精胺琥珀合成(酉每)的短髮夾核醣核酸(shRNA)於對基因合成精胺酸去亞胺(酉每)具有抗性之癌細胞株的效果

Abstract

Arginine is an important and versatile amino acid, serving as a precursor in many biosynthesis, such as proteins, nitric oxide, and polyamines. For some cancer cells, arginine is critical for their survival. One of strategies for cancer therapy is arginine deprivation; recombinant arginine deiminase (rADI), which can convert arginine to citrulline and ammonia, has been used for arginine deprivation. However, arginine can be regenerated from citrulline by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) in some rADI-resistant cancer cells. The former, AS, is the rate-limiting enzyme and plays a crucial role in the citrulline-arginine regeneration pathway. In order to inhibit AS expression, we established a permanent AS gene knockdown cell line via transduction of lentiviruses carrying shRNA against AS mRNA. The cell model we chose was MCF-7 which was resistant to rADI treatment with high amount of endogenous AS expression. At the first, lentiviruses were produced and the virus titer was measured to evaluate the appropriate amount of virus for transduction. No influence of lentiviruses transduction on cell growth was observed because cell growth rates were similar in the presence and absence of lentiviruses transduction. After puromycin selection for infected cells, ASshRNA was stable expression in ASshRNA-transduced MCF-7. It was required to determine the efficacy and efficiency of AS gene silencing, so AS mRNA and protein expression were measured by PCR and immunoblotting, respectively. Our results showed that both of AS mRNA and protein expression were significantly decreased and lower than 10% in ASshRNA-transduced MCF-7. The viable cells were significantly reduced when stablized ASshRNA-transduced MCF-7 cells were treated with rADI. The result was indicated that rADI would significantly decrease cell survival in ASshRNA-transduced MCF-7. The ASshRNA-transduced MCF-7 had only 22.78% cell viability while untranduced MCF-7 was still resistant to rADI treatment at concentration of 1 mU/mL. Furthermore, to understand the death pathway induced by rADI in ASshRNA-transduced MCF-7 cells, more than 50% of subG1 part was found in cell cycle using flow cytometry. It represented that cells had been DNA damaged and processed to apoptosis. In addition, the microarray data showed that the rADI regulated genes in cell cycle in AS silenced MCF-7. AS silencing could be a strategy to overcome the resistance, when cells were resistant to rADI due to AS protein expression. It may provide a potential method for the cancer therapy in the future. However, how to target to the cancer cells or how the effect on the normal cells should be further studied.