1,9-二取代β-咔啉在头颈部鳞癌中抗肿瘤机制的研究

Reseach on the 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：本实验选取两种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,以一定药物浓度分别相应处理两种细胞系后,分别采用CCK-8实验检测细胞活力,克隆形成实验检测细胞集落形成能力,Wound healing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 Blot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：药物处理后,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: In this study, two squamous cell carcinoma cell lines of head and neck, namely laryngeal carcinoma cell lines Tu177 and tongue carcinoma cell line Cal-27, were selected as the main objects of study. After corresponding treatment of the two cell lines with certain drug concentration, cell viability was detected by CCK-8 assay, and cell colony formation ability was detected by clonal formation assay. Wound Healing and Transwell assay were used to detect the invasion and migration of cells, flow cytometry was used to analyze cell apoptosis, Western Blot was used to detect the expression of cell related proteins, and immunofluorescence assay was used to detect the formation of intracellular autophagosomes.Results: After drug treatment, the growth and proliferation of squamous cell carcinoma lines of head and neck were inhibited to varying degrees in a dose-dependent manner, among which Tu177 and Cal-27 had more significant inhibition effect, and their clonogenesis ability was also significantly inhibited (P<0.05). The migration ability of Tu177 and Cal-27 was significantly inhibited (P<0.01). The invasive ability of Tu177 and CAL-27 was significantly inhibited (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).Conclusion: 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. Experiments have preliminarily verified that the drug mainly promotes the occurrence of autophagy through regulating ATG5 and thus plays an anti-tumor effect.