苦瓜三萜類化合物TCD對人類胃癌AGS細胞的抗腫瘤效應及增加化學治療的敏感性

Abstract

先前文獻報導葫蘆烷三萜類化合物透過延滯細胞週期及促進細胞凋亡而抑制乳癌、子宮頸癌及前列腺癌的增生，並且也能夠增強子宮頸癌細胞KB-V1對化療藥物vinblastine和paclitaxel的敏感度。關於3β, 7β, 25-trihydroxycucurbita-5, 23-dien-19-al (TCD)對於胃癌細胞之影響的文獻報告卻相當有限，因此本研究以人類AGS胃癌細胞模式，探討自山苦瓜葉萃取純化分離的TCD對於抑制胃癌細胞增殖和增強AGS細胞對於順鉑(cisplatin)敏感度的效應。 實驗結果顯示，由細胞存活率MTT分析及群落形成分析發現TCD (>20 µM) 單獨處理能顯著抑制AGS細胞增生與細胞聚落生成。由劉氏染色法觀察發現TCD改變AGS細胞的細胞核/細胞質比例和細胞型態，因此以流式細胞儀分析細胞凋亡(apoptotic assay)和細胞週期(cell cycle)，發現30 µM TCD會將細胞週期延滯於G1期並引致細胞凋亡。以西方轉漬法觀察發現30 µM TCD造成AGS細胞凋亡相關蛋白caspase 9、caspase 3、和PARP表現量上升。另外觀察caspase酵素活性發現30 µM TCD能提高AGS細胞caspase 3活性。為觀察TCD是否具有增強cisplatin的藥效，合併使用IC20濃度的TCD (20 µM)與cisplatin (2 µM) 有效抑制AGS細胞增生且具協同作用(synergism)。在核質比及細胞型態的部分，cisplatin會使細胞脹大、DNA斷裂溢出細胞核，TCD會使細胞核聚縮並死亡，TCD/cisplatin併用時AGS細胞兼具上述特質。TCD/cisplatin (20 µM/ 2 µM)併用時顯著增加AGS細胞的凋亡比例,並顯著抑制cisplatin誘導之TNF-α mRNA表現。 以AGS細胞異種移植模式，將裸鼠分為4組分別為控制組、TCD (20 mg/kg)組、cisplatin (4mg/kg)組和TCD/cisplatin併用組。給予藥物後經35天後觀察發現：和控制組比較，不論是單獨給予TCD或cisplatin和TCD/cisplatin併用都能顯著降低腫瘤體積和重量。於試驗第20~35天實驗期間TCD/cisplatin組的腫瘤體積平均值均低於其他3組，但僅於第30天和TCD組達統計上的顯著差異。然而投予TCD和TCD/cisplatin不會影響裸鼠體重和脾臟相對重量，但cisplatin組的體重和脾臟相對重量則顯著低於控制組和TCD組，因此推測TCD具有抑制AGS腫瘤生長的效用且副作用較低。 綜論，在細胞實驗TCD可透過延滯細胞週期、引起細胞凋亡和抑制細胞增生，導致AGS細胞存活率下降。當TCD與cisplatin併用具有協同性的抑制AGS增殖的效果。在動物實驗TCD能抑制AGS腫瘤生長，顯示TCD具有協助治療胃癌的潛力，未來仍有待更多的研究以了解TCD的抗腫瘤作用機制與化療藥物併用的效果。 關鍵字：胃癌、順鉑、三萜類化合物、抑制增殖、增強化療敏感Cucurbitane-type triterpenoids exert chemopreventive effects and cytotoxicities toward several cancer cells, such as prostate, cervical, and breast cancers. Kuguacin J, one of cucurbitane-type triterpenoids isolated from bitter melon, improved the sensitivity of chemotherapy drugs in cervical cancer cells. However, the knowledge concerning the anti-tumor effect of 3β, 7β, 25-trihydroxycucurbita-5, 23-dien-19-al (TCD) is limited. In the present study, we investigated the anti-proliferative and chemosensitizing effects of TCD, isolated from leaves of wild bitter melon, on human AGS gastric adenocarcinoma cells. AGS cells were treated with TCD alone or combined with cisplatin, a chemotherapeutic agent. Cell viability was assessed by MTT assay, cell cycle and apoptosis were assessed by flow-cytometric analysis, and the expression of major proteins regulating apoptosis was also detected. The results showed that TCD reduced cell viability, inhibited cell proliferation, and changed cellular morpgology of AGS cells. Flow cytometry revealed that TCD increased the sub-G1 population of of AGS cells. Treatment of TCD significantly increased caspase 3 activity. Western blotting showed that TCD treatment significantly increased the levels of pro-apoptotic proteins, including, caspase 9, caspase 3, and PARP, thus inducing apoptosis. It was also demonstrated that a combinational treatment of TCD and cisplatin synergistically inhibited cell growth and cisplatin-induced TNF-α expression, and increased apoptosis. Furthermore, a tumor xenograft model was generated by subcutaneous injection of AGS cells into the dorsum of nude mice. The growth of xenograft tumors was monitored and measured. Cisplatin (4mg/ kg·d), TCD (20 mg/ kg·d), or both combination significantly suppressed the tumor volume and weight from the 35th day after treatment, as compared to the vehicle control. However, the mice challenged with cisplatin showed body weight loss and splenic atrophy. No obvious damage to the organs was found in TCD group, indicating that TCD treatment (20 mg/ kg·d) showed no toxic effects. These findings suggested that the tumor-suppressing effect of TCD would be due to cell cycle inhibition, caspase activation and apotosis induction. TCD may be a promising candidate agent used in the treatment of gastric cancer.

Key word: gastric cancer, cisplatin, 3β, 7β, 25-trihydroxycucurbita-5, 23-dien-19-al, anti-proliferative and chemosensitizing effects