| Background:Macroautophagy (or autophagy) is a highly regulated cellular process that serves to remove and digest damaged proteins and organelles from the cell to fulfill its nutrition requirement under nutrient stress. Autopohagy could provide nutrition for the survival of stressed cells. For instance, it may promote the survival of cells detached from extracellular matrix. Overexpression of adaptor protein p66Shc in small lung cancer cells resulted in anoikis, thus blocking the tumor metastasis. Based on the reported functions of p66Shc, we wonder whether p66Shc plays an important role in autophagy and the specific mechanisms.Objective:In this study, we aimed at exploring the critical role and regulatory mechanism(s) of p66Shc on nutrient stress induced autophagy in lung cancer cells. In addition to the studies on cell lines (A549, BEAS-2B and MCF-7), we also expected to reveal the correlation between p66Shc expression level and autophagic level in clinical lung cancer specimens. These results may add more deep understanding on the regulation of autophagy in lung cancer cells.Methods:We first reduced the p66Shc expression by lentivirus infection in A549cells and BEAS-2B cells, and overexpressed p66Shc in MCF-7cells. Nutrient stress is regarded as a positive control of autophagy induction. Immunoblot was then performed to detect the autophagy marker LC3B, the related signaling pathway molecules such as Erkl/2, Akt, etc and several apoptosis related molecules. Plasmid DNA electroporation and confocal microscope were used to observe the florescent puncta of RFP-LC3B. Cell proliferation and death events were also assessed by BrdU incorporation assay and released DNA fragmentation, respectively; and were both normalized to cell numbers. Lastly, GST-pulldown assay was utilized to determine k-Ras activity after p66Shc silencing. Results:(1) Both protein and mRNA levels of p66shc were induced by nutrient stress in A549cells, also cell death was reduced after p66shc silence.(2) In A549cells, p66shc silence significantly reduced the RFP-LC3B puncta by nutritient stress for1day compared to the control. In whole medium culture condition, LC3B-â…¡/â… ratio decreased3folds after p66Shc silencing. However, after nutrient stress for3days, LC3B-â…¡/â… ratio showed no significant difference between p66Shc silencing and controls. Similarly, LC3B-II/I ratio was significantly reduced by p66Shc silencing after nutrition stress for8hours in BEAS-2B cell line. In MCF-7cells, overexpressed p66Shc led to an increased ratio of LC3B-â…¡/â… which was blocked by silenced Atg5. These data indicates that p66Shc is necessary for autophagy under nutrient stress.(3) In15out of24lung cancer tissues, p66Shc expression level is positively correlated to LC3-II expression levels.(4) p66Shc silencing reduced the proliferation of A549cells. Nutrient stress decreased the phosphorylation level of Erkl/2which was reversed by p66Shc silencing. Phospho-Akt but not phosphor-Erkl/2was activated after p66Shc overexpression in MCF-7.3-MA, an early-phase autophagy inhibitor, significantly enhanced the level of phospho-Erkl/2. Phospho-Akt but not phospho-p38decreased along with nutrient stess in A549cells. Silencing of p66shc increased the activity of k-Ras and reduced the level of Beclinl, but not affected p62degradation. These data indicate that k-Ras-Erkl/2-Beclinl signal pathway is partially involved in autophagic regulation by p66Shc.(5) Silencing of p66Shc by nutritien stress resulted in the reduced cell death as well as the decrease of cleaved caspase-7and PARP, but not cleaved caspase-6,-9. Thus p66Shcmay promote apoptosis resistance by the reduction of cleaved caspase-7and PARP.Conclusion:Under the nutritien stress, p66Shc silence delays autophagy level through k-Ras-Erkl/2-Beclinl pathway and increases apoptosis resistance which promotes lung cancer cells survival. The regulatory mechanism of autophagic pathway by adaptor protein p66Shc may shed lignt on the understanding of autophagic regulation in lung cancer progression. |
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