Tumor suppressor gene RBM5 delivered by attenuated Salmonella inhibits lung adenocarcinoma through diverse apoptotic signaling pathways

World Journal of Surgical Oncology

Ke Wang²  Jie Zhang²  Song Wang³  Lijing Zhao¹  Baoxue Yang⁴  Chen Shao² 

[1] Department of Pathophysiology, Norman Bethune College of Medicine of Jilin University, 18, Zhiqiang Street, Changchun, Jilin 130021, China;Department of Respiratory Medicine, the Second Affiliated Hospital of Jilin University, 18 Ziqiang Street, Changchun, Jilin 130041, China;Department of Urinary Surgery, the First Affiliated Hospital of Jilin University, 71 Xinmin Street, Changchun, Jilin 130041, China;Department of Pharmacology, School of Basic Medical Sciences of Peking University, 38 Xueyuan Road, Beijing 100191, China

关键词: Attenuated Salmonella;    Xenograft mice model;    A549;    Apoptosis;    Lung adenocarcinoma;    RBM5;   

Others  :  823921
DOI  :  10.1186/1477-7819-11-123

 received in 2013-02-03, accepted in 2013-05-16,  发布年份 2013

【 摘 要 】

Background

RBM5 (RNA-binding motif protein 5, also named H37/LUCA-15) gene from chromosome 3p21.3 has been demonstrated to be a tumor suppressor. Current researches in vitro confirm that RBM5 can suppress the growth of lung adenocarcinoma cells by inducing apoptosis. There is still no effective model in vivo, however, that thoroughly investigates the effect and molecular mechanism of RBM5 on lung adenocarcinoma.

Method

We established the transplanted tumor model on BALB/c nude mice using the A549 cell line. The mice were treated with the recombinant plasmids carried by attenuated Salmonella to induce the overexpression of RBM5 in tumor tissues. RBM5 overexpression was confirmed by immunohistochemistry staining. H&E staining was performed to observe the histological performance on plasmids-treated A549 xenografts. Apoptosis was assessed by TUNEL staining with a TUNEL detection kit. Apoptosis-regulated genes were detected by Western blot.

Results

We successful established the lung adenocarcinoma animal model in vivo. The growth of tumor xenografts was significantly retarded on the mice treated with pcDNA3.1-RBM5 carried by attenuated Salmonella compared to that on mice treated with pcDNA3.1. Overexpression of RBM5 enhanced the apoptosis in tumor xenografts. Furthermore, the expression of Bcl-2 protein was decreased significantly, while the expression of BAX, TNF-α, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and cleaved PARP proteins was significantly increased in the pcDNA3.1-RBM5-treated mice as compared to that in the control mice.

Conclusions

In this study, we established a novel animal model to determine RBM5 function in vivo, and concluded that RBM5 inhibited tumor growth in mice by inducing apoptosis. The study suggests that although RBM5’s involvement in the death receptor-mediated apoptotic pathway is still to be investigated, RBM5-mediated growth suppression, at least in part, employs regulation of the mitochondrial apoptotic pathways.

【 授权许可】

   
2013 Shao et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin 2012, 62:10-29.
• [2]Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA: Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 2008, 83:584-594.
• [3]Borczuk AC, Gorenstein L, Walter KL, Assaad AA, Wang LQ, Powell CA: Non-small-cell lung cancer molecular signatures recapitulate lung developmental pathways. Am J Pathol 2003, 163:1949-1960.
• [4]Menna C, Ciccone AM, Ibrahim M, Andreetti C, D'Andrilli A, Maurizi G, Cassiano F, Cavaliere I, Venuta F, Rendina EA: Pneumonectomy: quality of life and long-term results. Minerva Chir 2012, 67:219-226.
• [5]Lerman MI, Minna JD, Chromosome ILC: The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. Cancer Res 2000, 60:6116-6133.
• [6]Kok K, Naylor SL, Buys CH: Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv Cancer Res 1997, 71:27-92.
• [7]Ji L, Minna JD, Roth JA: 3p21.3 tumor suppressor cluster: prospects for translational applications. Future Oncol 2005, 1:79-92.
• [8]Wistuba II, Behrens C, Virmani AK, Mele G, Milchgrub S, Girard L, Fondon JW 3rd, Garner HR, McKay B, Latif F, Lerman MI, Lam S, Gazdar AF, Minna JD: High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res 2000, 60:1949-1960.
• [9]Wei MH, Latif F, Bader S, Kashuba V, Chen JY, Duh FM, Sekido Y, Lee CC, Geil L, Kuzmin I, Zabarovsky E, Klein G, Zbar B, Minna JD, Lerman MI: Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG. Cancer Res 1996, 56:1487-1492.
• [10]Sutherland LC, Edwards SE, Cable HC, Poirier GG, Miller BA, Cooper CS, Williams GT: LUCA-15-encoded sequence variants regulate CD95-mediated apoptosis. Oncogene 2000, 19:3774-3781.
• [11]Rintala-Maki ND, Sutherland LC: LUCA-15/RBM5, a putative tumour suppressor, enhances multiple receptor-initiated death signals. Apoptosis 2004, 9:475-484.
• [12]Mourtada-Maarabouni M, Sutherland LC, Williams GT: Candidate tumour suppressor LUCA-15 can regulate multiple apoptotic pathways. Apoptosis 2002, 7:421-432.
• [13]Mourtada-Maarabouni M, Sutherland LC, Meredith JM, Williams GT: Simultaneous acceleration of the cell cycle and suppression of apoptosis by splice variant delta-6 of the candidate tumour suppressor LUCA-15/RBM5. Genes Cells 2003, 8:109-119.
• [14]Sutherland LC, Lerman M, Williams GT, Miller BA: LUCA-15 suppresses CD95-mediated apoptosis in Jurkat T cells. Oncogene 2001, 20:2713-2719.
• [15]Sutherland KD, Lindeman GJ, Choong DY, Wittlin S, Brentzell L, Phillips W, Campbell IG, Visvader JE: Differential hypermethylation of SOCS genes in ovarian and breast carcinomas. Oncogene 2004, 23:7726-7733.
• [16]Oh JJ, Razfar A, Delgado I, Reed RA, Malkina A, Boctor B, Slamon DJ: 3p21.3 tumor suppressor gene H37/Luca15/RBM5 inhibits growth of human lung cancer cells through cell cycle arrest and apoptosis. Cancer Res 2006, 66:3419-3427.
• [17]Li P, Wang K, Zhang J, Zhao L, Liang H, Shao C, Sutherland LC: The 3p21.3 tumor suppressor RBM5 resensitizes cisplatin-resistant human non-small cell lung cancer cells to cisplatin. Cancer Epidemiol 2012, 36:481-489.
• [18]Oh JJ, Taschereau EO, Koegel AK, Ginther CL, Rotow JK, Isfahani KZ, Slamon DJ: RBM5/H37 tumor suppressor, located at the lung cancer hot spot 3p21.3, alters expression of genes involved in metastasis. Lung Cancer 2010, 70:253-262.
• [19]Akhtar MJ, Ahamed M, Khan MA, Alrokayan SA, Ahmad I, Kumar S: Cytotoxicity and apoptosis induction by nanoscale talc particles from two different geographical regions in human lung epithelial cells. Environ Toxicol 2012. [Epub ahead of print]
• [20]Niu ZY, Jin WX, Zhang LB, Li XL: Tumor suppressor RBM5 directly interacts with the DExD/H-box protein DHX15 and stimulates its helicase activity. FEBS Lett 2012, 586:977-983.
• [21]Fushimi K, Ray P, Kar A, Wang L, Sutherland LC, Wu JY: Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5. Proc Natl Acad Sci USA 2008, 105:15708-15713.
• [22]Zhao M, Yang M, Li XM, Jiang P, Baranov E, Li S, Xu M, Penman S, Hoffman RM: Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA 2005, 102:755-760.
• [23]Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M, Ash O, Carmichael E, Chakraborty A, Fischer J, Lin SL, Luo X, Miller SI, Zheng L, King I, Pawelek JM, Bermudes D: Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat Biotechnol 1999, 17:37-41.
• [24]Mu YM, Oba K, Yanase T, Ito T, Ashida K, Goto K, Morinaga H, Ikuyama S, Takayanagi R, Nawata H: Human pituitary tumor transforming gene (hPTTG) inhibits human lung cancer A549 cell growth through activation of p21(WAF1)/CIP1. Endocr J 2003, 50:771-781.
• [25]Zhang L, Gao L, Li Y, Lin G, Shao Y, Ji K, Yu H, Hu J, Kalvakolanu DV, Kopecko DJ, Zhao X, Xu DQ: Effects of plasmid-based Stat3-specific short hairpin RNA and GRIM-19 on PC-3M tumor cell growth. Clin Cancer Res 2008, 14:559-568.
• [26]Gao L, Zhang L, Hu J, Li F, Shao Y, Zhao D, Kalvakolanu DV, Kopecko DJ, Zhao X, Xu DQ: Down-regulation of signal transducer and activator of transcription 3 expression using vector-based small interfering RNAs suppresses growth of human prostate tumor in vivo. Clin Cancer Res 2005, 11:6333-6341.
• [27]Lin GM, Zhao LJ, Yin F, Lan RF, Li LB, Zhang XM, Zhang H, Yang BX: TCF3 inhibits F9 embryonal carcinoma growth by the down-regulation of Oct4. Oncol Rep 2011, 26:893-899.
• [28]Lerman MI, Minna JD: The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res 2000, 60:6116-6133.
• [29]Angeloni D, ter Elst A, Wei MH, van der Veen AY, Braga EA, Klimov EA, Timmer T, Korobeinikova L, Lerman MI, Buys CH: Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes. Genes Chromosomes Cancer 2006, 45:676-691.
• [30]Ramaswamy S, Ross KN, Lander ES, Golub TR: A molecular signature of metastasis in primary solid tumors. Nat Genet 2003, 33:49-54.
• [31]Oh JJ, West AR, Fishbein MC, Slamon DJ: A candidate tumor suppressor gene, H37, from the human lung cancer tumor suppressor locus 3p21.3. Cancer Res 2002, 62:3207-3213.
• [32]Liang H, Zhang J, Shao C, Zhao L, Xu W, Sutherland LC, Wang K: Differential expression of RBM5, EGFR and KRAS mRNA and protein in non-small cell lung cancer tissues. J Exp Clin Cancer Res 2012, 31:36. BioMed Central Full Text
• [33]King I, Itterson M, Bermudes D: Tumor-targeted Salmonella typhimurium overexpressing cytosine deaminase: a novel, tumor-selective therapy. Methods Mol Biol 2009, 542:649-659.
• [34]Gardlik R, Fruehauf JH: Bacterial vectors and delivery systems in cancer therapy. IDrugs 2010, 13:701-706.
• [35]Shao Y, Liu Y, Shao C, Hu J, Li X, Li F, Zhang L, Zhao D, Sun L, Zhao X, Kopecko DJ, Kalvakolanu DV, Li Y, Xu DQ: Enhanced tumor suppression in vitro and in vivo by co-expression of survivin-specific siRNA and wild-type p53 protein. Cancer Gene Ther 2010, 17:844-854.
• [36]Ji K, Wang B, Shao YT, Zhang L, Liu YN, Shao C, Li XJ, Li X, Hu JD, Zhao XJ, Xu DQ, Li Y, Cai L: Synergistic suppression of prostatic cancer cells by coexpression of both murine double minute 2 small interfering RNA and wild-type p53 gene in vitro and in vivo. J Pharmacol Exp Ther 2011, 338:173-183.
• [37]Li X, Zhang L, Shao Y, Liang Z, Shao C, Wang B, Guo B, Li N, Zhao X, Li Y, Xu D: Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion. Biochem Biophys Res Commun 2011, 416:270-276.
• [38]Israels LG, Israels ED: Apoptosis. Stem Cells 1999, 17:306-313.
• [39]Ashkenazi A, Dixit VM: Death receptors: signaling and modulation. Science 1998, 281:1305-1308.
• [40]Rintala-Maki ND, Abrasonis V, Burd M, Sutherland LC: Genetic instability of RBM5/LUCA-15/H37 in MCF-7 breast carcinoma sublines may affect susceptibility to apoptosis. Cell Biochem Funct 2004, 22:307-313.
• [41]Davis FA, Haque MS, Ulatowski TG, Towson JC: Asymmetric oxidation of ester and amide enolates using new (camphorylsulfonyl)oxaziridines. J Org Chem 1986, 51:2402-2404.
• [42]Dudley E, Hornung F, Zheng L, Scherer D, Ballard D, Lenardo M: NF-kappaB regulates Fas/APO-1/CD95- and TCR-mediated apoptosis of T lymphocytes. Eur J Immunol 1999, 29:878-886.