난소암 세포주에서 클러스터린의 항암제 결합과 항암제 내성의 연관성
The correlation of clusterin binding affinity to chemotherapeutic agents with chemoresistance in ovarian cancer cells
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목적: Chemo-resistance protein으로 알려진 clusterin의 항암약제와의 결합력 및 약제 내성과의 연관성을 분석하여 그 작용기전을 알아본다. 방법: 항암제를 coating buffer에 희석하여 96 well plate에 피막 시킨 후 정제된 clusterin과 반응시킨 후 세척하여 잉여 clusterin 을 제거시키고 mouse monoclonal human clusterin antibody와 peroxidase가 결합된 2차 항체를 붙여 측정한 Optical density (OD) 값으로 정량화하여 비교한다. 각각의 항암제와 clusterin간의 결합 정도가 항암제 내성과 연관성이 있는지 알아보기 위하여 Western blotting으로 clusterin 분비가 거의 없는 것으로 확인된 난소암 세포주 SKOV-3에 항암제를 단독으로 투여한 경우와 항암제와 clusterin을 동시 투여한 군 간의 난소암 세포주 SKOV-3의 생존율을 XTT를 이용하여 비교하였다. 결과: ELISA검사를 통한 결합력 검사에서 paclitaxel, cisplatin, carboplatin, topotecan, adriamycin, Etoposide, 5-Fluorouracil (5-FU) 등은 O.D. ratio가 각각 2.34, 2.40, 0.52, 2.44, 1.602, 1.14, 1.13으로 Topotecan, cisplatin, paclitaxel 등에서 높은 결합력을 보였다. 또한 이들 약제를 SKOV-3에 clusterin과 처치하였을 경우 항암내성이 증가되었다 (P<0.05). 결론: Clusterin의 항암내성은 항암약제와 결합과 연관성이 있을 것으로 추정된다.
Abstract
Objective: The purpose of this study was to determine the mechanism of action of clusterin—known as a chemo-resistance protein—by analyzing its binding with chemotherapeutic agents and elucidating its relation with drug resistance. Methods: Chemotherapeutic agents were diluted with coating buffer and coated onto 96 well plates. We then had these agents cross-react with purified clusterin and wash the wells to remove residual clusterin. We quantified the amount of clusterin with optical density (OD) measured by binding peroxidase-conjugated secondary antibody associated with mouse monoclonal clusterin antibody. To determine if anticancer drug-clusterin binding is related to chemotherapeutic agent resistance, we compared survival rates in the SKOV-3 cell line, which rarely secretes clusterin. We compared a group of SKOV-3 cells treated with a chemotherapeutic agent and a group treated with both the agent and clusterin, by means of XTT. Results: In binding tests using ELISA OD, ratios of paclitaxel, cisplatin, carboplatin, topotecan, Adriamycin, etoposide, and 5-fluoruracil (5-FU) were 2.34, 2.40, 0.52, 2.44, 1.602, 1.14, and 1.13, respectively. Topotecan, cisplatin, and paclitaxel showed relatively higher binding. In addition, when these drugs were treated with clusterin in SKOV-3 cells, anticancer resistance increased (P<0.05). Conclusions: The anticancer drug resistance endowed by clusterin is considered to be related to its binding with chemotherapeutic agents.
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