Recombinant arginine deiminase (rADI) and its synergism with chemotherapeutic drugs

化學藥物治療目前對於癌症治療是很重要的方式。目前臨床上使用的化療藥物可分為細胞週期專一性以及非專一性，治療方式常採用合併藥物治療。而併用針對不同細胞週期的化療藥物，可能會改變原本藥物的效果，如協同 (synergism)、加成 (additive effect) 或拮抗作用 (antagonism)。此外，使用不同的給藥順序也會可能影響到化療藥物併用的結果。 精胺酸去亞胺酶 (arginine deiminase; ADI) 已經在臨床試驗中被證實用於治療肝癌、黑色素癌；這類的癌細胞被稱為精胺酸缺乏型 (arginine auxotrophy)，因為精胺酸 (arginine) 的缺乏攸關其存亡。精胺酸在生化合成途徑中扮演重要角色，包括蛋白質、一氧化氮、多元胺、肌酸等生成。而精胺酸缺乏型癌細胞，因為本身缺乏精胺酸琥珀酸合成酶 (argininosuccinate synthetase; AS) 的表現，而無法將瓜胺酸 (citrulline) 轉換成精胺酸，以致於無法在缺乏精胺酸的環境中生存，但是正常成人細胞卻能在這樣的環境中生存，因此 ADI 能對此類細胞產生選擇性毒殺作用。然而某些癌細胞本身會表現 AS，因此對於 ADI 便會產生抗藥性。本研究中所使用的人類乳癌細胞株 MCF-7 即為此類細胞。 雖然 ADI 已經進入第三期臨床試驗，但是在與其他化療藥物併用的效果仍不清楚。而且 ADI 為蛋白質藥物，本研究希望藉由藥物併用的方式，來減少所使用的劑量，這對於蛋白質藥物在臨床使用上增加更多優勢。因此本研究合併使用基因重組精胺酸去亞胺酶 (recombinant arginine deiminase; rADI) 與五種臨床上常用的化療藥物，cisplatin、vincristine、paclitaxel、fluorouracil、gemcitabine，以三種不同給藥順序評估其療效，分別為同時給予化療藥物和 rADI (4-day C/T / 4-day rADI)、先給化療藥物再給 rADI (4-day C/T / 3-day rADI)、以及先給 rADI 再給化療藥物 (3-day C/T / 4-day rADI)。四天後以 SRB assay 觀察 rADI 與化療藥物併用的效果。 除了使用 MCF-7 細胞株之外，也同時使用穩定表現 AS 之短髮夾核醣核酸干擾 (ASshRNA) 的MCF-7 細胞株 (MCF-7-ASKD)，作為精胺酸缺乏型的細胞模型，rADI 對於此類細胞有明顯抑制生長效果。藥物併用的效果以 SRB 檢驗分析細胞存活率；並且使用 Chou-Talalay 所提出併用係數 (combination index; CI) 的公式來評估兩種藥物之間的反應關係，如果 CI 1，分別代表兩種藥物併用的效果是協同、加成或拮抗。 對於 MCF-7 給予 1 mU/mL rADI 仍無法達到抑制 50% 的細胞生長，而 MCF-7-ASKD 的 IC50 為 0.25 mU/mL。為了使得 MCF-7-ASKD 細胞在還未加化療藥物時仍有 50% 的存活率，在後續實驗中，選用 0.2 mU/mL rADI 併用化療藥物。而分別在兩株細胞給予不同的化療藥物，MCF-7-ASKD 的 IC50 在 cisplatin、vincristine、paclitaxel、fluorouracil及gemcitabine，相較這些藥物在 MCF-7 的 IC50 分別減少了 35%、32%、30%、15%、43%，表示 AS 蛋白的表現能增加癌細胞對於化療藥物的抗藥性。藥物併用實驗中，對於 MCF-7 細胞株，先給予 rADI 再給予 gemcitabine 的實驗中增強效果最明顯 (> 20%)，而同時給予 rADI 以及 vincristine 或 paclitaxel，則在部分濃度有增強效果 (> 15%)。對於 MCF-7-ASKD 細胞株，先用 cisplatin 再用 rADI，在部分濃度才有協同作用，若是給藥順序相反或同時給藥，在部分濃度則是產生拮抗作用。對於 vincristine而言，先使用 rADI 會有協同作用，若給藥順序相反或同時給藥，在部分濃度則是產生拮抗作用。然而，對於 paclitaxel 而言，無論何種給藥順序都是拮抗作用。對於 fluorouracil 而言，併用 rADI 並沒有明顯協同作用，而且同時給藥則有拮抗作用。對於 gemcitabine 而言，先給予 rADI 會有拮抗作用，而同時給予在部分濃度呈現拮抗作用，若之後給予 rADI 則沒有明顯拮抗作用。 總結來說，本研究中對照 MCF-7 以及 MCF-7 ASKD 的結果，發現 AS 會影響癌細胞對於化療藥物的感受性；而在使用 rADI 來治療癌症時，對於 rADI 具敏感性或不具敏感性的細胞而言，不同的併用藥物，有不同的效果。本研究提供 rADI 與其他化療藥物併用或者併用順序之參考。Chemotherapy has become an important treatment in cancer therapy. In currently clinincal use, chemotherapeutic agents can be divided into two categories, cell-cycle-specific and cell-cycle-nonspecific; combination therapy is usually used. To combine different chemotherapeutic agents which have different effects on cell cycle may change the drug’s original effect, such as synergism, additive effect, and antagonism. Nevertheless, by using different administration orders of chemotherapeutic agents may influence the therapeutic results and, sometimes even opposite. Arginine deiminase (ADI) for the treatment of hepatocellular carcinoma and malignant melanoma has been confirmed in clinical trials. This type of cancer is known as arginine auxotrophy because arginine is critical for their survival. Arginine plays an important role in biosynthesis, such as protein, nitric oxide, polyamines, and creatine etc. Arginine auxotrophy cancers can’t express argininosuccinate synthetase (AS), so they do not survive in the arginine-free medium. Normal cells in adults with AS expression can grow in such environment. Therefore, ADI produce selective cytotoxicity to arginine auxotrophy cells. However some cancer cells would express AS, which may result in rADI-resistance. The cell model we chose was the human breast cancer cell line MCF-7 known resistant to rADI. ADI has been in Phase III clinical trials, but its effect in combine with other chemotherapeutic agents is still unclear. It would gain more advantages of the reduction of rADI dose and clinical efficacy by the combination therapy. Therefore, this study combined recombinant arginine deiminase (rADI) with five clinically common used chemotherapeutic agents (cisplatin, vincristine, paclitaxel, fluorouracil, gemcitabine), and used three different schemes as following to evaluate their effects: (a) C/T and rADI were administered simultaneously (4-day C/T /4-day rADI), (b) C/T was administered one day earlier followed by rADI (4-day C/T / 3-day rADI), (c) rADI was administered one day ealiear followed by C/T. After a total of 4-day treatment, cell viability was evaluated by SRB assay. MCF-7-ASKD cells, with low AS by expressing ASshRNA are sensitive to rADI treatment, using as an arginine auxotrophy cell model. The effects of the drug combinations using the method of combination index (CI) described by Chou and Talalay. When the CI was 1, the combination was considered as synergism, additive, antagonism respectively. The IC50 of rADI at MCF-7 was greater than the tested concentration 1 mU/mL, but that of rADI at MCF-7-ASKD is 0.25 mU/mL. For keep 50% survival rate of MCF-7-ASKD with rADI alone, we used 0.2 mU/mL of rADI in the following combination experiments.The IC50 of rADI at MCF-7-ASKD was reduced in cisplatin, vincristine, paclitaxel, fluorouracil, gemcitabine about 35%, 32%, 30%, 15%, and 43%, comparing to that at MCF-7; it means that the AS expression could increase the resistance to C/T. Combination experiments showed that for MCF-7, the significant enhancement was shown in the administration order of rADI followed by gemcitabine (> 20%), and in simultaneously administration of certain concentrations of vincristine and paclitaxel (> 15%). For MCF-7-ASKD, synergism was shown in certain concentrations by the order of cisplatin followed by rADI, but antagonism was shown in certatin concentrations by other administration orders; for vincristine, synergism was shown in rADI-pretreatment, but antagonism was shown in other orders; for fluorouracil, antagonism was shown in co-treatment; for gemcitabine, antagonism was shown in rADI-pretreatment and in co-treatment of certain concentrations but not in rADI-post-treatment. In conclusion, we compared the results of MCF-7 and MCF-7-ASKD and found that AS may influence the sensitivity of chemotherapeutic agents at cancer cells. For rADI-sensitive or non-sensitive cancer cells, different drug combinations may have different effects with rADI. This study provides some evidence for drug combination and administration order when rADI combines with other chemotherapeutic agents