布比卡因介导PI3K/AKT/mTOR通路诱导结肠癌SW480细胞自噬和凋亡及抑制裸鼠移植瘤的形成

Bupivacaine-mediated PI3K/AKT/mTOR pathway in the induction of autophagy and apoptosis of colon cancer SW480 cells and inhibition of formation of xenografts in nude mice

目的探究布比卡因介导PI3K/AKT/mTOR通路诱导结肠癌SW480细胞自噬和凋亡的机制及其对裸鼠移植瘤形成的抑制作用。方法结肠癌SW480细胞分为对照组、布比卡因组(0.25、0.5、1 mM)。各组细胞处理24h后采用Edu染色检测细胞增殖;流式细胞术检测细胞凋亡;Western blot检测增殖、凋亡、自噬及信号通路相关蛋白表达。建立结肠癌裸鼠模型,随机分为布比卡因组(5、10、20 mg)及对照组,分别腹腔注射5、10、20 mg/kg的布比卡因及等体积生理盐水,1次/d,连续治疗30d后处死小鼠,取瘤、称重,免疫组化检测肿瘤组织Ki67蛋白阳性表达,并绘制各组裸鼠生存曲线。结果随着布比卡因浓度增加,SW480细胞增殖数及增殖相关蛋白Ki67、PCNA表达量逐渐降低(F=94.068、160.905、49.255,P<0.05),且均低于对照组(P<0.05);SW480细胞凋亡数及凋亡相关蛋白Caspase-3、Caspase-9表达量逐渐升高(F=30.948、110.730、233.550,P<0.05),且均高于对照组(P<0.05)。随着布比卡因浓度增加,SW480细胞Beclin1、ATG8蛋白相对表达量,LC3Ⅱ/LC3Ⅰ比值及LC3阳性数均逐渐升高(F=65.287、112.337、50.602、26.690,P<0.05),且高于对照组(P<0.05);p62蛋白相对表达量及p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR比值逐渐降低(F=33.276、98.246、102.488、58.051,P<0.05),且均低于对照组(P<0.05)。给药30d后,布比卡因给药组裸鼠肿瘤质量、Ki67阳性细胞占比及30d生存率均低于对照组(P<0.05)。结论布比卡因可抑制结肠癌SW480细胞增殖,诱导其发生自噬及凋亡,并抑制裸鼠移植瘤的形成,其作用机制可能与抑制PI3K/AKT/mTOR通路有关。

Objective To explore the mechanism of bupivacaine-mediated PI3 K/AKT/mTOR pathway in the induction of autophagy and apoptosis of colon cancer SW480 cells and its inhibitory effects on formation of xenografts in nude mice. Methods Colon cancer SW480 cells were divided into Control group and Bupivacaine groups(0.25, 0.5, 1 mM). After 24 h of cell treatment, cell proliferation was detected by Edu staining. Apoptosis was detected by flow cytometry. Western blot was used to detect the related proteins expressions of proliferation, apoptosis, autophagy and signaling pathway. Nude mice models of colon cancer were established and randomly divided into Bupivacaine groups(5, 10, 20 mg) and Control group, and they were intraperitoneally injected with 5, 10 and 20 mg/kg of Bupivacaine and an equal volume of normal saline once daily. After 30 d of continuous treatment, the mice were sacrificed, tumors were gotten to weigh. The positive expression level of Ki67 protein in tumor tissues was detected by immunohistochemistry. The survival curves of nude mice in each group were drawn. Results With the increase of Bupivacaine concentration, the proliferation quantity of SW480 cells and expression levels of proliferation-related proteins Ki67 and PCNA were decreased gradually(F=94.068, 160.905, 49.255,P<0.05), and were lower than those in Control group(P<0.05). The apoptosis quantity of SW480 cells and expression levels of apoptosis-related proteins Caspase-3 and Caspase-9 were increased gradually(F=30.948, 110.730, 233.550,P<0.05), and were higher than those in Control group(P<0.05). With the increase of Bupivacaine concentration, the relative protein expression levels of Beclin1 and ATG8 in SW480 cells, LC3 II/LC3 I ratio and positive number of LC3 were increased gradually(F=65.287, 112.337, 50.602 and 26.690,P<0.05), and were higher than those in Control group(P<0.05). The relative protein expression level of p62 and ratios of p-PI3 K/PI3 K, p-AKT/AKT and p-mTOR/mTOR were gradually decreased(F=33.276, 98.246, 102.488 and 58.051,P<0.05), and were lower than those in Control group(P<0.05). After 30 days of administration, The tumor mass, Ki67 positive cell ratio and 30 d survival rate in Bupivacaine administration groups were lower than those in Control group(P<0.05). Conclusion Bupivacaine can inhibit the proliferation of colon cancer SW480 cells, induce the occurrence of autophagy and apoptosis, and inhibit the formation of xenografts in nude mice. Its role mechanism may be related to the inhibition of PI3 K/AKT/mTOR pathway.