title: RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells.
abstract: 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.
authors: School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors: Fe-Lin Lin
authors: FL
authors: Wu
authors: Yu-Fen
authors: YF
authors: Liang
authors: Yuan-Chen
authors: YC
authors: Chang
authors: Hao-Hsin
authors: HH
authors: Yo
authors: Ming-Feng
authors: MF
authors: Wei
authors: Li-Jiuan
authors: LJ
authors: Shen
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000235:genetics*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D002956:Citrulline / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D006867:Hydrolases / Q000494:pharmacology*; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase; D001124:Argininosuccinate Synthase