succinyl-alanylalanyl-prolyl-phenylalanine-chloromethylketone and Uterine-Cervical-Neoplasms

The chymotrypsin-like serine protease inhibitor, succinyl-alanine-alanine-proline-phenylalanine chloromethyl ketone (AAPF(CMK)), has been shown to have anticarcinogenic activity in a number of model systems and to be relatively selective for a nuclear protease. This inhibitor also has substantial effects on growth of tumorigenic human papillomavirus (HPV)-infected keratinocytes in organotypic raft cultures. Here, we examined the effects of AAPF(CMK) on cell growth, cell-cycle kinetics, apoptosis induction, and DNA synthesis in two human cervical carcinoma cell lines: SiHa cells, which have integrated high-risk HPV-16; and C33a cells, which do not contain HPV DNA. AAPF(CMK) inhibited growth of both cell lines in a time- and dose-dependent manner. Apoptosis studies showed no significant difference in drug-treated versus vehicle-treated cells in the C33a cell line. However, a significant dose-dependent increase in apoptosis occurred at a late time point in SiHa cells. Cell-cycle progression and DNA synthesis assays showed that the cellular mechanisms of growth inhibition by AAPF(CMK) differ between the HPV16-positive and HPV-negative tumorigenic cell lines. Drug-treated C33a cells showed a significant accumulation of cells in the G(2) phase of the cell cycle. In SiHa cells, growth inhibition produced by AAPF(CMK) seemed to result from a global arrest of the cell cycle. Although the molecular mechanisms involved in AAPF(CMK)-induced growth inhibition are distinct between the two tumorigenic cell lines, such differences may ultimately prove to have therapeutic utility. Novel therapies for treating established HPV infections are needed, because HPV is a causative agent in the development of multiple types of cancer.

Topics: Amino Acid Chloromethyl Ketones; Cell Line, Tumor; Female; Growth Inhibitors; Humans; Oligopeptides; Papillomaviridae; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone; Uterine Cervical Neoplasms

Protease inhibitors have been known to exhibit anticarcinogenic activity in a variety of model systems, although the biological target(s) and mechanism remain enigmatic. Human papillomavirus (HPV) is the primary etiological agent of cervical cancer. Here we show that a nuclear chymotrypsin-like protease activity (NCLPA), which appears to be involved in transformation in several different experimental models, is significantly elevated in keratinocytes infected with high-risk HPV. Further, we demonstrate a marked growth inhibition of organotypic raft cultures, which is specific for cells infected with high-risk HPV types, using a chloromethyl ketone inhibitor previously shown to be relatively selective for the NCLPA. Surprisingly, this HPV-dependent inhibitory effect is independent of any alterations in the NCLPA. This finding has clear implications for the development of novel therapeutics specifically targeted to cervical dysplasias with HPV-infected cells.

Topics: Amino Acid Chloromethyl Ketones; Carcinoma; Cell Culture Techniques; Cell Nucleus; Cell Proliferation; Cells, Cultured; Chymotrypsin; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Female; Genome, Viral; Humans; Keratinocytes; Male; Oligopeptides; Papillomaviridae; Papillomavirus Infections; Peptide Hydrolases; Protease Inhibitors; Uterine Cervical Neoplasms