RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

Journal of Biomedical Science

Li-Jiuan Shen¹  Ming-Feng Wei²  Hao-Hsin Yo³  Yuan-Chen Chang³  Yu-Fen Liang³  Fe-Lin Lin Wu¹ 

[1] Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan;National Center of Excellence for Clinical Trial and Research Center, National Taiwan University Hospital, Taipei, Taiwan;School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan

关键词: RNA interference;    resistance;    arginine deiminase;    argininosuccinate synthetase;   

Others  :  833616
DOI  :  10.1186/1423-0127-18-25

 received in 2010-04-06, accepted in 2011-04-01,  发布年份 2011

【 摘 要 】

Background

Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively.

Methods

We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated.

Results

AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway.

Conclusions

Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing.

【 授权许可】

   
2011 Wu et al; licensee BioMed Central Ltd.

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