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

精胺酸扮演多元且重要的角色，其做為許多生化合成的前驅物，包含一氧化氮與多元胺等等。對於某些癌症細胞，精胺酸的缺乏攸關其存亡。因此可利用精胺酸缺乏做為可能有效的抗癌方式，其中精胺酸去亞胺酉每（arginine deiminase；ADI）可達到精胺酸缺乏的目的，其可有效將精胺酸轉化成瓜胺酸和胺類。然而某些癌細胞中的精胺琥珀合成酉每（argininosuccinate synthetase；AS）與精胺琥珀水解酉每（argininosuccinate lyase；AL）卻可以將瓜胺酸再度轉化成精胺酸，而產生抗藥性；其中AS為關鍵酵素，為反應過程中的速率決定步驟。因此為了增加癌細胞對ADI的抗藥性，利用核醣核酸干擾技術（RNA interference；RNAi）使該AS基因永久沉默，設計合適的核醣核酸序列，藉由高感染性的慢病毒帶入細胞內，以達到穩定表現的效果。而本研究所使用的細胞株為人類乳癌細胞株（MCF-7），其對ADI具有抗性且有內生性AS的表現。 首先，我們製造慢病毒並測試其感染力以做為決定慢病毒感染量的依據。同時考慮到慢病毒感染對於細胞生長的影響，故檢驗其生長情形，發現到無論感染慢病毒與否，其生長情形類似而無明顯變異。而在利用嘌呤黴素（puromycin）篩選出被慢病毒感染的細胞之後，AS之短髮夾核醣核酸干擾（ASshRNA）將穩定表現於細胞株中。為了確立慢病毒帶入ASshRNA的效力及效能，AS的訊息核醣核酸與蛋白質層次將利用聚合酉每連鎖反應（polymerase chain reaction；PCR）與免疫印跡法（immunoblotting）檢驗之。而本研究的結果也顯示在被具有ASshRNA的慢病毒感染後的細胞，無論是訊息核糖核酸或是蛋白質的表現都可以有效抑制到原先表現量的一成以下。 而在確立穩定AS基因沉默的細胞株建立之後， 給予基因重組精胺酸去亞胺酉每（recombinant arginine deiminase；rADI）後，發現細胞存活率僅剩下二成左右，同時以流式細胞儀檢驗而發現有超過一半的細胞為SubG1，意謂著細胞中的去氧核醣核酸斷裂，而發生自我凋亡的作用。利用微陣列分析，則指出細胞在加入rADI治療後，影響細胞週期之基因表現。 總結，我們建立了穩定AS基因沉默的細胞株，給予rADI後可將原先對rADI具抗藥性之細胞株轉為敏感性之細胞株，此抗癌策略可能具有潛力發展。然而如何將目標鎖定於腫瘤細胞，以及在對於一般細胞有何影響，都需要更進一步的研究。

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.