Ursolic acid에 의한 자궁 경부암 세포주에서 항증식, 항바이러스 작용 기전 분석 = Antiproliferative and Antiviral Mechanism of Ursolic Acid in Cervical Carcinoma Cell Lines

국문 초록 (Abstract)

목적 : 동양의 전통적인 약제로 사용되고 있는 ursolic acid는 항암, 항염증 효과가 있다고 알려져 있다. 본 연구는 ursolic acid의 자궁경부암 세포주에 대한 증식 억제와 HPV에 관련된 항바이러스 효과를 관찰하고자 하였다.
연구 방법 : Ursolic acid와 Dexamethasone을 자궁경부암 세포주에 처리한 후 세포 증식 억제 효과를 관찰하기 위하여 MTT assay를 시행하였으며, DNA fragmentation, DAPI staining과 FACS 분석을 통해 apoptosis의 특징을 관찰하였으며, apoptosis와 연관된 단백질들로 western blotting을 시행하였다. 또한, 항바이러스 효과를 관찰하기 위하여 HPV E6, E7의 발현 정도를 RT-PCR로 분석하였다.
결과 : Ursolic acid를 HPV가 함유된 자궁경부암 세포주(HeLa, CaSki, SiHa)에 투여하면 농도와 시간에 의존적으로 비례하여 세포증식이 억제되었으나, HPV를 함유하지 않은 자궁경부암 세포주(C33A)에서는 세포 증식억제 효과는 없었다. HeLa 세포주에서 ursolic acid의 농도를 증가시킴에 따라 apoptosis의 특징을 보여주는 DNA fragmentation, DAPI staining, FACS 분석 소견이 뚜렷하게 나타났으며, westem blottiong을 통하여 Fsa 단백질의 발현이 유도되고, caspase-8, caspase-3, PARP 단백질이 절단됨을 관찰할 수 있었다. HeLa 세포에 ursolic acid를 처리함에 따라 HPV-18 E6, E7 유전자 발현이 감소되었으나, p53과 Rb 단백질의 변화는 관찰할 수 없었다.
결론 : 자궁경부암 세포에 ursolic acid를 투여하면 세포증식 억제가 apoptosis에 의하여 일어나며, HPV E6/E7의 발현저하로 인한 항바이러스 효과도 기여할 것으로 사료된다. 이 연구 결과는 ursolic acid에 의한 항증식, 항바이러스 기전을 이해하는데 도움을 주었으며, 효과적인 약제로서의 사용 가능성을 제시하였다.

다국어 초록 (Multilingual Abstract)

Objective : Ursolic acid which has been used as a herb medication, was reported for anti-cancer and anti-inflammatory effects. We tried to analyze the anti-proliferative and anti-viral effects of ursolic acid in Human papillomavirus (HPV) associated cervical carcinoma cell lines.
Methods : We treated ursolic acid or dexamethasone to cervical carcinoma cell lines. We carried out MTT assay to observe the anti-proliferative effects and tried to find out the characteristics of apoptosis by DNA fragmentation, DAPI staining and FACS analysis. By means of westem blotting, we investigated proteins related with apoptosis. After treating of ursolic acid, we used RT-PCR for the expression of HPV E6 and E7 gene to observe the anti-viral effects. Through proteomics analysis including two-dimensional electrophorosis (2DE) and matrix-associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we found the interaction between cell cycle regulatory factor and apoptosis related protein, and also demonstrated anti-cancer and anti-viral mechanism.
Results : While the growth of HPV-positive cervical carcinoma cell lines (HeLa, CaSki, SiHa) is suppressed by ursolic acid in a dose-and time-dependent manner, that of HPV-negative cervical carcinoma cell line (C33A) did not have any inhibitory effects on proliferation. As we increased the concentrations of ursolic acid in HeLa cell, the typical characteristics of apoptosis were noted in DNA fragmentation, DAPI staining and FACS analysis, The expression of Fas protein was induced and caspase-8, caspase-3 and PARP proteins changed into cleavaged forms after treating ursolic acid. HPV-18 E6/E7 gene expression decreased after treating ursolic acid in HeLa cells, but the levels of p53 and Rb proteins did not changed. Total 30 spots were detected by proteomics analtsis, along with 20 up-regulated, 6 down-regulated and 4 unknown protein were founded.
Conclusion : These results suggest that ursolic acid inhibit the cell proliferation through apoptosis in HPV-associated cervical carcinoma cell lines. The apoptotic mechanism of ursolic acid in HeLa cells might be through the signal pathway "Fas→caspase-8→PARP". In addition, the antiviral effect of ursolic acid through suppression of HPV E6/E7 gene expression would contribute to growth inhibition in HeLa cells. These results suggest that ursolic acid could be useful for an effective anticancer drug in treatment of HPV-associated cervical neoplasia.