Abstract
BackgroundDysfunctions in autophagy and apoptosis are closely interacted and play an important role in cancer development. RNA binding motif 5 (RBM5) is a tumor suppressor gene, which inhibits tumor cells’ growth and enhances chemosensitivity through inducing apoptosis in our previous studies. In this study, we investigated the relationship between RBM5 overexpression and autophagy in human lung adenocarcinoma cells.MethodsHuman lung adenocarcinoma cancer (A549) cells were cultured in vitro and were transiently transfected with a RBM5 expressing plasmid (GV287-RBM5) or plasmid with scrambled control sequence. RBM5 expression was determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Intracellular LC-3 I/II, Beclin-1, lysosome associated membrane protein-1 (LAMP1), Bcl-2, and NF-κB/p65 protein levels were detected by Western blot. Chemical staining with monodansylcadaverine (MDC) and acridine orange (AO) was applied to detect acidic vesicular organelles (AVOs). The ultrastructure changes were observed under transmission electron microscope (TEM). Then, transplanted tumor models of A549 cells on BALB/c nude mice were established and treated with the recombinant plasmids carried by attenuated Salmonella to induce RBM5 overexpression in tumor tissues. RBM5, LC-3, LAMP1, and Beclin1 expression was determined by immunohistochemistry staining in plasmids-treated A549 xenografts.ResultsOur study demonstrated that overexpression of RBM5 caused an increase in the autophagy-related proteins including LC3-I, LC3-II, LC3-II/LC3-I ratio, Beclin1, and LAMP1 in A549 cells. A large number of autophagosomes with double-membrane structure and AVOs were detected in the cytoplasm of A549 cells transfected with GV287-RBM5 at 24 h. We observed that the protein level of NF-κB/P65 was increased and the protein level of Bcl-2 decreased by RBM5 overexpression. Furthermore, treatment with an autophagy inhibitor, 3-MA, enhanced RBM5-induced cell death and chemosensitivity in A549 cells. Furthermore, we successfully established the lung adenocarcinoma animal model using A549 cells. Overexpression of RBM5 enhanced the LC-3, LAMP1, and Beclin1 expression in the A549 xenografts.ConclusionsOur findings showed for the first time that RBM5 overexpression induced autophagy in human lung adenocarcinoma cells, which might be driven by upregulation of Beclin1, NF-κB/P65, and downregulation of Bcl-2. RBM5-enhanced autophagy acts in a cytoprotective way and inhibition of autophagy may improve the anti-tumor efficacy of RBM5 in lung cancer.
Highlights
Dysfunctions in autophagy and apoptosis are closely interacted and play an important role in cancer development
Ectopic expression of RNA binding motif 5 (RBM5) enhanced autophagic vacuoles formation in A549 cells We previously reported that RBM5 overexpression induced apoptosis in human lung adenocarcinoma A549 cells [19, 20]
With acridine orange (AO) staining, we observed that red fluorescent spots were increased in RBM5 overexpressed A549 cells compared to the control cells (Fig. 1e), indicating that RBM5 overexpression enhanced autophagic vacuoles formation
Summary
Dysfunctions in autophagy and apoptosis are closely interacted and play an important role in cancer development. RNA binding motif 5 (RBM5) ( called Luca or H37), the tumor suppressor gene, maps to the human chromosomal locus 3p21.3, which is strongly associated with lung cancer [9]. The ectopic expression of RBM5 suppresses the growth of human lung cancer [11], breast cancer [13], fibrosarcoma [14], and hematopoietic cells [15,16,17]. Our previous study demonstrated that exogenous expression of RBM5 by the pcDNA3.1-RBM5 inhibited the cell growth of human prostate cancer and lung cancer in vivo and in vitro and resensitized the response of A549/DDP cells (cisplatin resistant counterparts of A549 cells) to cisplatin [8, 18,19,20]. The relationship between RBM5 and autophagy in human lung cancer is still unknown
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