Resveratrol modulates the apoptosis and autophagic death of human lung adenocarcinoma A549 cells via a p53‑dependent pathway: Integrated bioinformatics analysis and experimental validation

Abstract

Resveratrol (RSV) has been reported to exhibit cytotoxic activity in multiple types of malignant cells; however, the mechanisms underlying the antitumor effects of RSV in non-small-cell lung cancer (NSCLC) cells remain undetermined. Combining bioinformatics analysis with experimental validation, the present study aimed to examine the effects of RSV on the apoptosis and autophagy of A549 NSCLC cells, and to determine the potential underlying molecular mechanisms. Bioinformatics analysis was used to determine the differentially expressed genes (DEGs) and identify the enriched biological functions and pathways associated with these DEGs following RSV treatment. Cell viability was determined by MTT assay, and flow cytometry and TUNEL assay were used to evaluate cell apoptosis. Monodansylcadaverine staining combined with a transmission electron microscope were used to evaluate the extent of autophagy. The expression levels of apoptosis-, autophagy-, or pathway-associated molecular markers were measured by reverse transcription-quantitative PCR and/or western blot analysis. By bioinformatics analysis, a total of 1,031 DEGs were identified in the RSV-treated A549 cells, which were enriched in apoptosis-, or autophagy-related biological functions and the p53 signaling pathway. In validation experiments, RSV significantly reduced cell viability and initiated apoptosis, with an increase in the number of apoptotic cells; it also upregulated cleaved caspase-3 expression and Bax expression, and downregulated the Bcl-2 expression levels. Additionally, there was an increase in the accumulation of green dot-like structures, indicative of autophagic vesicles, observed under a fluorescence microscope, and an increase in the presence of autophagic vacuoles observed using a transmission electron microscope following RSV treatment. Furthermore, the expression levels of the autophagy-related proteins, LC3-II/LC3-I and Beclin-1, were increased and p62 expression was decreased. 3-methyladenine (3-MA), an inhibitor of autophagy, partially reversed the RSV-induced cytotoxic effects, but did not significantly alter the number of apoptotic cells. RSV elevated the p53 levels and decreased the phosphorylated (p-)Mdm2 and p-Akt levels. Pifithrin-α, an inhibitor of p53, partially reduced RSV-induced apoptosis and autophagy. On the whole, the results of the present study demonstrated that RSV initiates the apoptosis and autophagic death of A549 cells via the activation of the p53 signaling pathway, further highlighting the potential of RSV for the treatment of NSCLC.

Modulation of Autophagy in Cancer Cells by Dietary Polyphenols

“…On the one hand, autophagy may play a cytoprotective role [ 7 ], and on the other hand, it may promote the development of tumors [ 8 , 9 ]. It is worth emphasizing that autophagy also may play a role in promoting chemoresistance [ 30 , 31 , 32 ]. The impact of autophagy on the reduction or progression of neoplastic processes is strictly dependent on the type and stage of the tumor, microenvironment, applied therapeutics, or the genetic context [ 8 , 9 ].…”

“…The effect of RSV on A549 autophagy was assessed by RT-qPCR, Western blot analysis, transmission electron microscopy (TEM), and monodansylcadaverine (MDC) staining [ 32 ]. The treatment of cells with 50 and 100 µM RSV for 48 h, resulted in the appearance of mature autophagous vacuoles with some of them containing fragments of a damaged endoplasmic reticulum and mitochondria as evidenced by TEM [ 32 ]. RT-qPCR analysis showed decreased expression of p62 mRNA in A549 cells, while expression of beclin-1 mRNA was increased.…”

“…RT-qPCR analysis showed decreased expression of p62 mRNA in A549 cells, while expression of beclin-1 mRNA was increased. 3-methyladenine was used to determine the viability of A549 cells and it was found that resveratrol decreased the viability of the cells [ 32 ]. These results clearly indicate that RSV plays a significant role in inducing autophagy in A549 cells [ 32 ].…”

Status and Challenges of Plant-Anticancer Compounds in Cancer Treatment

“…Several action mechanisms of resveratrol have been described: positive regulation of p53 and BAX proteins (related with pro-apoptotic pathways) and negative regulation of NF-κB, AP-1, hypoxia-inducible factor 1-alpha (HIF-1α), matrix metalloproteases, Bcl-2 protein, COX-2, cytokines and CDK [ 109 ]. Some pre-clinical studies performed in vitro demonstrated that resveratrol was able to suppress the cell proliferation through cell cycle arrest, induce apoptosis and modulate autophagy in different cancer cell lines, including ovarian cancer cell line, resistant human leukemia cells, non-small-cell lung cancer and human lung adenocarcinoma [ 110 , 111 , 112 ]. Regarding in vivo studies, the anticancer properties of this compounds were also significant.…”

Plant-Derived Natural Products in Cancer Research: Extraction, Mechanism of Action, and Drug Formulation

“…The nanohybrid's anticancer ability was investigated in human lung cancer cells (A549) and indicated higher activity comparing to bare resveratrol [64]. Moreover, resveratrol initiates the apoptosis and autophagic death of lung cancer cells by stimulating p53 signaling pathway [65].…”