探討新合成 arylsulfonamide 類的抗細胞分裂藥物 MPT0G066 誘發細胞凋亡與加強抗癌藥物 cisplatin 於人類卵巢癌細胞之體外和體內的作用機轉

Abstract

卵巢癌在台灣婦女的婦科惡性腫瘤中是好發率很高的癌症，由於此癌症不容易在早期診斷出來，所以患者的存活率極低。本研究發現一個化學合成具有arylsulfonamide 之結構的新藥物 MPT0G066 ，在 SRB 和 MTT 實驗中能夠有效抑制卵巢癌細胞的生長與存活；接著利用流式細胞儀分析發現 MPT0G066 能讓卵巢癌細胞 SKOV3 以及 A2780 分別停滯在 multipolyploidy (MP) 以及 G2/M 期，並且伴隨明顯的 Sub G1 增加。由於 SKOV3 是 p53 null 的癌細胞株，A2780 則具有 wild type p53，所以在藥物處理過後細胞易行成多倍體 (polyploidy) 進入 MP 期而非 G2/M 期。透過細胞外微管聚合�去聚合作用測定搭配細胞免疫螢光染色發現 MPT0G066 的作用類似 colchicine 和 vincristine 也會抑制微管聚合，並使 M 期指標蛋白 MPM2 磷酸化增加。同時發現 MPT0G066 在卵巢癌細胞中會抑制 STAT3 的磷酸化，但此作用對於 MPT0G066 造成的細胞週期停留與細胞凋亡並無相關。另MPT0G066 也會活化 JNK造成 bcl-2 family 蛋白改變，如：p-Bcl-2、Bax、Bim、PUMA 增加，Bcl-2、Mcl-1減少，因此改變粒線體通透性促進內生性細胞凋亡路徑活化，透過 caspase-3、-7、-9 以及 PARP 引起細胞凋亡。使用JNK抑制劑SP600125 會使細胞停留在 G2 期不進入 mitosis，並且反轉 MPT0G066 造成的細胞凋亡現象。此外，在 rhodamine 123 搭配流式細胞儀分析 p-glycoprotein 的功能中發現 MPT0G066 並非 p-glycoprotein 的受質，因此可以作用在多重抗藥性的卵巢癌細胞 NCI/ADR-RES。最後，在小鼠腫瘤移植實驗中證實 MPT0G066 能抑制人類卵巢癌細胞 A2780 的生長；並且於體外和體內都能加強抗癌藥物 cisplatin 於人類卵巢癌的作用。另外，我們在腫瘤組織的蛋白分析發現 MPT0G066 在 in vivo 也能抑制 STAT3、活化 JNK 及 caspase-9，這些作用和 in vitro 結果符合。因 MPT0G066 造成的細胞凋亡過程中 JNK 扮演重要的角色，因此我們利用免疫組織化學染色確認在腫瘤組織切片中，證明 JNK 確實有活化參與了細胞凋亡的過程。綜合以上結果，本研究發現 MPT0G066 能夠改變微管動態使細胞週期停留在 MP 以及 G2/M 期，活化JNK且影響Bcl-2 family 蛋白，最後經由內生性凋亡路徑造成細胞凋亡。因此 MPT0G066 為具有潛力發展成為新的抗癌藥物。

Ovarian cancer is a well-known increase of incidence in gynecological malignancy among women in Taiwan. Ovarian cancer patients are always diagnosed at an advanced stage resulting in poor survival rate. This study showed that MPT0G066, a novel synthetic arylsulfonamide compound, inhibited cell growth and viability in human ovarian cancer SKOV3 and A2780 cells using sulphorodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell cycle distribution was determined by flow cytometry, we demonstrated that MPT0G066 induced arrest of cell cycle at multipolyploidy (MP) in SKOV3 cells and at G2/M phase in A2780 cells, with increase of cell proportion at Sub G1 phase which may be caused by tubulin depolymerization effects of MPT0G066. Since SKOV3 cells were homozygous deleted p53 and A2780 cells have wild type p53, cell cycle arrest occured at MP instead of G2/M phase in SKOV3 cells. Then, tubulin binding assay and immunofluorescence stain showed that MPT0G066 was similar to colchicine and vincristine in causing depolymerization of tubulin. These results showed that MPT0G066 could disrupt microtubule dynamics and induce mitotic marker, MPM2. Furthermore, MPT0G066 inhibited STAT-3 tyrosine phosphorylation, but the effect was not related to cycle arrest and apoptosis. MPT0G066 induced JNK activation and influenced bcl-2 family proteins by increasing p-Bcl-2, BAX, Bim, PUMA and decreasing Bcl-2 and Mcl-1. Disruption of mitochondrial proteins caused intrinsic apoptotic pathways through the cleavage of caspase-3, -7, -9, and poly (ADP-ribose) polymerase (PARP). Treatment with SP600125, a JNK inhibitor, could prevent the entry of cells into mitosis and led to endoreplication from G2 phase, and then reversed MPT0G066-induced apoptosis. Flow cytometric analysis of p-glycoprotein function using rhodamine 123 showed that MPT0G066 was not a substrate of p-glycoprotein. Accordingly, MPT0G066 could inhibited cell viability in multiple-drug resistant human ovarian cancer cells. Finally, we used xenograft mouse models to demonstrate that MPT0G066 supressed ovarian carcinoma A2780 growth and potentiated antineoplastic effects of cisplatin in vivo. Protein analysis of tumor tissue discovered that MPT0G066 inhibited p-STAT3 and activated JNK and caspase-9 in vivo. Immunohistochemitry data confirmed MPT0G066-induced JNK activation in tumor sections. In summary, the results suggested that MPT0G066 could change microtubule dynamics, and lead to MP or G2/M arrest of the cell cycle. MPT0G066 induced JNK activation and altered bcl-2 family proteins which ultimately caused apoptosis through the intrinsic apoptotic pathways. These finding indicated that MPT0G066 can be a potential anticancer agent worthy for further development.