treated with only the pristimerin, the combined treatment of the pristimerin with the autophagy activator rapamycin （RAPA） enhanced the cell activity of CAL-27 cells （P < 0.05）, and the expression levels of PCNA, BECLIN1, and LC3B-II/LC3B-I proteins were significantly upregulated （P < 0.05）. Conclusion Pristimerin significantly inhibits the proliferation of CAL-27 oral squamous carcinoma cells in vitro, which may be associated with downregulation of autophagy-related genes BECLIN1, LC3B-II, and LC3B-I. -
Key words:
- Pristimerin /
- Oral squamous cell carcinoma /
- Proliferation /
- Autophagy
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图 1 扁塑藤素抑制口腔鳞状癌Cal-27细胞增殖
A：扁塑藤素化学式和分子结构[17]；B：CCK-8测定不同浓度扁塑藤素处理Cal-27细胞对细胞增殖的影响；C：CCK-8定扁塑藤素处理Cal-27细胞24 h、48 h、72 h对细胞增殖的影响。与0 μmol/L组相比，*P < 0.05，***P < 0.0005，****P < 0.0001。
Figure 1. Pristimerin suppresses proliferation of oral squamous cell carcinoma cell
图 2 扁塑藤素对口腔鳞状癌CAL-27细胞克隆形成的影响
Figure 2. The effect of Pristimerin on clonal formation of oral squamous cell carcinoma cell
图 3 扁塑藤素抑制相关增殖指标PCNA
A：Western blot实验检测Cal-27细胞增殖指标PCNA蛋白表达情况；B：PCNA蛋白相对表达量量化图。与0 μmol/L组相比，*P < 0.05，**P < 0.01，***P < 0.0001。
Figure 3. Effect of Pristimerin on PCNA expression
图 4 扁塑藤素抑制自噬相关自噬蛋白BECLIN1、LC3B-II\LC3B-I
A：Western blot实验检测Cal-27细胞自噬相关BECLIN1、LC3B-I、LC3B-II蛋白表达情况；B：BECLIN1蛋白相对表达量量化图；C：LC3B-II/LC3B-I蛋白相对表达量量化图。与0 μmol/L组相比，*P < 0.05，**P < 0.01。
Figure 4. Pristimerin inhibits apoptosis：BECLIN1and LC3B-II/LC3B-I protein expression
图 5 自噬激活剂RAPA对口腔鳞状癌细胞的影响
A：雷帕霉素对Cal-27细胞活性影响量化图；B：0.6 μmol/L扁塑藤素与不同浓度雷帕霉素分别及共同作用后对Cal-27细胞活性影响量化图。与对照组相比，**P < 0.01，***P < 0.005，****P < 0.001；与0.6 μmol/L相比，#P < 0.05。
Figure 5. Effect of autophagy activator RAPA on oral squamous carcinoma cells
图 6 扁塑藤素联合雷帕霉素处理对口腔鳞状癌CAL-27细胞克隆形成的影响
Figure 6. The effect of Pristimerin combined with RAPA on clonal formation of oral squamous carcinoma cells
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