ACSS2通过PI3K/AKT信号通路调控食管鳞癌细胞的顺铂敏感性

目的: 探讨乙酰辅酶A合成酶2（acetyl-CoA synthetase 2，ACSS2）表达对食管鳞癌细胞顺铂（cisplatin，DDP）敏感性的影响和可能机制。方法: 通过免疫组化和蛋白质印迹法检测40对食管鳞癌瘤体和癌旁组织中ACSS2蛋白的表达。蛋白质印迹法验证食管鳞癌细胞株TE-1、ECA-109和KY-SE150与正常食管鳞状细胞Het-1A中ACSS2的表达差异；利用脂质体转染siRNA-ACSS2或者慢病毒转染LV-ACSS2至TE-1细胞构建ACSS2下调或过表达细胞株；CCK8实验检测下调ACSS2对顺铂IC50值的改变；顺铂处理（5 μg/mL）前后，流式细胞术检测ACSS2干扰处理对TE-1细胞凋亡水平的影响；蛋白质印迹检测PI3K/AKT信号通路及凋亡相关蛋白cleaved-Caspase-3的表达。结果： 免疫组化及蛋白质印迹结果显示，食管鳞癌组织中ACSS2蛋白表达显著高于癌旁组织（P<0.001），食管鳞癌细胞株中ACSS2表达水平明显高于正常鳞状上皮细胞Het-1A。CCK8结果显示siRNA-ACSS2处理显著下调TE-1细胞对顺铂的IC₅₀值（P值均<0.05）；流式细胞术结果证实，抑制ACSS2表达可显著上调食管鳞癌细胞凋亡率（P <0.01）；结合5 μg/mL顺铂处理，ACSS2干扰后TE-1细胞凋亡率明显上升（P值均<0.001）；蛋白质印迹结果表明，顺铂处理上调TE-1细胞中ACSS2及p-PI3K、p-AKT的表达；顺铂作用下，靶向抑制ACSS2后可见p-PI3K、p-AKT的显著降低而凋亡相关蛋白cleaved-Caspase-3表达量明显升高，过表达ACSS2在维持PI3K/AKT信号活化的同时可有效逆转cleaved-Caspase-3的形成。结论： ACSS2通过调控PI3K/AKT信号通路活化以及cleaved-Caspase-3表达参与食管鳞癌细胞顺铂敏感性调节。

ACSS2 mediates the chemosensitivity of human esophageal squamous cell carcinoma via PI3K/AKT pathway

Objective: To explore the effect of the expression change of acetyl-CoA synthetase 2 (ACSS2) on the sensitivity of esophageal squamous cell carcinoma (ESCC) to cisplatin (DDP) and relevant mechanism. Methods: ACSS2 expression was detected in tumor and paracancerous tissues collected from 40 cases of ESCC by immunohistochemistry and western blotting. In vitro verification of ACSS2 expression difference was performed by Western blotting in ESCC cell line and normal esophageal squamous Het-1A cell line. ACSS2 downregulated and overexpressed cell strains were constructed by liposome transfection of siRNA-ACSS2 or lentivirus transfection of LV-ACSS2 into TE-1 cells. CCK8 assay was used to detect the change of IC50 value of DDP by downregulating the expression of ACSS2. Meanwhile, the effect of ACSS2 interference on TE-1 cell apoptosis was tested by flow cytometry before and after DDP treatment (5 μg/mL). In addition, Western blotting was carried out to detect the expression of proteins related to PI3K/AKT signaling pathway and apoptosis-related protein cleaved-Caspase-3. Results: Immunohistochemistry and Western blotting results showed that the expression intensity of ACSS2 in ESCC tissue was significantly higher than that in paracancerous normal tissue (P<0.001). In vitro test indicated that the expression of ACSS2 in ESCC cell line was obviously higher than that of normal esophageal squamous Het-1A cell line. CCK8 results showed that IC50 of TE-1 cells was remarkedly downregulated after siRNA-ACSS2 treatment (all P<0.05). Flow cytometry results showed that the apoptosis rate of ESCC cells was obviously decreased by inhibiting ACSS2 expression (P<0.01), which, however, increased significantly under the combined treatment of 5 μg/mL DDP and ACSS2 interference (all P<0.001). Furthermore, according to the results of Western blotting, expression levels of ACSS2, p-PI3K and p-AKT were upregulated in TE-1 cells after DDP treatment. Simultaneously, under DDP treatment, targeted inhibition of ACSS2 expression resulted in significant decrease of p-PI3K and p-AKT expression, but obvious increase of apoptosis related protein of cleaved-Caspase-3; while overexpression of ACSS2 could maintain the activation of PI3K/AKT signaling pathway and reverse the expression of cleaved-Caspase-3 effectively. Conclusion: ACSS2 is involved in regulating the sensitivity of ESCC to DDP by regulating activation of PI3K/AKT signaling pathway and expression of cleaved-Caspase-3. ［Key words］acetyl-CoA synthetase 2； cisplatin； esophageal squamous cell carcinoma； chemosensitivity； PI3K/AKT