Antiproliferative and Antiviral Mechanism of Ursolic Acid in Cervical Carcinoma Cell Lines

Lee, Keun Ho; Yim, Eun Kyoung; Yoon, Joo Hee; Um, Soo Jong; Namkoong, Sung Eun; Park, Jong Sup

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 western 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 analysis, 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.