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PAK4-relevant proliferation reduced by cell autophagy via p53/mTOR/p-AKT signaling
Qing Li1, Su-Jie Wang1, Wen-Jia Wang2
1Institute of Pathophysiology, College of Basic Medical Sciences, Lanzhou University, Lanzhou, China.
Background:
P21-activated kinase 4 (PAK4) involves in cell proliferation in cancer and mutually regulates with p53, a molecule is demonstrated to control cell autophagy by mammalian target of rapamycin (mTOR)/protein kinase B (AKT) signaling. Since the signaling exhibits an association with PAK family members in cell autophagy, it implies that PAK4-relevant proliferation may be impacted by autophagy via p53 with a lack of evidence in cancer cells.
Methods:
In this research, transient and stable PAK4-knockdown human hepatocarcinoma cell lines (HepG2) were constructed by transfection of PAK4-RNA interference (RNAi) plasmid and lentivirus containing PAK4-RNAi plasmid, respectively. We investigated cell proliferation using methyl thiazolyl tetrazolium (MTT) and Cell Counting Kit 8 (CCK8) assays, cell cycle by flow cytometry (FCM) and cell autophagy by monodansylcadaverine (MDC) staining and autophagic biomarker's expression, and detected the expressions of p53, mTOR, phosphorylated-AKT (p-AKT) and AKT by immunofluorescence and western blot to explore the mechanism.
Results:
We successfully constructed transient and stable PAK4-knockdown HepG2 cell lines, and detected dysfunction of the cells' proliferation. An increased expression of p53, as a molecule of cell-cycle-surveillance on G1/S phase, was demonstrated in the cells although the cell cycle blocked at G2/M. And then, we detected increased autophagosome and autophagic biomarker LC3-II, and decreased expressions in p-AKT and mTOR.
Conclusions:
The proliferation is reduced in PAK4-knockdown HepG2 cells, which is relative to not only cell cycle arrest but also cell autophagy, and p53/mTOR/p-AKT signaling involves in the cell progress. The findings provide a new mechanism on PAK4 block in cancer therapy.
Insights
P21-activated kinase 4 (PAK4) knockdown reduces cancer cell proliferation by inducing cell cycle arrest and autophagy. This process involves the p53/mammalian target of rapamycin (mTOR)/protein kinase B (AKT) signaling pathway, offering a new target for cancer therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- P21-activated kinase 4 (PAK4) is implicated in cancer cell proliferation and interacts with p53.
- p53 regulates autophagy via the mammalian target of rapamycin (mTOR)/protein kinase B (AKT) signaling pathway.
- The interplay between PAK4, p53, and autophagy in cancer remains underexplored.
Purpose of the Study:
- To investigate the role of PAK4 in hepatocellular carcinoma (HepG2) cell proliferation.
- To elucidate the mechanism by which PAK4 influences cell cycle and autophagy.
- To explore the involvement of the p53/mTOR/AKT signaling pathway.
Main Methods:
- Constructed transient and stable PAK4-knockdown HepG2 cell lines using RNA interference.
- Assessed cell proliferation via MTT and CCK8 assays.
- Analyzed cell cycle progression using flow cytometry and autophagy via monodansylcadaverine staining and biomarker expression (LC3-II).
- Detected protein expression levels of p53, mTOR, p-AKT, and AKT using immunofluorescence and western blot.
Main Results:
- PAK4 knockdown resulted in impaired HepG2 cell proliferation.
- Cell cycle analysis revealed arrest at the G2/M phase, with increased p53 expression.
- Autophagy markers, including autophagosomes and LC3-II, were elevated, while p-AKT and mTOR expression decreased.
- Confirmed the involvement of the p53/mTOR/p-AKT signaling pathway.
Conclusions:
- PAK4 knockdown inhibits hepatocellular carcinoma cell proliferation through cell cycle arrest and enhanced autophagy.
- The p53/mTOR/p-AKT signaling pathway is a key mediator in PAK4-regulated cancer cell progression.
- These findings suggest a novel mechanism for targeting PAK4 in cancer therapy.
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