Mechanism(s) of growth inhibition of Human Papillomavirus positive and negative human cervical cancer cells by a chymotrypsin-like serine protease inhibitor

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The purpose of this study was to characterize the mechanisms by which the chloromethyl ketone protease inhibitor AAPF_CMK (an inhibitor of serine chymotrypsin-like activities) inhibits the growth of human cervical cancer-derived cell lines. Increased nuclear protease activity in cancer cells has been established by previous research, and inhibitors of chymotrypsin-like serine proteases have shown potent anticarcinogenic activity. AAPF_CMK was shown previously to be relatively selective for a nuclear protease, and a number of non-protease targets were identified. To explore potential mechanisms of inhibition, we used 2 human cervical cancer cell lines: SiHa cells, which have integrated high-risk Human Papillomavirus (HPV), and C33a which have no HPV DNA. Effects of AAPF_CMK on cell growth, cell cycle kinetics, apoptosis induction and DNA synthesis were examined in both cell lines. AAPF_CMK inhibited growth almost completely in both the C33a and SiHa cell lines. Growth-inhibited C33a cells showed a significant accumulation of cells in the G2 phase of the cell cycle. In SiHa cells, growth inhibition produced by AAPF_CMK is due to a global arrest of the cell cycle. BrdU incorporation was used to determine S phase content of the cell populations. Results show a significant decrease in BrdU incorporation which was time- and dose-dependent in the SiHa cells, an effect that was not observed in the C33a cells. Apoptosis also appears to be a contributing factor in the SiHa cells but not the C33a cells. These results demonstrate that the underlying mechanism of growth inhibition is different between the two cell lines. Understanding the mechanism(s) of AAPF_CMK-induced growth inhibition is necessary to determine its efficacy as a potential therapeutic, and we hypothesize that expression of HPV E6/E7 proteins may differentially alter susceptibility of cervical cells to AAPF_CMK effects. The prevalence of HPV in cancers makes finding novel therapies a high priority.