| Lung cancer is one of the diseases that seriously threatens human health. It was reported that lung cancer is the leading cause of cancer-related mortality and morbidity in our country in the ’2012 Chinese Cancer Registration Report’. Nearly 85% of these are non-small cell lung cancer(NSCLC), which typically has a poor prognosis with the overall 5-year survival rate of ~17%, despite innovations in diagnostic testing, surgical technique and development of new chemotherapeutic agents. As most of the cases are at the advanced stage when they are diagnosed and lost che chance for surgery resection, platinum-based treatment, especially cisplatin-based chemotherapy is the first-line chemotherapy recommended. But the therapeutic efficacy is limited due to the primary or acquired cisplatin resistance. Therefore, looking for more effective anti-tumor treatment, a better understanding of the molecular mechanisms underlying chemoresistance, and more effective strategies to exclusive drug resistance are of high importance and urgency for the clinical treatment of lung cancer patients.RNA binding motif 5(RBM5)(also called Luca15 or H37) is a newly confirmed tumor suppressor gene in recent years, which maps to the human chromosomal locus 3p21.3. Through pre-m RNA splicing of multiple target genes, RBM5 regulates gene expression of various target genes, cell signal pathways and cell biological behaviours. RBM5 is closely related to lung cancer genesis and development. Its expression levels are obviously lower in lung cancer tissues than in normal lung tissues. The ectopic expression of RBM5 suppresses the growth of human lung cancer, breast cancer, fibrosarcoma and prostate cancer, and resensitized the response of cisplatin resistant counterparts of human lung adenocarcinoma cells to cisplatin. Recent studies suggest that RBM5 acts an anti-tumor role through regulation of the mitochondrial apoptotic pathway, Bcl-2 family expression, EGFR and cell cycle proteins expression to induce appotosis and cell cycle arrest. However, the mechanisms of RBM5-mediated tumor suppression and reversal of cisplatin resistance are still not clear.Autophagy is an evolutionarily conserved process in all eukaryotic cells, which is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. Deregulation of autophagy is implicated in several human diseases including cancers. Recently, increasing evidence has shown that autophagy plays an important role in the resistance of cancer cells to chemotherapy and radiotherapy. Recent evidence suggests that autophagy plays a role in acquired cisplatin resistance in lung cancer cells. However, the molecular mechanism of the regulation of autophagy in lung cancer, is largely unknown. It has been shown that EGFR and Bcl-2 family members function as autophagic regulators via their interaction with the core autophagy factor Beclin1, which plays an important role in the initiation of autophagosome formation. EGFR and Bcl-2 family members have been found to be regulated by RBM5. However, whether RBM5 plays a role on autophagy regulation, and the role autophagy played in the process of RBM5 mediated anti-tumor and cisplatin resistance reversal in human lung adenocarcinoma cells has yet to be elucidated.In this study, we focused our research specifically on lung adenocarcinoma A549 cells and their cisplatin resistant variant A549/DDP cells. We investigated the regulation of autophagy gene expression and autophagy level by RBM5 and its effect on response to cisplatin in lung adenocarcinoma cells. These findings provide scientific basis for clarifing the mechanisms of RBM5 mediated anti-tumor, cisplatin resistance reversal and autophagy regulation, as well as for the development of targeted anti-tumor treatment and chemoresistance reversal strategy.Methods1) MTT assay was used to evaluate chemosensitivity of human lung adenocarcinoma A549 cells and cisplatin resistant A549(A549/DDP) cells to cisplatin. Phase-contrast microscopy was used to observe the cells’ appearance.2) Monodansylcadaverine(MDC) staining, acridine orange(AO) staining and transmission electron microscopy(TEM) were applied to detect acidic vesicular organelles(AVOs) morphologically. Intracellular microtubule-associated protein1 light chain3- I/II(LC3- I/II), Beclin1, lysosome associated membrane protein-1(LAMP1), expression levels were detected by Western blot analysis.3) A549 cells were cultured in vitro and were transiently transfected with a RBM5 expressing plasmid(GV287-RBM5) using lipofectamin2000, with or without 3-Methyladenine(3-MA) treatment. RBM5 m RNA and protein levels were determined by semi-quantitative RT-PCR and Western blot analyses, respectively. MDC staining, AO staining, TEM and Western blot analyses were performed to access autophagy activity. MTT assay was used to evaluate cell viability. Expression levels of autophagy regulators, Beclin1, NF-κB/p65, and Bcl-2 were detected by Western blot analysis.4) Transplanted tumor models of A549 cells on BALB/c nude mice were established and treated with the GV287-RBM5 plasmids carried by attenuated Salmonella to induce RBM5 overexpression in tumor tissues. The mice were sacrificed on day 42 and the tumors were removed and fixed in formalin for immunohistochemistry analysis. RBM5, LC3, LAMP1 and Beclin1 expression levels were determined by immunohistochemistry staining in the xenografts.5) A549/DDP cells were cultured in vitro and were transiently transfected with a RBM5 expressing plasmid(GV287-RBM5) using lipofectamin 2000, with or without 3-Methyladenine(3-MA) treatment. RBM5 m RNA and protein levels were determined by semi-quantitative RT-PCR and Western blot analyses, respectively. MDC staining, AO staining, TEM and Western blot analyses were performed to access autophagy activity. MTT assay was used to evaluate cell viability.Results1) A549/DDP cells are highly resistant to cisplatin-induced cell death compared with A549 cells. Using phase-contrast microscopy, the A549/DDP cells were observed as having a regular, round shape, and were markedly larger as compared with the A549 cells.2) MDC staining, AO staining and TEM examination showed that formation of autophagic vacuoles in the cytoplasm of A549/DDP cells was obviously increased compared with A549 cells. Western blot analysis demonstrated that autophagy related proteins including LC3-I, LC3-II, LC3-II/LC3-I ratio, Beclin1 and LAMP1 were increased significantly in A549/DDP cells compared with A549 cells.3) The m RNA and protein levels of RBM5 were significantly increased in the RBM5 transfected A549 cells. MDC staining, AO staining, TEM examination and Western blot analysis showed that autophagy activity was enhanced in RBM5 overexpression group than in control group. When co-treated with 3-MA, RBM5 overexpression-mediated autophagy was inhibited and early stage apoptosis increased. MTT assay showed that RBM5 overexpression inhibited the proliferation of A549 cells, while co-treated with 3-MA further enhanced RBM5 overexpression-mediated cell proliferation inhibition and chemosensitivity. The expression levels of Beclin1 and NF-κB/p65 were increased significantly, and the levels of Bcl-2 were decreased in RBM5 overexpression group, and these were inhibited by co-treatment of 3-MA.4) Lung adenocarcinoma animal models were successfully established using A549 cells. Overexpression of RBM5 enhanced the LC3, LAMP1 and Beclin1 expression in the A549 xenografts.5) Transfection of A549/DDP cells with GV287-RBM5 resulted in significant increase of RBM5 m RNA and protein levels. Exogenous expression of RBM5 further enhanced the autophagy activity as demonstrated by MDC staining, AO staining, TEM and Western blot analysis. Administration of 3-MA inhibited RBM5-induced autophagy and increased RBM5-induced early stage apoptosis. MTT assay showed that RBM5 overexpression inhibited the proliferation of A549/DDP cells. Pharmacological inhibition of autophagy by 3-MA resulted in enhanced RBM5 overexpression-mediated decrease of cell viability and increase of chemosensitivity.Conclusions:1) In cisplatin sensitivie/resistant human lung adenocarcinoma cells RBM5 overexpression activated autophagy, which may act in a cytoprotective way and weaken the anti-tumor effect of RBM5. Upregulation of Beclin1, NF-κB/P65 and downregulation of Bcl-2 by overexpression of RBM5 may be one of the mechanisms.2) Autophagy inhibitor, 3-MA could so effectively inhibit RBM5 overexpression-induced autophagy, that it enhanced apoptosis, tumor supression and chemotherapy sensitization induced by RBM5 overexpression. Hance, a combination of autophagy inhibitor and RBM5 overexpression may be a promising therapy to treat human lung adenocarcinoma and resensitize tumor cells to chemotherapy. |
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