3-MA 通过抑制 Beclin-1 介导的自噬增强顺铂耐药下咽鳞癌细胞的化疗敏感性。

3-MA Enhanced Chemosensitivity in Cisplatin Resistant Hypopharyngeal Squamous Carcinoma Cells via Inhibiting Beclin -1 Mediated Autophagy.

机构信息

Division of ENT & HN Surgery, Shanghai General Hospital of Nanjing Medical University, Key Laboratory of Head and Neck, Shanghai, 200080, China.

出版信息

Curr Pharm Des. 2021;27(7):996-1005. doi: 10.2174/1381612826666201221150431.

DOI:10.2174/1381612826666201221150431

PMID:33349212

Abstract

BACKGROUND

Hypopharyngeal carcinoma is characterized by a high degree of malignancy. The most common pathological type is squamous cell carcinoma (HSCC). Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most widely used chemotherapeutic drugs nowadays and cisplatin resistance is a major problem in current treatment strategies. Clinical researchers have reported that high autophagy levels often caused insensitivity to chemotherapy, a common phenomenon that greatly reduces the therapeutic effect in cisplatin- resistant tumor cell lines. 3-methyladenine (3-MA), an inhibitor of PI3K, plays a vital role in forming and developing autophagosomes. Therefore, we speculate that the use of 3-MA may reduce cisplatin resistance in hypopharyngeal squamous cell carcinoma (HSCC).

METHODS

Part I: Cisplatin-resistant FaDu cell line (Human hypopharyngeal squamous cell carcinoma cells) was established and cultured. Cell counting kit-8 was used to detect drug resistance. An inverted microscope was used to observe the morphological changes at different concentrations, then the survival rate was calculated. After MDC staining, the autophagic vacuoles were observed by fluorescence microscopy. The expression of Beclin1 from each group was confirmed by RT-PCR and Western blot method. Part II: 3-MA was applied for cisplatin-resistant cells intervention, Beclin1 was knocked down by plasmid transfection. Cell cycle was detected using flow cytometry assay, apoptosis with necrosis was detected by staining with propidium iodide (PI). CCK-8 was used to observe the cell survival rate in each group. The expression of autophagy-related protein Beclin1, LC3I, LC3II, Atg-5 and P62 in each group was verified by Western blot analysis.

RESULTS

Cisplatin-resistant FaDu cell line can be stably constructed by cisplatin intervention. Compared with normal group, autophagy and its related protein Beclin1 expression were enhanced in cisplatin resistant FaDu cells. Autophagy inhibition group showed significant cell cycle changes, mainly manifested by G1 arrest, increased apoptosis rate and significantly decreased survival rate at 24h level. The number of autophagy vacuoles were significantly reduced in the 3-MA group. Furthermore, Western blot showed that expression of Beclin1, lc3-I, lc3-II, atg-5 protein decreased significantly after 3-MA intervention, while the expression of p62 upregulated, which also confirmed autophagy flow was blocked.

CONCLUSION

Our work confirmed that enhanced autophagy is an important cause of cisplatin resistance in FaDu cells. The use of 3-MA can significantly reduce autophagy level and arresting its cell cycle, promote apoptosis and reverse the cisplatin resistance condition, this effect is partly mediated by inhibition of Beclin-1 expression. Our data provide a theoretical basis for the application of 3-MA in overcoming cisplatin resistance in hypopharyngeal cancer.

摘要

背景

下咽癌的恶性程度较高，最常见的病理类型为鳞状细胞癌（HSCC）。顺铂（cis-diamminedichloroplatinum，CDDP）是目前应用最广泛的化疗药物之一，而顺铂耐药是当前治疗策略中的主要问题。临床研究人员报告称，高自噬水平常导致化疗不敏感，这是在顺铂耐药肿瘤细胞系中极大降低治疗效果的常见现象。3-甲基腺嘌呤（3-MA）是一种 PI3K 的抑制剂，在自噬小体的形成和发育中起着至关重要的作用。因此，我们推测使用 3-MA 可能会降低下咽鳞癌（HSCC）中的顺铂耐药性。

方法

第一部分：建立并培养顺铂耐药 FaDu 细胞系（人下咽鳞癌细胞）。用细胞计数试剂盒-8 检测药物耐药性。倒置显微镜观察不同浓度下的形态变化，然后计算存活率。MDC 染色后，用荧光显微镜观察自噬小泡。通过 RT-PCR 和 Western blot 方法确认各组 Beclin1 的表达。第二部分：用 3-MA 对顺铂耐药细胞进行干预，用质粒转染敲低 Beclin1。用流式细胞术检测细胞周期，用碘化丙啶（PI）染色检测凋亡和坏死。用 CCK-8 观察各组细胞的存活率。用 Western blot 分析各组自噬相关蛋白 Beclin1、LC3I、LC3II、Atg-5 和 P62 的表达。

结果

顺铂干预可稳定构建顺铂耐药 FaDu 细胞系。与正常组相比，顺铂耐药 FaDu 细胞中的自噬及其相关蛋白 Beclin1 表达增强。自噬抑制组细胞周期发生明显变化，主要表现为 G1 期阻滞，24 小时水平时凋亡率增加，存活率明显降低。3-MA 组自噬小泡数量明显减少。此外，Western blot 显示，3-MA 干预后 Beclin1、lc3-I、lc3-II、atg-5 蛋白表达明显下降，而 p62 表达上调，这也证实了自噬流被阻断。