不同藥物組合在人類肺癌細胞之加成性與拮抗性療效研究與機制探討

Study of various drug combinations with synergistic and antagonistic anticancer effects in human lung cancer cells

顏坤煌(Kun-Huang Yan)

指導教授 ： 張晃猷 ； 莊雙恩

國立清華大學/生命科學院/生命科學系/博士(2009年)

摘要

Troglitazone (TGZ) 是屬於過氧化小體增生活化受體丙型的增敏劑，由於可回復胰島素敏感，曾做為第二型糖尿病的口服治療用藥。一般也認為具有抑制多種型態的癌細胞增生、改變細胞週期調控、以及引起癌細胞的凋亡的效用。阿斯匹靈 (aspirin, ASA) 可有效抑制環氧化酵素活性而普遍的做為抗發炎的藥物。研究發現，阿斯匹靈的抗腫瘤作用是來自於抑制癌細胞週期的進行、引發癌細胞凋亡、以及抑制血管新生作用。另外、滅殺除癌錠 (methotrexate, MTX) 很早以前就被用來廣泛治療癌症，除此之外，MTX 也被應用在治療類風濕性關節炎。此篇研究我們首次發現以非小細胞肺癌細胞株 (NSCLC cells ； CL1-0, A549) 為實驗對象時，TGZ 合併使用 ASA 處理肺癌細胞可以經由調控細胞週期停滯在 G1 週期的作用達到抑制生長的加成效果。然而，我們也發現當 MTX 合併使用 ASA 處理肺癌細胞時，ASA 會經由降低 MTX 調控細胞停留在 S 週期的比例和恢復 G1 週期的作用來拮抗 MTX 的抗癌效用。在 CL1-0 與 A549 兩種肺癌細胞株的單細胞聚落形成實驗也顯示 TGZ 加上 ASA 的組合發揮強大的抗癌加成作用，而 MTX 和 ASA 的組合則呈現明顯的藥物拮抗作用。以西方墨點法研究證明 TGZ 與 ASA 合併使用比單一藥物處理肺癌細胞更能降低 phospho-Rb/Rb 的比例，以及加強向下調控 E2F-1, Cdk2, cyclin B1, cyclin D3 這些與細胞週期調控相關的蛋白質。更重要的是 TGZ 與 ASA 合併處理 CL1-0 細胞株還能藉由 caspase-3 的活化、PARP 的斷化作用、p27 的表現、PI3K/Akt 活性的抑制和 Rac1 在 ser71 位置的去磷酸化而引發細胞凋亡作用。另外、我們的研究也證明 ASA 利用細胞週期的調控與 Bcl-2 的表現而拮抗 MTX 的抗癌功效。綜合以上的實驗結果，我們的研究顯示使用在臨床上可達到的濃度範圍， TGZ 和 ASA 的藥物組合所表現的加成作用值得進一步研究，以期運用在癌症治療上。然而，我們的結果也認為 MTX 與 ASA 的合併使用對於臨床治療的病人可能有不利的效果。

關鍵字

加成作用 ； 拮抗作用

並列摘要

Troglitazone (TGZ) is a group of potent PPARγ (peroxisome proliferator-activated receptor γ) agonists known to inhibit proliferation, alter cell cycle regulation, and induce apoptosis in various cancer cell types. TGZ is also an oral anti-type II diabetes drug that can reverse insulin resistance. Aspirin (ASA), a nonselective cyclooxygenase (COX) inhibitor, has been successfully used as an anti-inflammatory drug. It has been observed that ASA’s anti-tumor effect can be attributed to inhibition of cell cycle progression, induction of apoptosis, and inhibition of angiogenesis. Methotrexate (MTX) has been widely used for the treatment of cancer. MTX was also applied to be a potent and effective therapy for rheumatoid arthritis (RA). Above all, we demonstrate for the first time that, when administered in combination, TGZ and ASA can produce a strong synergistic effect in growth inhibition and G1 arrest in lung cancer CL1-0 and A549 cells. Whereas we observed that ASA concomitance decreased the MTX-mediated S phase arrest and, surprisingly, increased the proportion of cells at the G1 phase in MTX+ASA. Examination by colony formation assay revealed an even more profound synergy in combination treatment of TGZ+ASA. On the other hand, marked antagonistic effects were observed in both CL1-0 and A549 cells when the two drugs of MTX and ASA were combined. In Western blot, combined TGZ and ASA also could down-regulate Cdk2, E2F-1, cyclin B1, cyclin D3 protein, and the ratio of phospho-Rb/Rb. Importantly, apoptosis was 4
synergistically induced by the combination treatment, as evidenced by caspase-3 activation and PARP cleavage. The involvements of PI3K/Akt inhibition and p27 up-regulation, as well as hypophosphorylation of Rac1 at ser71, were demonstrated. Additionally, our investigation proved that ASA regulates cell cycle progression and Bcl-2 expression, as well as subsequently antagonizes MTX efficiency. Taken together, these results suggest that clinically achievable concentrations of TGZ and ASA used in combination may produce a strong anticancer synergy that warrants further investigation for its clinical applications. However, the results also suggest that ASA may have adverse consequences in patients who are receiving MTX therapy.

參考文獻

1. Jalving M, Koornstra JJ, De Jong S, De Vries EG, Kleibeuker JH. Review article: the potential of combinational regimen with non-steroidal anti-inflammatory drugs in the chemoprevention of colorectal cancer. Alimentary pharmacology & therapeutics 2005;21(4):321-339.

2. Yao CJ, Lai GM, Chan CF, Cheng AL, Yang YY, Chuang SE. Dramatic synergistic anticancer effect of clinically achievable doses of lovastatin and troglitazone. International journal of cancer 2006;118(3):773-779.

3. Keshamouni VG, Reddy RC, Arenberg DA et al. Peroxisome proliferator-activated receptor-gamma activation inhibits tumor progression in non-small-cell lung cancer. Oncogene 2004;23(1):100-108.

4. Chang TH, Szabo E. Induction of differentiation and apoptosis by ligands of peroxisome proliferator-activated receptor gamma in non-small cell lung cancer. Cancer research 2000;60(4):1129-1138.

5. Feinstein DL, Spagnolo A, Akar C et al. Receptor-independent actions of PPAR thiazolidinedione agonists: is mitochondrial function the key? Biochemical pharmacology 2005;70(2):177-188.