Pro-apoptotic effects of splice-switching oligonucleotides targeting Bcl-x pre-mRNA in human glioma cell lines

Li,Zhaohui; Li,Qingwei; Han,Liang; Tian,Nan; Liang,Qianlei; Li,Yanzhe; Zhao,Xingli; Du,Chao; Tian,Yu

doi:10.3892/or.2015.4465

Pro-apoptotic effects of splice-switching oligonucleotides targeting Bcl-x pre-mRNA in human glioma cell lines

Authors:
- Zhaohui Li
- Qingwei Li
- Liang Han
- Nan Tian
- Qianlei Liang
- Yanzhe Li
- Xingli Zhao
- Chao Du
- Yu Tian
View Affiliations

Affiliations: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China, Department of Neurosurgery, Heilongjiang Provincial Hospital, Harbin, Heilongjiang 150036, P.R. China, Department of Cell Biology, College of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China
Published online on: November 30, 2015 https://doi.org/10.3892/or.2015.4465
Pages: 1013-1019

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Alternative splicing is a near-ubiquitous phenomenon with important roles in human diseases, including cancers. Splice-switching oligonucleotides (SSOs) have emerged as a class of antisense therapeutics that modulate alternative splicing by hybridizing to the pre-mRNA splice site. The Bcl-x gene is alternatively spliced to express anti‑apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL expression is upregulated in many cancers and is considered a general mechanism by which cancer cells evade apoptosis. By redirecting Bcl-x pre-mRNA splicing from Bcl-xL to Bcl-xS, SSO exerted pro-apoptotic and chemosensitizing effects in various cancer cell lines. In this study, we investigated the effects of SSO targeting Bcl-x pre-mRNA in human glioma cell lines. First, we performed reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine the mRNA and protein expression levels of Bcl-xL in glioma cell lines (U87 and U251) and a normal human astrocyte cell line (HA1800). Then, the Bcl-x SSO was designed to bind to the downstream 5' alternative splice site of exon 2 in Bcl-x pre-mRNA and was modified using 2'-O-methoxyethyl-phosphorothioate. An oligonucleotide targeting aberrantly spliced human β-globin intron was used as a negative control. The SSOs were delivered with a cationic lipid into glioma and astrocyte cell lines. The antitumor effects of the SSOs were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and flow cytometry, and the switch in production from Bcl-xL to Bcl-xS was analyzed by RT-PCR and western blotting. Bcl-xL mRNA and protein were highly expressed in both glioma cell lines. The Bcl-x SSO modified Bcl-x pre-mRNA splicing and had pro-apoptotic effects on the glioma cell lines. By contrast, the lipid alone and the control SSO did not affect Bcl-xL expression or induce apoptosis. Our study demonstrated the antitumor activity of an SSO that targets Bcl-x pre-mRNA splicing in glioma cell lines. Bcl-x SSO may be a potential strategy for treating gliomas.

View Figures

View References

1	Nilsen TW and Graveley BR: Expansion of the eukaryotic proteome by alternative splicing. Nature. 463:457–463. 2010. View Article : Google Scholar : PubMed/NCBI
2	Gamazon ER and Stranger BE: Genomics of alternative splicing: Evolution, development and pathophysiology. Hum Genet. 133:679–687. 2014. View Article : Google Scholar : PubMed/NCBI
3	Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, Barretina J, Boehm JS, Dobson J, Urashima M, et al: The landscape of somatic copy-number alteration across human cancers. Nature. 463:899–905. 2010. View Article : Google Scholar : PubMed/NCBI
4	Kalsotra A and Cooper TA: Functional consequences of developmentally regulated alternative splicing. Nat Rev Genet. 12:715–729. 2011. View Article : Google Scholar : PubMed/NCBI
5	Barta A and Schümperli D: Editorial on alternative splicing and disease. RNA Biol. 7:388–389. 2010. View Article : Google Scholar : PubMed/NCBI
6	Oltean S and Bates DO: Hallmarks of alternative splicing in cancer. Oncogene. 33:5311–5318. 2014. View Article : Google Scholar
7	Pal S, Gupta R and Davuluri RV: Alternative transcription and alternative splicing in cancer. Pharmacol Ther. 136:283–294. 2012. View Article : Google Scholar : PubMed/NCBI
8	Hanahan D and Weinberg RA: Hallmarks of cancer: The next generation. Cell. 144:646–674. 2011. View Article : Google Scholar : PubMed/NCBI
9	Mercatante DR, Bortner CD, Cidlowski JA and Kole R: Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. Analysis of apoptosis and cell death. J Biol Chem. 276:16411–16417. 2001. View Article : Google Scholar : PubMed/NCBI
10	Palve V, Mallick S, Ghaisas G, Kannan S and Teni T: Overexpression of Mcl-1L splice variant is associated with poor prognosis and chemoresistance in oral cancers. PLoS One. 9:e1119272014. View Article : Google Scholar : PubMed/NCBI
11	Vegran F, Boidot R, Oudin C, Riedinger JM and Lizard-Nacol S: Distinct expression of Survivin splice variants in breast carcinomas. Int J Oncol. 27:1151–1157. 2005.PubMed/NCBI
12	Bojesen SE, Pooley KA, Johnatty SE, Beesley J, Michailidou K, Tyrer JP, Edwards SL, Pickett HA, Shen HC, Smart CE, et al Australian Cancer Study; Australian Ovarian Cancer Study; Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab); Gene Environment interaction and Breast Cancer (GENICA); Swedish Breast Cancer Study (SWE-BRCA); Hereditary Breast and Ovarian Cancer Research group Netherlands (HEBON); Epidemiological study of BRCA1 and BRCA2 Mutation Carriers (EMBRACE); Genetic Modifiers of Cancer Risk in BRCA1/2 Mutation Carriers (GEMO): Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer. Nat Genet. 45:371–384. e1–e2. 2013. View Article : Google Scholar : PubMed/NCBI
13	Babic I, Anderson ES, Tanaka K, Guo D, Masui K, Li B, Zhu S, Gu Y, Villa GR, Akhavan D, et al: EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer. Cell Metab. 17:1000–1008. 2013. View Article : Google Scholar : PubMed/NCBI
14	Singh A, Karnoub AE, Palmby TR, Lengyel E, Sondek J and Der CJ: Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation. Oncogene. 23:9369–9380. 2004. View Article : Google Scholar : PubMed/NCBI
15	Mayer S, Hirschfeld M, Jaeger M, Pies S, Iborra S, Erbes T and Stickeler E: RON alternative splicing regulation in primary ovarian cancer. Oncol Rep. 34:423–430. 2015.PubMed/NCBI
16	Matsuyama M, Chijiwa T, Inoue Y, Abe Y, Nishi M, Miyazaki N, Furukawa D, Mukai M, Suemizu H, Sekido Y, et al: Alternative splicing variant of vascular endothelial growth factor-A is a critical prognostic factor in non-small cell lung cancer. Oncol Rep. 22:1407–1413. 2009.PubMed/NCBI
17	David CJ, Chen M, Assanah M, Canoll P and Manley JL: HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature. 463:364–368. 2010. View Article : Google Scholar :
18	Kim YJ and Kim HS: Alternative splicing and its impact as a cancer diagnostic marker. Genomics Inform. 10:74–80. 2012. View Article : Google Scholar : PubMed/NCBI
19	Schuster J, Lai RK, Recht LD, Reardon DA, Paleologos NA, Groves MD, Mrugala MM, Jensen R, Baehring JM, Sloan A, et al: A phase II, multicenter trial of rindopepimut (CDX-110) in newly diagnosed glioblastoma: The ACT III study. Neuro Oncol. 17:854–861. 2015. View Article : Google Scholar : PubMed/NCBI
20	Chen J and Weiss WA: Alternative splicing in cancer: Implications for biology and therapy. Oncogene. 34:1–14. 2015. View Article : Google Scholar
21	Bonomi S, Gallo S, Catillo M, Pignataro D, Biamonti G and Ghigna C: Oncogenic alternative splicing switches: Role in cancer progression and prospects for therapy. Int J Cell Biol. 2013:9620382013. View Article : Google Scholar : PubMed/NCBI
22	Kinali M, Arechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C, Guglieri M, Ashton E, Abbs S, Nihoyannopoulos P, et al: Local restoration of dystrophin expression with the morpholino oligomer AVi-4658 in Duchenne muscular dystrophy: A single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol. 8:918–928. 2009. View Article : Google Scholar : PubMed/NCBI
23	Moore MJ, Wang Q, Kennedy CJ and Silver PA: An alternative splicing network links cell-cycle control to apoptosis. Cell. 142:625–636. 2010. View Article : Google Scholar : PubMed/NCBI
24	Miura K, Fujibuchi W and Unno M: Splice variants in apoptotic pathway. Exp Oncol. 34:212–217. 2012.PubMed/NCBI
25	Mercatante DR, Mohler JL and Kole R: Cellular response to an antisense-mediated shift of Bcl-x pre-mRNA splicing and anti-neoplastic agents. J Biol Chem. 277:49374–49382. 2002. View Article : Google Scholar : PubMed/NCBI
26	Bauman JA, Li SD, Yang A, Huang L and Kole R: Anti-tumor activity of splice-switching oligonucleotides. Nucleic Acids Res. 38:8348–8356. 2010. View Article : Google Scholar : PubMed/NCBI
27	Bauman J, Jearawiriyapaisarn N and Kole R: Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides. 19:1–13. 2009. View Article : Google Scholar : PubMed/NCBI
28	Weiler M, Bähr O, Hohlweg U, Naumann U, Rieger J, Huang H, Tabatabai G, Krell HW, Ohgaki H, Weller M, et al: BCL-xL: Time-dependent dissociation between modulation of apoptosis and invasiveness in human malignant glioma cells. Cell Death Differ. 13:1156–1169. 2006. View Article : Google Scholar
29	Alexandru-Abrams D, Jadus MR, Hsu FP, Stathopoulos A and Bota DA: Therapeutic targeting of malignant glioma. Anticancer Agents Med Chem. 14:1075–1084. 2014. View Article : Google Scholar : PubMed/NCBI
30	Li Z, Tian Y, Tian N, Zhao X, Du C, Han L and Zhang H: Aberrant alternative splicing pattern of ADAR2 downregulates adenosine-to-inosine editing in glioma. Oncol Rep. 33:2845–2852. 2015.PubMed/NCBI
31	Fontana L, Rovina D, Novielli C, Maffioli E, Tedeschi G, Magnani I and Larizza L: Suggestive evidence on the involvement of polypyrimidine-tract binding protein in regulating alternative splicing of MAP/microtubule affinity-regulating kinase 4 in glioma. Cancer Lett. 359:87–96. 2015. View Article : Google Scholar : PubMed/NCBI
32	Disterer P, Kryczka A, Liu Y, Badi YE, Wong JJ, Owen JS and Khoo B: Development of therapeutic splice-switching oligo-nucleotides. Hum Gene Ther. 25:587–598. 2014. View Article : Google Scholar : PubMed/NCBI
33	Sazani P, Astriab-Fischer A and Kole R: Effects of base modifications on antisense properties of 2′-O-methoxyethyl and PNA oligonucleotides. Antisense Nucleic Acid Drug Dev. 13:119–128. 2003. View Article : Google Scholar
34	Adkin C, Fletcher S and Wilton SD: Optimizing splice-switching oligomer sequences using 2′-O-methyl phosphorothioate chemistry. Methods Mol Biol. 867:169–188. 2012. View Article : Google Scholar
35	Wan J, Bauman JA, Graziewicz MA, Sazani P and Kole R: Oligonucleotide therapeutics in cancer. Cancer Treat Res. 158:213–233. 2013. View Article : Google Scholar : PubMed/NCBI
36	Shchelkunova A, Ermolinsky B, Boyle M, Mendez I, Lehker M, Martirosyan KS and Kazansky AV: Tuning of alternative splicing - switch from proto-oncogene to tumor suppressor. Int J Biol Sci. 9:45–54. 2013. View Article : Google Scholar :