Phosphoinositide specific phospholipase Cγ1 inhibition-driven autophagy caused cell death in human lung adenocarcinoma A549 cells in vivo and in vitro

Abstract

Our previous studies indicated that phosphoinositide specific phospholipase Cγ1 (PLCγ1) was involved in autophagy induction in colon and hepatic carcinoma cells. However, whether and how PLCγ1 regulation in human lung adenocarcinoma is linked to autophagy remains unclear. Here, we assessed the protein expression of PLCγ1 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 PLCγ1 and autophagy-related signal molecules was investigated in human lung adenocarcinoma A549 cells treated with different inhibitors or transduction with lentivirus-mediated PLCγ1 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/PLCγ1 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 PLCγ1 expression occurred frequently in human lung adenocarcinoma tissue with higher grades of T in TNM staging classification. PLCγ1 significantly enriched in autophagic process and regulation, which negatively regulating autophagy was enriched in higher expression of PLCγ1. PLCγ1 inhibition partially reduced cell proliferation and migration of A549 cells, with an increased autophagic flux involving alterations of AMPKα, mTOR, and ERK levels. However, PLCγ1 inhibition-driven autophagy led to cell death without depending on Caspase-3 and RIP1. Additionally, the abrogation of PLCγ1 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 PLCγ1 inhibition could be partially attributed to PLCγ1 inhibition-driven autophagic cell death (ACD). It highlights the potential role of a combination between targeting PLCγ1 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.

A Correlation Study of Prognostic Risk Prediction for Colorectal Cancer Based on Autophagy Signature Genes

“…In a previous report, the initiation of autophagy in sorafenib-resistant hepatocellular carcinoma cells enhanced the resistance of cancer cells to sorafenib ( 14 ). In addition, it has also been found that when autophagy dies, it reduces the proliferation and migration of lung adenocarcinoma cells to the extent that reducing increased tumor autophagy may be an effective therapeutic strategy ( 15 ). Based on these findings, we speculate that ARGs play a multifaceted effect in cancer.…”

Reticulocalbin 1 is required for proliferation and migration of non‐small cell lung cancer cells regulated by osteoblast‐conditioned medium

“…NSCLC cells were seeded in 96‐well plates and cultured in different types of medium for 48 h. Cell viability was measured by 3‐(4, 5‐Dimethylthiazol‐2‐y)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay at the end of the assay period 16,17 . Clonogenicity was also used to assess cell proliferation.…”

“…Clonogenicity was also used to assess cell proliferation. Cells cultured in 6‐well plates for 48 h were fixed with 100% methanol and stained with 0.1% crystal violet, followed with observation of colonies under an Olympus BX41 microscope equipped with a digital camera (Olympus, Tokyo, Japan) at 4x magnification 16 …”

“…NSCLC cells were seeded in 96‐well plates and cultured in different types of medium for 48 h. Cell viability was measured by 3‐(4, 5‐Dimethylthiazol‐2‐y)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay at the end of the assay period. 16 , 17 Clonogenicity was also used to assess cell proliferation. Cells cultured in 6‐well plates for 48 h were fixed with 100% methanol and stained with 0.1% crystal violet, followed with observation of colonies under an Olympus BX41 microscope equipped with a digital camera (Olympus, Tokyo, Japan) at 4x magnification.…”

“… 17 Transwell migration assay was also used to assess cell migration. According to the previous studied, 16 , 17 cells in serum‐free RPMI‐1640 were placed into the top chambers of Transwell inserts set with 8 μm pore filters and different types of media were added to the bottom chamber, respectively. Cells on the top of the membrane were removed with a cotton swab.…”