1,9-二取代β-咔啉在头颈部鳞状细胞癌中抗肿瘤机制的研究

Antitumor mechanism of 1, 9-dissubstituted β-carboline in head and neck squamous cell carcinoma

摘要:

目的 初步探索1，9-二取代β-咔啉（CZY1-4）在头颈部鳞状细胞癌中抗肿瘤机制的研究。方法 CZY1-4是一种新合成的1，9-二取代β-咔啉，具有抗肿瘤潜力。采用CCK-8实验经过CZY1-4浓度梯度（0、2、4、6、10、15、20、25、30、40 μmol/L）处理24 h后检测细胞活力，筛选出两种对药物较为敏感的头颈部鳞状细胞癌细胞系：喉癌细胞系Tu177与舌癌细胞系Cal-27为主要研究对象。以浓度梯度（0、2、4、6、8 μmol/L）进行克隆形成实验检测细胞集落形成能力；随后分别以2 μmol/L与4 μmol/L的CZY1-4处理Tu177与Cal-27 24 h后进行划痕实验和Transwell实验检测细胞侵袭和迁移能力，流式细胞术分析细胞凋亡情况，Western blotting检测细胞相关蛋白表达量变化，免疫荧光实验检测细胞内自噬小体形成情况等。结果 经过上述浓度的CZY1-4处理24 h后，各头颈部鳞状细胞癌细胞系的生长增殖均受到不同程度的抑制，且呈剂量依赖性，其中Tu177与Cal-27抑制效果更为显著，两者的克隆形成能力也被显著抑制（P<0.05）；Tu177与Cal-27的迁移能力被显著抑制（P<0.01）；Tu177与Cal-27的侵袭能力被显著抑制（P<0.001）；Tu177与Cal-27凋亡率变化无显著差异（P>0.05）；自噬相关蛋白ATG5与LC3B，Benclin1蛋白表达量增加，呈剂量依赖性；自噬小体形成显著增加（P<0.01）；药物对被敲除ATG5后的Cal-27生长、增殖的抑制作用显著减弱（P<0.01）。结论 CZY1-4能显著抑制头颈部鳞状细胞癌相关细胞系的生长、增殖作用，并抑制细胞克隆形成、侵袭和迁移，具有一定的抗肿瘤活性，通过实验初步验证该药物主要通过调控ATG5促进细胞自噬的发生进而发挥抗肿瘤效果。

Abstract:

Objective To preliminarily explore the antitumor mechanism of 1,9-disubstituted β-carbolines (CZY1-4) in the head and neck squamous cell carcinoma.Methods CZY1-4 is a newly synthesized 1,9-disubstituted β-carboline with anti-tumor potential. In the experiment, the CCK-8 experiment was used to detect cell viability after CZY1-4 concentration gradient (0, 2, 4, 6, 10, 15, 20, 25, 30, 40 μmol/L) treatment for 24 hours. Two sensitive head and neck squamous cell carcinoma cell lines (laryngeal carcinoma cell line Tu177 and tongue cancer cell line Cal-27) were selected as the main research objects. Colony formation experiments under the concentration gradient (0,2,4,6,8μmol/L) were used to detect the cell colony-forming ability. After treatment of Tu177 and Cal-27 with 4 μmol/L and 2 μmol/L CZY1-4 for 24 h, wound healing test and Transwell test were conducted to detect cell invasion and migration ability, flow cytometry to analyze cell apoptosis, Western blotting to detect changes in the expression of cell-related proteins, and immunofluorescence experiments to detect the formation of autophagosomes in cells.Results After 24 h treatment with the above-mentioned concentrations of CZY1-4, the growth and proliferation of squamous cell carcinoma lines of head and neck were inhibited to varying degrees in a dose-dependent way, among which Tu177 and Cal-27 had more significant inhibition effect, and their colony forming abilities were also significantly inhibited (P<0.05). The migration abilities and invasive abilities of Tu177 and Cal-27 were significantly inhibited (P<0.01) (P<0.001). There was no significant difference in apoptosis rate between Tu177 and CAL-27 (P>0.05). The expression levels of autophagy related proteins ATG5, LC3B and Benclin1 increased in a dose-dependent manner. The formation of autophagosome was significantly increased (P<0.01). The inhibitory effect of the drug on the growth and proliferation of CAL-27 after ATG5 knockout was significantly weakened (P<0.01).Conclusions CZY1-4 can significantly inhibit the growth and proliferation of squamous cell carcinoma related cell lines, and inhibit the formation, invasion and migration of cell clones. It has certain anti-tumor activity by mainly promoting the occurrence of autophagy through regulating ATG5.