Phosphoinositide specific phospholipase Cγ1 inhibition-driven autophagy caused cell death in human lung adenocarcinoma A549 cells in vivo and in vitro
Phosphoinositide specific phospholipase Cγ1 inhibition-driven autophagy caused cell death in human lung adenocarcinoma A549 cells in vivo and in vitro
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DOI：
10.7150/ijbs.42962
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发表时间：
2020-01-01
影响因子：
9.2
通讯作者：
Zhang, Bing
中科院分区：
文献类型：
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作者：
Lu, Xiaohong;Fu, Haijing;Zhang, Bing
我们的前期研究表明，磷脂酰肌醇特异性磷脂酶C γ 1（PLC γ 1）参与了结肠癌和肝癌细胞的自噬诱导。然而，在人类肺腺癌中PLC γ 1调节是否以及如何与自噬相关联仍不清楚。本研究采用免疫组化方法检测人肺腺癌组织中PLC γ 1蛋白的表达，并采用斯皮尔曼相关分析和GSEA软件在癌症基因组图谱网络（TCGA）中分析PLC γ 1与自噬的关系。此外，在用不同抑制剂处理或用慢病毒介导的PLC γ 1基因短发夹RNA（shRNA）载体转导的人肺腺癌A549细胞中，使用MTT、克隆形成、Transwell迁移、RT-PCR、Caspase-3、线粒体跨膜电位和蛋白质印迹分析研究PLC γ 1和自噬相关信号分子之间的相互作用。以及透射电镜技术。此外，在A549异种移植裸鼠模型中使用免疫组织化学和测定来测量单独的shRNA/PLC γ 1或与自噬激活剂氯化锂（LiCl）组合对肿瘤生长和转移的影响。结果表明，PLC γ 1在TNM分期中T分级较高的肺腺癌组织中表达增高。PLC γ 1在自噬过程和调节中显著富集，其负调节自噬在PLC γ 1的较高表达中富集PLC γ 1抑制部分地降低了A549细胞的细胞增殖和迁移，同时增加了自噬通量，涉及AMPK α、mTOR和ERK水平的改变。然而，PLC γ 1抑制驱动的自噬导致细胞死亡而不依赖于Caspase-3和RIP]。此外，通过shRNA消除PLC γ 1信号传导并与自噬激活剂LiCl组合可以有效地抑制A549异种移植裸鼠中的肿瘤生长和转移，同时降低P62水平。这些发现共同表明，PLC γ 1抑制细胞增殖和迁移的减少可能部分归因于PLC γ 1抑制驱动的自噬细胞死亡（ACD）。它突出了靶向PLC γ 1和自噬通路之间的组合在抗肿瘤治疗中的潜在作用，这可能是克服肿瘤的自噬增加和对当前治疗的获得性抗性的有效新策略。
Our previous studies indicated that phosphoinositide specific phospholipase C gamma 1 (PLC gamma 1) was involved in autophagy induction in colon and hepatic carcinoma cells. However, whether and how PLC gamma 1 regulation in human lung adenocarcinoma is linked to autophagy remains unclear. Here, we assessed the protein expression of PLC gamma 1 in human lung adenocarcinoma tissue using immunohistochemistry assay and the relationship between PLCG1 and autophagy in The Cancer Genome Atlas Network (TCGA) using Spearman correlation analysis and GSEA software. Furthermore, the interaction between PLC gamma 1 and autophagy-related signal molecules was investigated in human lung adenocarcinoma A549 cells treated with different inhibitors or transduction with lentivirus-mediated PLC gamma 1 gene short-hairpin RNA (shRNA) vectors using MTT, clonogenicity, Transwell migration, RT-PCR, Caspase-3, mitochondrial transmembrane potential, and western blotting assays, as well as transmission electron microscope technique. Additionally, the effect of shRNA/PLC gamma 1 alone or combined with autophagic activator Lithium Chloride (LiCl) on tumor growth and metastasis was measured using immunohistochemistry and assays in A549 xenograft nude mouse model. The results showed that increased PLC gamma 1 expression occurred frequently in human lung adenocarcinoma tissue with higher grades of T in TNM staging classification. PLC gamma 1 significantly enriched in autophagic process and regulation, which negatively regulating autophagy was enriched in higher expression of PLC gamma 1 PLC gamma 1 inhibition partially reduced cell proliferation and migration of A549 cells, with an increased autophagic flux involving alterations of AMPK alpha, mTOR, and ERK levels. However, PLC gamma 1 inhibition-driven autophagy led to cell death without depending on Caspase-3 and RIP]. Additionally, the abrogation of PLC gamma 1 signaling by shRNA and combination with autophagic activator LiCl could efficaciously suppress tumor growth and metastasis in A549 xenograft nude mice, in combination with a decrease in P62 level. These findings collectively suggest that reduction of cell proliferation and migration by PLC gamma 1 inhibition could be partially attributed to PLC gamma 1 inhibition-driven autophagic cell death (ACD). It highlights the potential role of a combination between targeting PLC gamma 1 and autophagy pathway in anti-tumor therapy, which may be an efficacious new strategy to overcome the autophagy addition of tumor and acquired resistance to current therapy.