Molecular Mechanisms of Growth Inhibition by the Chymotrypsin-like Serine Protease Inhibitor, AAPFcmk
Open Access
- Author:
- Duncan, Kimberly JoAnne
- Graduate Program:
- Molecular Toxicology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 16, 2010
- Committee Members:
- Kristin Eckert, Dissertation Advisor/Co-Advisor
Kristin Ann Eckert, Committee Chair/Co-Chair
Gary Alan Clawson, Committee Chair/Co-Chair
Neil David Christensen, Committee Member
Thomas E Spratt, Committee Member
John Peter Richie Jr., Committee Member - Keywords:
- HPV
AAPFcmk
cervical cancer
serine protease inhibitor - Abstract:
- Cervical cancer is the second most prevalent cancer in woman worldwide (Kamangar et al., 2006). Infection with one of the 15 known high-risk human papillomavirus (HPV) types is etiologically linked to 95% of cervical cancer (zur Hausen, 1996). Multiple studies have also causally linked oncogenic HPV infection to 25% of head and neck squamous cell carcinomas (HNSCC). Recently, preventative virus-like particle (VLP) vaccines against HPV-16 and HPV-18, two of the most common high-risk HPVs (Munoz et al., 2003), have been approved. However, the participation of HPV in the development of multiple types of cancer, the prevalence of infected women in less developed countries and the current negative reproductive consequences of treatment makes finding novel and affordable therapies critical to treating established HPV infections.<BR><BR> AAPFcmk is a chymotrypsin-like serine protease inhibitor that has been shown to inhibit the growth of organotypic raft cultures containing cells with high-risk HPV types, with no effect observed on uninfected keratinocytes (Drubin et al. 2006). Since many proteases have been shown to be overexpressed in cancer, protease inhibitors have become candidates for use in cancer therapeutics (Turk, 2006). The Bowman-Birk serine protease inhibitor (which has mainly chymotrypsin-specific activity), has been in clinical trials for its anticarcinogenic activity against oral cancer. AAPFcmk has not only shown the ability to inhibit the growth of high-risk HPV cells but also has demonstrated anticarcinogenic activity in a number of model systems and is relatively selective for a nuclear protease (Clawson et al., 1992b). Investigation into potential binding targets of AAPFcmk found multiple targets which are not proteases including ATP-dependent helicases (Dhamne et al., 2006). AAPFcmk was shown to inhibit the ATP-dependent helicase activity of the SV40 Large T-antigen in vitro. However, the molecular mechanisms of inhibition induced in HPV-infected cervical cancer cells by AAPFcmk have not yet been determined. Understanding how AAPFcmk inhibits the growth of human cervical cancer cells at a molecular level could identify possible treatment options, including its prospect as a topical therapeutic, and applications in combination therapies.<BR><BR> We hypothesized that AAPFcmk would arrest the growth of cervical cancer cell lines and cell lines immortalized or transformed by the integration of the SV40 and HPV small DNA tumor viruses through inhibition of the cell cycle based on previous studies (Clawson et al., 1995) (Drubin et al., 2006). We also hypothesized that AAPFcmk growth arrests cell lines infected by the small DNA tumor viruses, SV40 and HPV-16, by blocking the ability of the SV40 Large T-antigen (SV40 LTag), HPV-16 E6 and/or HPV-16 E7 oncoproteins from interacting with the tumor suppressor proteins, p53 and pRb. Both the SV40 LTag and HPV-16 E6 protein functionally inactivate p53 and the downstream cyclin-dependent kinase inhibitor p21, however the mechanism of p53 inactivation is different (Munger and Howley, 2002). The HPV-16 E7 protein and the SV40 LTag both inactivate the hypophosphorylated form of pRb which binds to E2F1 inhibiting cell cycle progression and transcription of the DNA replication factor PCNA. Mechanistically both the SV40 LTag and HPV-16 E7 proteins target the LxCxE motif located in the pRb binding pocket. Previous research with serine protease inhibitors demonstrated that the binding of HPV-18 E7 to pRb could be disrupted at the LxCxE motif after protease inhibitor treatment (Stoppler et al., 1996b).<BR><BR> In this dissertation, we investigated mechanisms of AAPFcmk growth inhibition, using cell culture model systems involving three human cervical carcinoma cell lines and the THLE-2 liver epithelial cell line immortalized with the SV40 LTag to test the hypotheses. C33a cells are cervical cancer cells with no HPV DNA while SiHa and CaSki cervical cancer cells have integrated HPV-16. Using this model system we examined the effects of AAPFcmk on cellular growth, cell cycle kinetics, DNA synthesis, and p53 and pRb protein expression. Growth of the three cervical cancer cell lines was inhibited after AAPFcmk treatment. C33a cells arrested at the G2/M phase of the cell cycle after AAPFcmk treatment. The HPV-16 SiHa and CaSki cells also exhibited an arrest of the cell cycle after AAPFcmk treatment; however, CaSki cells exhibited an arrest at the G1/S phase of the cell cycle while SiHa cells exhibited a significant decrease in cellular proliferation resulting in a global arrest of the cell cycle.<BR><BR> Western analysis of HPV-16 E7 and the E7 protein downstream target E2F1 showed no significant difference in protein expression levels after AAPFcmk treatment in both the CaSki and SiHa cells. However, mRNA expression levels of the E2F1 target gene, PCNA, showed a significant decrease in both CaSki and SiHa cells after AAPFcmk treatment. AAPFcmk treatment also inhibited the growth of the SV40 LTag immortalized THLE-2 cell line, but the inhibition of growth was not associated with a change in p53 protein levels, or the downstream target p21. The protein levels of pRb, and the downstream target proteins E2F1 and PCNA, were also unchanged in THLE-2 cells after AAPFcmk treatment. However, cell cycle arrest, decreased cellular proliferation and inhibition of PCNA transcription observed experimentally after AAPFcmk treatment in both CaSki and SiHa cells indicate that AAPFcmk may potentially inhibit the HPV-16 E7 protein binding of p21 and/or inhibit E2F1 from transcribing PCNA.<BR><BR> Western analysis of p53 and p21, the downstream targets of the HPV-16 E6 protein, demonstrated a significant increase of both p53 and p21 protein expression in CaSki cells but no change was observed in the SiHa cells after AAPFcmk treatment. The results of the western analysis were verified by p53 and p21 mRNA expression levels after AAPFcmk treatment in both cell lines. Changes in p53 and p21 expression in SiHa cells should not be dismissed entirely as high levels of p53 and p21 expression in SiHa cells (as compared to CaSki cells), may make any changes difficult to detect. Therefore, AAPFcmk may also potentially interfere with either HPV-16 E6 protein binding to E6AP or with the E6/E6AP complex from binding to and/or ubiquitinating p53. Future research into the potential binding targets of AAPFcmk elucidated in this dissertation may be necessary to utilize AAPFcmk as a potential HPV therapeutic.