白藜蘆醇併用抗癌藥物Temozolomide於腦癌細胞療效之研究

Study for the Adjuvant Therapeutic Effect of Resveratrol and Temozolomide in Malignant Glioma Cells

林千如(Chien-Ju Lin)

指導教授 ： 施純明

臺北醫學大學/醫學院/醫學科學研究所/博士(2012年)

https://doi.org/10.6831/TMU.2012.00126

摘要

惡性膠質細胞瘤(malignant gliomas)為最常見之成人原發性惡性腦瘤，然因腦瘤好發於腦部重要位置，無法完全以手術切除，因此給予化療藥物是手術之外主要的治療方式。Temozolomide (TMZ)是DNA烷化劑，為目前臨床上治療惡性腦瘤唯一有效的化療藥物，然而其抗癌分子機制仍尚未完全釐清。白藜蘆醇(resveratrol)是在葡萄、紅酒、堅果類等食品中富含的多酚類成分，具有抗氧化能力，且可作為抗癌藥物，因此本論文擬探討白藜蘆醇與TMZ併用對腦癌細胞之毒殺作用及機轉。腦癌細胞株經TMZ處理後，利用MTT、colony formation assay及LDH release assay等實驗證實TMZ降低細胞存活率及增加細胞毒性。利用流式細胞儀搭配acridine orange及annexin V/PI染劑偵測中autophagy及apoptosis比例，並利用immunoblotting方法偵測caspase 3、PARP與autophagy marker蛋白LC3-II，皆證實TMZ可導致腦癌細胞同時進行autophagy及caspase-dependent apoptosis。Autophagy inhibitor (3-methyladenine)及inducer (rapamycin)分別與TMZ併用後偵測細胞死亡現象，結果顯示TMZ所誘導之autophagy扮演保護細胞角色。利用ROS專一性染劑分別偵測細胞內ROS量，結果顯示superoxide anion (O2•-)、intracellular H2O2及 mitochondrial H2O2皆隨TMZ處理時間而增加。抗氧化劑如白藜蘆醇、vitamin C及tiron，或電子傳遞鏈抑制劑如rotenone (Complex I inhibitor)、sodium azide (Complex IV inhibitor)及oligomycin (Complex V inhibitor)則可降低ROS量及TMZ所誘導的autophagy，並增加apoptosis比例及減少細胞存活率。Immunoblotting分析之結果證實TMZ可透過ROS以活化ERK，且ERK抑制劑PD98059及U0126可降低TMZ誘導之autophagy並增加細胞死亡。這些結果顯示TMZ藉ROS/ERK訊息傳遞路徑誘導autophagy以保護細胞免於進行apoptosis。MTT assay配合isobolographic analysis分析法更證實白藜蘆醇與TMZ併用對於抗癌效果具有協同作用(synergistic effect)。此外，mouse xenograft之動物實驗模式亦證實resveratrol與TMZ併用可降低腫瘤生長，且其機轉與細胞模式相同。利用流式細胞儀搭配rhodamine 123或calcein AM/CoCl2染劑分別偵測粒線體膜電位及mitochondrial permeability transition pore (MPTP)證實TMZ會導致腦癌細胞之粒線體受損。利用10-N-nonyl-acridine orange (NAO)染劑偵測mitochondrial mass，亦發現其隨TMZ處理時間而減少。加入電子傳遞鏈抑制劑或MPTP抑制劑cyclosporine A皆可回復粒線體受損現象，因而降低autophagy比例，增加TMZ所誘導之apoptosis及LDH release百分比，並降低colony數量。此外，抗氧化劑如白藜蘆醇、vitamin C及tiron可減低粒線體受損。這些實驗結果證明TMZ透過ROS的產生以導致粒線體受損，並誘導腦癌細胞進行保護性autophagy使細胞免於死亡。此外，ER stress相關蛋白GADD153及GRP78表現量隨TMZ處理時間而增加，並減少pro-caspase 12蛋白表現量，顯示ER stress可導致細胞產生apoptosis。然而ER stress抑制劑4-phenylbutyrate (4-PBA)則可降低autophagy而增加TMZ之細胞毒性。進一步利用immunoblotting及Fluo-3 AM染劑配合流式細胞儀之實驗發現ER stress可活化c-Jun N-terminal kinases (JNK)，並增加細胞內鈣離子濃度。給予JNK抑制劑SP600125或鈣離子清除劑EGTA/BAPTA-AM與TMZ併用之結果則證實JNK的活化與鈣離子增加可促進腦癌細胞進行autophagy。這些結果顯示TMZ可導致內質網受損並活化其下游訊息傳遞因子而參與autophagy之進行。綜合上述，本論文證實抗腦癌之化療藥TMZ可透過不同機制，包括ROS/ERK、粒線體受損、ER stress/JNK及Ca2+等訊息傳遞路徑來誘導細胞進行保護性autophagy使細胞避免走向死亡。併用白藜蘆醇或其他針對粒線體及內質網受損的藥物則可增加TMZ之細胞毒性。因此應用在臨床上可提供一個新的抗癌方針，給予TMZ合併這些藥物治療腦癌病人，期望可降低腦癌細胞生長而提升病人存活率。

關鍵字

細胞凋亡 ； 細胞自噬 ； 腦癌 ； 粒線體 ； ER

並列摘要

Malignant gliomas are the primary malignant brain tumors which are difficult to be treated with surgery completely. Adjuvant chemotherapy is one of the alternative treatments in addition to surgery. Temozolomide (TMZ), an alkylating agent, is the major chemotherapeutic agent used clinically in treating malignant gliomas. However, the detail mechanisms are not completely studied. Resveratrol, a polyphenol derived from grapes, red wine and nuts, has antioxidative effect and is considered as a chemoprotective agent. Accordingly, we aimed to investigate the chemotherapeutic effects and mechanisms of resveratrol combined with TMZ in glioma cells. MTT, colony formation, and LDH release assays revealed the decrease of cell survival rate and increase of cytotoxicity after TMZ treatment. Using flow cytometry with annexin V/PI and acridine orange staining to measure apoptosis and autophagy, respectively, or using immunoblotting to detect caspase 3, PARP, and LC3-II, we revealed that TMZ induced apoptosis and autophagy in glioma cells. The combination of TMZ and autophagy inhibitor (3-methyladenine) or inducer (rapamycin) indicated that TMZ-induced autophagy played a pro-survival role to block apoptosis. Using flow cytometry with ROS specific dyes, we found that the levels of ROS, including superoxide anion (O2•-), intracellular H2O2, and mitochondrial H2O2, were all increased after TMZ treatment, and could be suppressed by the antioxidants, such as resveratrol, vitamin C or tiron, or electron transport chain (ETC) inhibitors, such as rotenone (a Complex I inhibitor), sodium azide (a Complex IV inhibitor), and oligomycin (a Complex V inhibitor). Moreover, the antioxidants were able to decrease TMZ-induced autophagy and increase apoptosis. As revealed by immunoblotting, ROS induced the activation of extracellular signal-regulated kinase (ERK). The percentage of autophagy and cell viability were decreased by ERK inhibitor, PD98059 and U0126, suggesting that TMZ induced autophagy through ROS/ERK pathway to block apoptosis. MTT assay and the isobolographic analysis demonstrated that the combination of TMZ and resveratrol had a synergistic effect. Moreover, in vivo mouse xenograft study has also shown that co-administration of resveratrol and TMZ reduced tumor volumes through the same pathway as in vitro. After TMZ treatment, mitochondrial depolarization and MPTP opening were induced, and mitochondrial mass was deceased as revealed by flow cytometry with rhodamine 123, calcein AM/CoCl2, and 10-N-nonyl-acridine orange (NAO) staining, respectively. Pretreatment of ETC inhibitors or the MPTP inhibitor, cyclosporine A, decreased the phenomena of mitochondrial damage and TMZ-induced autophagy, increased apoptosis and LDH release, and suppressed the number of colony formation. Moreover, combination of antioxidants and TMZ decreased mitochondrial damage. All of these results strongly suggested that ROS-mediated mitochondrial damage were considered as a prelude to TMZ-induced autophagy to protect glioma cells. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and decreased the level of pro-caspase 12 protein, suggesting that ER stress induces caspase-dependent apoptosis. However, the combination of TMZ and 4-phenylbutyrate (4-PBA), an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Continuously, the activation of c-Jun N-terminal kinases (JNK) and the concentration of intracellular Ca2+ were increased after TMZ treatment as detected by immunoblotting and flow cytometry with Fluo-3 AM, respectively. The effects of JNK inhibitor (SP600125) and Ca2+ chelators (EGTA/BAPTA AM) on TMZ-induced cytotoxicity demonstrated that ER stress induced autophagy through JNK and Ca2+ signaling pathways. Taken together, our results indicated that TMZ induced autophagy to protect glioma cells from apoptosis through ROS/ERK, mitochondrial damage, ER stress/JNK and Ca2+ signaling pathways. Moreover, the combination of TMZ with resveratrol or other agents targeting on mitochondrial damage and ER stress could potentiate the efficacy of chemotherapy for brain tumors. Therefore, this study provides evidence that TMZ combined with these agents may be potential anticancer strategies in clinical.

並列關鍵字

apoptosis ； sutophagy ； brain tumor ； mitochondria ； ER

參考文獻

Wang, S. H.; Shih, Y. L.; Lee, C. C.; Chen, W. L.; Lin, C. J.; Lin, Y. S.; Wu, K. H.; Shih, C. M. The role of endoplasmic reticulum in cadmium-induced mesangial cell apoptosis. Chem. Biol. Interact. 181:45-51; 2009.

Ahmad, K. A.; Clement, M. V.; Hanif, I. M.; Pervaiz, S. Resveratrol inhibits drug-induced apoptosis in human leukemia cells by creating an intracellular milieu nonpermissive for death execution. Cancer Res. 64:1452-1459; 2004.

Austin, R. C. The unfolded protein response in health and disease. Antioxid Redox Signal 11:2279-2287; 2009.

Azad, M. B.; Chen, Y.; Gibson, S. B. Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid. Redox Signal. 11:777-790; 2009.

Bauvy, C.; Gane, P.; Arico, S.; Codogno, P.; Ogier-Denis, E. Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29. Exp. Cell Res. 268:139-149; 2001.