干擾精胺酸琥珀酸合成酶之核糖核酸對於基因合成精胺酸去亞胺酶具抗藥性癌細胞株之作用

Abstract

精胺酸（L-arginine）除了為蛋白質合成所需的胺基酸之外，同時它也可以藉由不同的酵素轉換成其他的胺基酸。另外，精胺酸代謝後也會產生許多與調節細胞生化功能有關的非蛋白質化合物，如聚胺、一氧化氮等。基因合成精胺酸去亞胺酶（recombinant arginine deiminase，rADI）由體外細胞實驗證實，當用於對精胺酸缺乏不具耐受性的惡性腫瘤細胞是具有抑制其生長的效果，因為它會將環境中的L-arginine降解變成瓜胺酸（L-citrulline），使細胞得不到養分而無法繼續生長；而且在體內動物實驗已經證明它具有抗腫瘤的活性。目前，rADI也已經進入臨床試驗，分別用於無法切除肝腫瘤的病人以及轉移性黑色素瘤的病人。然而，並非所有的惡性腫瘤細胞均對rADI具敏感性。造成某些癌細胞株對rADI具有抗藥性的原因是來自細胞內的一個內生性蛋白質，精胺酸琥珀酸合成酶（argininosuccinate synthetase，AS），當rADI將L-arginine降解成L-citrulline後，L-citrulline可以藉由AS再轉變回L-arginine供細胞正常使用。因為將L-citrulline轉變成L-arginine的速率決定步驟酵素為AS，於是在本研究中，我們想要利用核糖核酸干擾（RNA interference，RNAi）的策略來干擾AS基因的表現量，再併用rADI之後，觀察是否能使具有抗藥性的癌細胞株對於rADI的治療敏感度提高。 在本研究中我們使用MCF-7（人類乳癌細胞株）及HeLa（人類子宮頸癌細胞株）當做體外實驗的模式，因為它們均為對rADI不具有敏感性的癌細胞株。我們將設計好且對AS具有專一性的21個核苷酸小片段干擾核糖核酸（AS small interference RNA，AS-siRNA）及隨機排序的小片段干擾核糖核酸（NC-siRNA）以LipofectamineTM 2000轉染進入細胞內。接著，利用此體外實驗模式把AS-siRNA / NC-siRNA轉染進入細胞內，再併用rADI，將細胞培養24-96小時後，分別以MTT assay及流式細胞儀分析細胞的存活率及細胞週期分佈情形。另外再以含有10％透析過FBS的L-arginine缺乏培養基培養MCF-7細胞1-7天後，以MTT assay測定其細胞存活率。 結果顯示，我們所設計的AS-siRNA對於AS基因具有顯著的抑制效果。當MCF-7及HeLa細胞分別給予AS-siRNA培養四天後，其AS蛋白質的表現量分別是對照組的37.8±7.3％ 及0.2±0.3％。即時定量聚合酶連鎖反應的結果顯示，當給予MCF-7及ＨeLa細胞AS-siRNA / NC-siRNA培養四天之後，其AS訊息核糖核酸的表現量分別是對照組的22.3±2.9％ 及49.3±5.2％。以AS-siRNA併用rADI給予MCF-7及HeLa細胞分別培養24，48，72，96小時，對MCF-7細胞而言，其細胞存活率並無明顯的變化。相反地，對HeLa細胞而言，其細胞存活率分別是90.1±5.0％，64.9±0.4％，13.1±1.4％，7.7±0.2％。另外，我們以AS-siRNA併用rADI培養四天後，以流式細胞儀分析MCF-7及HeLa細胞的細胞週期分佈情形，其細胞凋亡的比例分別是8.1±3.4％ 及63.4±4.7％。另外，以L-arginine缺乏的培養基培養MCF-7細胞，結果顯示MCF-7細胞並不會因為L-arginine的缺乏而影響其細胞存活率。 從我們的實驗結果可知，雖然AS-siRNA併用rADI均可以降低HeLa及MCF-7細胞株的AS蛋白質及訊息核糖核酸的表現量，但是，我們觀察到只有HeLa細胞株對於這樣的合併療法才具有敏感性，同時會使HeLa細胞株走向凋亡的途徑。另外，觀察到MCF-7細胞可以在L-arginine缺乏的培養基中存活及增殖，這可能意謂著L-arginine對於MCF-7細胞的生長並非必需的胺基酸。在我們的研究中，我們也觀察到HeLa及MCF-7細胞的內生性AS蛋白質表現量是截然不同的。對HeLa細胞而言，它們的內生性AS蛋白質表現量微弱，但是當給予rADI培養四天後，其AS蛋白質的表現量會被誘導增至為原來的5倍左右。對MCF-7細胞而言，當給予rADI培養四天後，其AS蛋白質的表現量只有少量的被誘導產生（約1.1倍）。因此，藉由核糖核酸干擾來降低AS基因的表現量或許可以當做治療某些對rADI具有抗藥性癌細胞株（如HeLa細胞株）的策略。未來，我們將更進一步的探討造成對這樣的合併療法具有抗藥性的癌細胞株（如MCF-7細胞株）之可能機轉。

L-arginine is not only one of the essential amino acids for protein synthesis, but also the substrate for the conversion of other amino acids, and several non-protein compounds relating to the biochemical functions of cells, such as polyamines and nitric oxide. It has been demonstrated that recombinant arginine deiminase (rADI), a protein starving arginine-auxotrophic malignant cells by the degradation of arginine to citrulline in vitro and in vivo as well, has anti-tumoral activity. rADI is currently in clinical trials, used in patients with unresectable hepatocellular carcinoma and metastatic melanoma. However, not all malignant cells are sensitive to rADI. Endogenous argininosuccinate synthetase (AS), a rate-limiting enzyme in the arginine regeneration from citrulline, has been reported playing a crucial role in the resistance of malignant cells to rADI. Therefore, we would like to use RNA interference (RNAi) to down-regulate the expression level of AS gene and it combines with rADI to increase the sensitivity of resistant cells to rADI-treatment. Human breast cancer cell line MCF-7 and cervical cancer cell line HeLa, both of the resistant cancer cell lines to rADI-treatment, were used in our experiments. Firstly, the 21-nucleotide sequences of small interference RNA (siRNA) of AS gene and negative control (NC) were designed. MCF-7 and HeLa cells were transfected AS-siRNA and NC-siRNA, respectively, with lipofectamineTM 2000. Subsequently, cells were transfected AS-siRNA / NC-siRNA with lipofectamineTM 2000 and treated with rADI concurrently in this in vitro model. After 24-96 hours treatment, the cell viability and cell cycle distribution were analyzed by MTT assay and flow cytometry. Additionally, MCF-7 cells were incubated in L-arginine-free medium with 10% dialyzed FBS for 1-7 days to measure their cell viability by using MTT assay.

The designed AS-siRNA significantly down-regulated AS gene in mRNA and protein levels in both cell lines, but not NC-siRNA. Four days after the treatment of AS-siRNA and NC-siRNA, the AS protein expression level in MCF-7 and HeLa cells were 37.8±7.2% and 0.2±0.3%, respectively, compared to each control group by Western blotting. We also measured the AS mRNA expression level in MCF-7 and HeLa cells after the treatment of AS-siRNA and NC-siRNA at day 4, they were 22.3±2.9% and 49.3±5.2%, respectively, compared to each control group. After 24-96 hours treatment of the combination of AS-siRNA and rADI in MCF-7, the cell viability was not significantly affected by MTT assay. On the contrary, the percentage of cell viability in HeLa were 90.1±5.0%, 64.9±0.4%, 13.1±1.4%, and 7.7±0.2%, respectively, after 24, 48, 72, 96 hr treatment of the combination. Four days after the combination of AS-siRNA and rADI, the percentage of apoptosis in MCF-7 and HeLa cells were 8.1±3.4% and 63.4±4.7%, respectively, by the flow cytometry. In addition, when MCF-7 cells were cultured in L-arginine-free medium, the cell viability was not affected by the absence of L-arginine. From our results, although the combination of AS-siRNA and rADI decreased the AS protein expression and AS mRNA level in both HeLa and MCF-7 cell lines, only HeLa cells were sensitive to the combination treatment via the apoptotic pathway. In addition, the MCF-7 can survive and proliferate in the L-arginine depletion medium. It may indicate the L-arginine is not the essential amino acid for MCF-7 cells. In our study, it is known that the endogenous AS protein expression level are different between HeLa cells and MCF-7 cells. For HeLa cells, their endogenous AS protein expression level is low, but it is induced to 5 fold of the AS expression in the control group after 4 days treatment of rADI. For MCF-7 cells, the induction of AS protein expression level is only minimal (1.1 fold) of it in the control after 4 days treatment of rADI. Therefore, down-regulation of AS gene by RNAi could be a strategy to overcome the resistance of rADI in some malignant cells, such as HeLa cells. However, it may need further studies to understand the mechanism of the resistance of the combination treatment of AS-siRNA and rADI in other cells, such as MCF-7 cells.