Expression Of Human Papillomavirus Research Articles

TNF-alpha, IL-4 and IL-10 cytokines induces changes in HO-1 and HDC expression in cervical cancer cells

Event Abstract Back to Event TNF-alpha, IL-4 and IL-10 cytokines induces changes in HO-1 and HDC expression in cervical cancer cells Paulina Gomez-Lomeli1, Pablo C. Ortiz-Lazareno1*, Alejandro Bravo-Cuellar1, Georgina Hernandez-Flores1, Jorge R. Dominguez-Rodriguez1, Luis F. Jave-Suarez1 and Adriana Aguilar-Lemarroy1 1 Instituto Mexicano del Seguro Social, Mexico Background Chronic infection with human papilloma virus (HPV) has been associated as an important factor in generating cancer. E6 and E7 viral proteins are involved in cell transformation. Likewise tumor cells generate escape mechanisms as the expression of molecules that regulate the immune response. HDC and HO-1 exert anti-apoptotic functions. These enzymes may represent another mechanism of resistance in cervical cancer cells. Objectives. We analyzed the gene and protein expression of heme oxygenase (HO-1) and histidine decarboxylase (HDC) enzymes in HPV+ HeLa and SiHa cervical cancer cell lines and HPV- C33A cancer cells treated with TNF-α, I-4 and IL-10 cytokines. Methods HeLa, SiHa and C33A cervical cancer cells were treated with pro and anti-inflammatory cytokines; mRNA was extracted and analyzed expression of HO1 and HDC by real-time PCR. Protein expression was assessed by flow cytometry. Results Cytokines treatment induces a downregulation in HDC gene expression in HPV+ cancer cells in comparison with untreated tumor cells. In HeLa and SiHa cells this gene is downregulated whereas in C33A cancer cells we observed upregulation of HDC gene. In general HDC and HO-1 protein expression was reduced in cancer cells treated with pro and anti-inflammatory cytokines. Conclusions HDC and HO-1 protein expression is modified when the cells were treated with cytokines. In HeLa, SiHa and C33 cancer cells the expression of these enzymes is similar, however at the gene level the behavior is different between cancer cell lines, and this could be due to the presence of HPV infection in some cancer cells. References Moody, C. A. and L. A. Laimins (2010). "Human papillomavirus oncoproteins: pathways to transformation." Nat Rev Cancer 10(8): 550-560. Kareva, I. and P. Hahnfeldt (2013). "The Emerging "Hallmarks" of Metabolic Reprogramming and Immune Evasion: Distinct or Linked?" Cancer Res Gozzelino, R., et al. (2010). "Mechanisms of Cell Protection by Heme Oxygenase-1." Annual Review of Pharmacology and Toxicology 50(1): 323-354. Ichikawa, A., et al. (2010). "Molecular biology of histidine decarboxylase and prostaglandin receptors." Proceedings of the Japan Academy, Series B 86(8): 848-866. Keywords: Heme-oxynenase 1, Histidine Decarboxylase, Cervical cancer cells, tNF-alpha, Anti-inflammatory cytokines Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Gomez-Lomeli P, Ortiz-Lazareno PC, Bravo-Cuellar A, Hernandez-Flores G, Dominguez-Rodriguez JR, Jave-Suarez LF and Aguilar-Lemarroy A (2013). TNF-alpha, IL-4 and IL-10 cytokines induces changes in HO-1 and HDC expression in cervical cancer cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00634 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Pablo C Ortiz-Lazareno, Instituto Mexicano del Seguro Social, Guadalajara, Mexico, pablolazareno@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Paulina Gomez-Lomeli Pablo C Ortiz-Lazareno Alejandro Bravo-Cuellar Georgina Hernandez-Flores Jorge R Dominguez-Rodriguez Luis F Jave-Suarez Adriana Aguilar-Lemarroy Google Paulina Gomez-Lomeli Pablo C Ortiz-Lazareno Alejandro Bravo-Cuellar Georgina Hernandez-Flores Jorge R Dominguez-Rodriguez Luis F Jave-Suarez Adriana Aguilar-Lemarroy Google Scholar Paulina Gomez-Lomeli Pablo C Ortiz-Lazareno Alejandro Bravo-Cuellar Georgina Hernandez-Flores Jorge R Dominguez-Rodriguez Luis F Jave-Suarez Adriana Aguilar-Lemarroy PubMed Paulina Gomez-Lomeli Pablo C Ortiz-Lazareno Alejandro Bravo-Cuellar Georgina Hernandez-Flores Jorge R Dominguez-Rodriguez Luis F Jave-Suarez Adriana Aguilar-Lemarroy Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Human papillomavirus in oropharyngeal squamous cell carcinoma: Assessing virus presence in normal tissue and activity in cervical metastasis

Human papillomavirus (HPV) has been established as an etiologic and prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV oncogenesis involves expression of E6/E7 oncoproteins, with downstream p53 degradation and pRb inhibition. Although much research has focused on HPV's oncogenic behavior in primary OPSCC, minimal information exists about HPV in adjacent normal and metastatic tissue. Retrospective cohort study Patient-matched tumor, normal, and metastatic tissue was gathered from 42 OPSCC patients and tested with real-time quantitative polymerase chain reaction (RT-qPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). RT-qPCR was performed using total RNA from fresh-frozen tissues and primers for HPV16 E6, E7, and p16 transcripts. HPV ISH was performed to detect the presence of HPV DNA and IHC to detect p16 protein. Primary tumor, adjacent normal tissue, and tumor metastasis from 17 OPSCC patients were analyzed. When comparing the presence of HPV16 DNA in tumor, metastatic, and normal tissue by ISH, perfect correlation is found at all subsites (P < .0001). However, active infections determined by HPV16 E6 and E7 expression using quantitative polymerase chain reaction or p16 detection by IHC, were present only in primary and metastatic tissue (P = .0012, E6; P = .02, E7). No such correlation was found in normal tissue when compared to primary or metastatic tissue. There is a clear pattern of active HPV expression that correlates to disease course. In HPV-positive patients, all sites including primary, metastatic, and normal tissues are DNA positive. Transcriptionally active infections were detected in primary and metastatic tissues, whereas normal tissues appear to have latent infections.

1018O - Safety and Efficacy of Cisplatin Plus 5-Fu and Cetuximab in HPV-Positive and HPV-Negative Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck (R/M SCCHN): Analysis of the Phase III Extreme Trial

ABSTRACT Background Tumor human papillomavirus (HPV) status is a strong predictor of survival and response to treatment in patients (pts) with early and locally advanced oropharyngeal cancer, but its effects in R/M SCCHN remain to be clarified. This retrospective analysis of data from the EXTREME trial assessed the role of tumor HPV status in pts with R/M SCCHN receiving cisplatin + 5-FU chemotherapy (CT) alone or in combination with cetuximab. Material and methods: Immunohistochemical detection of p16INK4A was used to determine HPV status: p16-positive and p16-negative disease is referred to as HPV-positive (HPV+) and HPV-negative (HPV-) disease, respectively. Results 196/222 (88%) pts in the CT + cetuximab and 185/220 (84%) pts in the CT arm had tissue evaluable for p16. Of these, 91% and 88%, respectively, had HPV- tumors. Among HPV- pts, CT + cetuximab (n = 178) improved overall survival (OS), progression-free survival (PFS) and overall response rate (ORR), compared with CT (n = 162) (Table). A similar pattern was observed among HPV+ pts, between CT + cetuximab (n = 18) and CT (n = 23) for OS, PFS and ORR. Interaction tests between treatment and HPV status for OS and PFS confirmed that the treatment effect of CT + cetuximab vs CT was independent of tumor HPV status (p = 0.482 and p = 0.430, respectively: no significant interaction was noted). Within the CT + cetuximab and CT arms, HPV (p16) expression is associated with a more favorable outcome than HPV- (Table). In the CT + cetuximab arm, the overall incidence of grade 3/4 adverse events was slightly higher among HPV+ than HPV- pts. Conclusions Although the number of HPV+ pts in this analysis was low, the results suggest that in R/M SCCHN pts in the EXTREME trial, the survival benefits ofCT + cetuximab vs CT are independent of tumor HPV status and HPV (p16) expression is associated with a more favorable outcome than HPV-. Table: 1018O: Tumor HPV status: effects on prognosis and efficacy of CT + cetuximab and CT in R/M SCCHN. Population Comparison OS PFS ORR CT + cetuximab vs CT HPV- (n = 340) Median (months)/rate (%) 9.7 7.3 5.7 3.1 37 17 HR/odds ratio 95% CI p 0.822 (0.647–1.043) 0.1064 0.486 (0.376–0.628) 2.753 (1.655–4.579) HPV+ (n = 41) Median (months)/rate (%) 12.6 9.6 5.6 3.6 50 22 HR/odds ratio 95% CI p 0.628 (0.295–1.338) 0.2241 0.730 (0.363–1.469) 0.3761 3.600 (0.929–13.953) 0.0614 HPV+ vs HPV- CT + cetuximab (n = 196) Median (months)/rate (%) 12.6 9.7 5.6 5.7 50 37 HR/odds ratio 95% CI p 0.592 (0.319–1.098) 0.0925 1.172 (0.685–2.005) 0.5624 1.738 (0.657–4.600) 0.2620 CT (n = 185) Median (months)/rate (%) 9.6 7.3 3.6 3.1 22 17 HR/odds ratio 95% CI p 0.827 (0.504–1.355) 0.4492 0.871 (0.529–1.434) 0.5870 1.329 (0.455–3.880) 0.6025 Data are medians and hazard ratios (HR) for PFS and OS and rates and odds ratios for ORR. p: log-rank test for OS/PFS; Cochran-Mantel-Haenszel test for ORR. Disclosure B. De Blas: The author is an employee of Merck KGaA. I. Celik: The author is an employee of Merck KGaA J.B. Vermorken: I have participated in advisory boards of Merck-Serono, Amgen, Boehringer-Ingelheim, Genentech and Sanofi-aventis and have lectured (compensated) for Merck-Serono, Amgen, Bristol-Myers Squibb and Sanofi-aventis All other authors have declared no conflicts of interest.

Human Papilloma Virus infection in sexually active adolescent girls

PurposeAdolescents are a vulnerable group with regard to sexually transmitted infections, including Human Papilloma Virus (HPV). This is thought to be both because of their more liberal sexual behavior and also the relative immaturity of their genital tract.The aim of the study was to examine trends in HPV infection among sexually active adolescents attending for a sexual health screen. MethodsSexually active adolescents were offered cervical screening, HPV typing and conventional genital cultures as part of a sexual health prevention protocol. Participating adolescents also completed a sexual health questionnaire. ResultsBetween January 2008 and July 2011, 149 sexually active girls were examined for reproductive health issues (mean age 17.3years). 62 (42%) tested positive for HPV DNA, 30 were infected by multiple types and 54 had at least one high risk type identified. The most commonly identified HPV DNA was for low risk type 42 (15 cases, 7.8%), followed by high risk types 51 (14 cases, 7.3%) and 59 (11 cases, 5.7%). E6–E7 mRNA expression was detected in 16 girls (11%), of which half were for HPV 16. Girls who tested positive for HPV DNA and mRNA had similar age at sexual debut with those that tested negative (15.1years in all groups) but had on average more sexual partners (4.1 vs1.9 p=0.007).Only 9 girls in this cohort had been vaccinated against HPV. Approximately half of the girls reported using inadequate or no contraception. ConclusionsHPV infection rate was high in our sample. Furthermore a high percentage was infected with high risk types and a proportion of girls demonstrated mRNA HPV expression. As age at first sexual intercourse drops, vaccination against HPV and sexual education should be instituted in early adolescence, in order to reduce long term reproductive sequelae of unsafe sexual practices.

HPV Positive Squamous Cell Carcinoma of the Oropharynx. Are we Observing an Unusual Pattern of Metastases?

To describe the clinical, histopathologic, immunohistochemical and molecular features of human papilloma virus (HPV)+ squamous cell carcinomas of the oropharynx that had an atypical clinical course. Four patients with HPV+ oropharyngeal squamous cell carcinoma (OPSCC) were identified retrospectively based on unanticipated clinical behavior. The histopathology, immunohistochemistry and molecular studies of both the primary tumor and the metastases were analyzed to look for any predictors to explain the clinical course. The four patients were all male (average age 55) who presented initially with a neck mass and had stage IVA disease. Three of the primary tumors were nonkeratinizing squamous cell carcinoma and one case was a hybrid tumor of both high-grade squamous cell carcinoma and nonkeratinizing type, and all were p16 and HPV 16/18 positive. All patients received concurrent chemoradiation as primary therapy and had a complete response. Disease-free survival ranged from 7 to 15 months and metastases in 3 patients occurred only in bone, including the sternum, humerus, clavicle, and vertebrae. In one patient, distant metastases were identified in the pancreas, liver, lung and skull base. All metastatic lesions were nonkeratinizing morphology and were p16 and/or HPV positive. Two patients died of their disease, one patient is alive with disease and one patient is disease-free. Although infrequent, unanticipated clinical outcomes can occur in HPV-related OPSCC including distant metastases and bone-only metastases. The tumor morphology of the metastases was comparable to the primary and retained p16 and HPV expression.

Abstract 2882: Therapeutic effects of ormeloxifene on cervical cancer

Abstract Background: Cervical cancer is one of the most common and deadly cancers among women worldwide and is strongly associated with persistent Human Papillomavirus (HPV) infection. HPV E6/E7 oncoproteins are well known to interfere with p53 and retinoblastoma (Rb) proteins and drive the development of cervical cancer. While screening for cervical cancer can reduce the incidence and mortality, treatment of advanced stage cervical cancer is difficult and often unsuccessful. Therefore, there is an urgent need for the development of additional treatment modalities. Ormeloxifene is a non-hormonal anti-estrogen oral contraceptive for human use and it effectively suppresses the highly proliferative decidual cells in the endometrium to inhibit egg implantation. Considering its strong anti-proliferative effect, we hypothesized that ormeloxifene may inhibit the growth of cervical cancer cells and may suppress the expression of HPV oncoproteins E6/E7. Methods: The effect of ormeloxifene on the proliferation of various cervical cancer cell lines (Caski, SiHa, C33A, and HT3) was determined by MTS and colony formation assay. Annexin V/7AAD and propidium iodide (PI) staining was analyzed by flow cytometry to determine ormeloxifene's effect on apoptosis and cell cycle inhibition, respectively. The expression of apoptotic proteins such as poly (ADP-ribose) polymerase (PARP), Caspase 3 and Caspase 9 was determined by immunoblotting. The potential of ormeloxifene to repress the expression of HPV E6/E7 oncogenes was determined by quantitative RT-PCR and immunoblotting analyses. Further, the efficacy of ormeloxifene to restore tumor suppressor proteins, such as p53 and Rb, was also determined by immunoblotting. Results: In all 4 cell lines, ormeloxifene effectively inhibited cervical cancer cell growth. Ormeloxifene treatment induced apoptosis in cervical cancer cells as determined by staining for Annexin V/7AAD and immunoblotting for cleaved PARP, Caspase 3, and Caspase 9. PI staining also clearly revealed that ormeloxifene treatment arrests Caski cells in the G0-G1 phase. Additionally, ormeloxifene treatment caused a marked decrease in the levels of HPV E6 and E7 oncogenes and restored the levels of p53 and Rb proteins in Caski cells. Conclusion: Our findings show that ormeloxifene significantly inhibits cervical cancer cell growth and down-regulates HPV E6/E7 oncogenes, therefore; ormeloxifene may be novel chemo-preventive and treatment method for the management of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2882. doi:1538-7445.AM2012-2882

Abstract LB-445: Human papillomavirus (HPV) status is a long-term survival indicator for patients with oropharyngeal cancers in British Columbia

Abstract The incidence of oropharyngeal squamous cell carcinoma (OpSCC) is increasing worldwide. In British Columbia, similar trend is observed. High-risk HPV and p16 have been used as prognostic markers in OpSCC; however, due to the difference in HPV detection methods, the results have been indeterminate. In BC, we do not routinely stain for p16 or investigate tumoral HPV status. This is the first time to link these data with treatment and outcome information. Objectives: To determine HPV status and expression of cyclin D1, p16 and EGFR of OpSCC and their impact on survival. Methods: Patients (N = 151) with primary OpSCC and treated with intent-to-cure at the Vancouver Centre from 2001 to 2007 were retrospectively identified from Pathology database. Outcome data was collected through electronic chart review. Among these patients, 100 (66 %) patients had no recurrence or died without disease at last follow-up (better outcome, Group1) and 50 (33%) patients had persistent disease or died of disease (poorer outcome, Group 2). The high-risk HPV status was determined using Chromogenic in situ hybridization (CISH) (DAKO, Denmark) and expression of p16, EGFR, and cyclin D1 were analyzed using immunohistochemistry. Results: There was no difference in gender, smoking habit, and tumor staging between Groups 1 and 2. However, Group 1 had a significant longer follow-up time comparing to those of Group 2 (50 ± 20 vs. 29 ± 21 months; P &amp;lt; 0.0001). Group1 was 5 years younger than Group 2 (55.5 ± 9.4 vs. 60.1 ± 9.7 years, P= 0.001). HR-HPV had been found in 70% of all cases (N = 105). Group 1 showed significantly higher HR-HPV infection (77% vs. 54%, P = 0.005), more p16 overexpression (80% vs. 64%, P = 0.05), and less cyclin D1 overexpression (16% vs. 36%, P = 0.007). There was no difference observed in EGFR expression. Using Cox Proportional Hazard model for age, sex, treatment type (Surgery and/or radiation, radiation only, combined chemo and radiation therapy), tumor staging (early and late), HPV status, p16, cyclin D1, and EGFR expression, only HPV status (p = 0.0003) and age (p = 0.011) were found to be significant predictors of 5- year survival. After adjusting the age variable, HPV positive patients were 65% less likely to die compared to those HPV negative patients. Those patients with cyclinD1 positive tumors have a 2.8 time elevated risk to die of disease. Conclusion: High frequency of high-risk HPV is identified in OpSCC in British Columbia. Presence of HPV and younger ages have better outcome. The presence of high-risk HPV is an independent factor to long-term survival. (Canadian Cancer Society CCSRI-20336 and BC Cancer Foundation) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-445. doi:1538-7445.AM2012-LB-445

Correlation of human papilloma virus presence with precancerous and cancerous lesions of uterine cervix by immunohistochemistry

Background : Cancer of cervix is the most common form of cancer in females of developing countries. Cervical cancer is the best example of common human malignancy with a proven infectious etiology. The data linking human papilloma virus (HPV) infection with the epidemiology and pathogenesis of cervical neoplasia is convincing. There are various methods for detection of HPV like immunohistochemistry, polymerase chain reaction, liquid phase hybridization (hybrid capture test), in situ hybridization etc. Materials and Methods: We studied the profile of precancerous and cancerous lesions of uterine cervix and correlated human papilloma virus (HPV) presence with precancerous and cancerous lesions of uterine cervix by immunohistochemistry. Total 50 cases were first studied, analyzed, and classified histologically and then immunohistochemistry was done. Results : Majority of the cases 36/50 (72%) under study are squamous cell carcinomas, which is the most common carcinoma found in the uterine cervix. HPV positivity for all cases of cervical carcinoma was found to be 34.7% (16/46 cases), for carcinoma in situ, it was 50% (2/4 cases). It can be thus hypothesized that with decreasing differentiation, there is loss of expression of HPV in the cervical epithelial cells. Conclusion: To conclude, though this study confirms the correlation of HPV presence with precancerous and cancerous lesions of uterine cervix, it also suggests that there must be other co-factors involved in cervical carcinogenesis as well.

Abstract B78: Evaluation of letrozole for the prevention and inhibition of tumor growth in human cervical cancer xenograft mouse models

Abstract Purpose: To evaluate the efficacy of letrozole, a third generation aromatase inhibitor, for the prevention or delay of cervical cancer recurrence in either human papillomavirus (HPV) positive (HeLa) or HPV negative (C-33a) cervical cancer xenograft mouse models. Method: In this three arm study with two xenograft cervical cancer mouse models, HeLa (HPV 16 positive), and C-33a (HPV negative), the efficacy of letrozole was evaluated in prevention and tumor growth inhibition. Sixty mice were divided into six groups of ten mice each of treatment arm, vehicle control arm and no treatment arm for two cell lines. Mice in the treatment arm received letrozole 10 μg orally once daily every day beginning seven days before the inoculation with respective cancer cells and continued until day 90 of the study. Mice were injected subcutaneously on eighth day of the study with 0.5 million cells of respective cell line. Tumor growth was assessed three times a week. After 90 days of letrozole treatment, a 30 day period without drug was observed to evaluate tumor growth. At the end of the study, tumors were extracted and the expression of estrogen receptor alpha (ERα and estrogen receptor beta (ERβ was evaluated from the extracted protein by immunoblotting and RT-PCR was completed on DNA samples from extracted tumors to evaluate the HPV expression. Results: After 90 days of continuous treatment, letrozole inhibited microscopic cervical tumor growth in the HeLa mouse models demonstrated by 45% reduction in tumor volume in the HeLa letrozole treated mice compared to the no treatment control (p &amp;lt; 0.05). Off treatment, the HeLa letrozole treated mice also had slower rate of tumor progression by 38% compared to untreated group (p&amp;lt; 0.05). In addition, overall survival (OS) was improved by 75% in HeLa letrozole arm with (OS of 40% in the letrozole arm compared to 10% in the no treatment arm). While there was only an 18% reduction (p= 0.63) in tumor volume in the C-33a letrozole treated mice compared to the no treatment control, once treatment was stopped, the mice that had been receiveing letrozole had a slower rate of tumor progression by 37% (p &amp;lt; 0.07). There was no survival benefit observed in the C-33a tumor model. In both studies, no change in ERa, ERb, or HPV expression was observed in the letrozole treatment group compared to no treatment group. Conclusion: Although there was no change in ERα, ERβ, or HPV expression observed in this study, the response data in the HPV 16 positive HeLa tumor model compared to the HPV negative C-33a tumor model suggests HPV expression is associated with the potential activity of letrozole for the prevention of cervical cancer recurrence/progression. Additional studies are needed to elucidate the differences of letrozole activity in presence and absence of HPV expression. Overall, this preliminary data indicates a potential agent for the prevention and treatment of cervical cancer recurrence. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B78.

Abstract B79: Evaluation of active hexose correlated compound (AHCC) for the prevention or delay of tumor growth in human cervical cancer xenograft model

Abstract Purpose: Active hexose correlated compound (AHCC) is a mixture of polysaccharides, amino acids, lipids and minerals extracted from the culture of the basidiomycete mushroom Lentinula edodes (shiitake) that has been proposed to have many health benefits including both immunomodulatory and anti-tumor effects. In clinical studies AHCC has demonstrated numerous immunomodulating and potential restorative effects on natural killer (NK) cells, macrophages and cytokines. The objectives of this study were to evaluate if daily treatment with AHCC would eradicate human papillomavirus (HPV) 16/18 expression and prevent or delay cervical tumor growth using human xenograft mouse model. Methods: Selected cervical cancer cells, SiHa (HPV 16/18 positive), and C-33A (HPV negative) were treated in vitro with a single dose AHCC 0.42 mg/mL and incubated for 72 hours. In the second study AHCC dose was repeated once every 24 hours for total of seven days. This was followed by a three arm in vivo study in two xenograft cervical cancer mouse models, SiHa (HPV 16/18 positive), and C-33A (HPV negative), in which each cell line had ten mice for the treatment arm, vehicle control arm and no treatment arm. Mice in the treatment arm received 50 mg/kg AHCC in 0.25 mL of sterile water every day for seven days before the injection of the tumor cells and until the completion of the study. Tumors were measured three times per week. After 90 days of treatment, there was a 30 day observation period to evaluate the potential for recurrence of the HPV infection and the impact on tumor growth. At the end of the study, tumors were extracted and RT-PCR was completed on DNA samples from extracted protein to evaluate the HPV expression. Results: In vitro treatment with a single dose of AHCC for 72 hour incubation suppressed HPV expression in the first 24 hours but then HPV expression recovered by 48 hours. However, with continuous in vitro exposure, sustained HPV suppression was observed. In the in vivo animal studies, expression of HPV was eradicated with once daily AHCC dosing for 90 days and no detection of HPV expression was sustained after 30 days off treatment. In addition, AHCC daily treatment was associated with a 15.9% decrease in SiHa (HPV 16/18 positive) tumor growth compared to the untreated control (P&amp;lt; 0.05). AHCC did impact the growth rate of the C-33A (HPV negative) tumors. Conclusion: In conclusion, these data suggest daily dosing of AHCC will eradicate HPV 16/18 infections and may have a role in the prevention of HPV-related cervical cancer. Furthermore, there is a potential for the addition of AHCC to primary treatment regimens for cervical cancer, which may potentially improve response rates and prevent recurrence. A confirmatory pilot study in HPV positive women is underway. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B79.

Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells

Cervical cancer is caused by human papilloma virus (HPV) expressing E6 and E7 oncoproteins, which are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Repression of HPV oncoproteins would therefore result in reactivation of tumor suppressor pathways and cause apoptosis in cancer cells. Withaferin A (WA), the active component of the medicinal plant Withania Somnifera, has exhibited inhibitory effects against several different cancers. We examined the activity of WA on human cervical cancer cells in vitro and in vivo. WA potently inhibited proliferation of the cervical cancer cells, CaSki (IC(50) 0.45 ± 0.05 μM). Mechanistically, WA was found to (i) downregulate expression of HPV E6 and E7 oncoproteins, (ii) induce accumulation of p53, (iii) increase levels of p21(cip1/waf1) and its interaction with proliferating cell nuclear antigen (PCNA), (iv) cause G(2)/M cell cycle arrest, associated with modulation of cyclin B1, p34(cdc2) and PCNA levels, (v) decrease the levels of STAT3 and its phosphorylation at Tyr(705) and Ser(727) and (vi) alter expression levels of p53-mediated apoptotic markers-Bcl2, Bax, caspase-3 and cleaved PARP. In vivo, WA resulted in reduction of nearly 70% of the tumor volume in athymic nude mice with essentially similar trend in the modulation of molecular markers as in vitro. This is the first demonstration indicating that WA significantly downregulates expression of HPV E6/E7 oncogenes and restores the p53 pathway, resulting in apoptosis of cervical cancer cells. Together, our data suggest that WA can be exploited as a potent therapeutic agent for the treatment and prevention of cervical cancer without deleterious effects.

Abstract 3859: Defining groupings of oropharyngeal squamous cell carcinomas by expression patterns of human papillomavirus oncogenes

Abstract There is considerable clinical interest in the role that human papillomavirus (HPV) plays in the development of oropharyngeal cancers. A variety of assays are currently available to stratify patients based on HPV status prior to treatment. The most frequently utilized are PCR and In Situ hybridization (ISH) to detect the presence of HPV DNA sequences. The increasing clinical interest in determining HPV patient status relates to compelling evidence that a subset of patients with HPV positive tumors have better clinical outcomes. Evidence also suggests that patients with HPV positive tumors should be further divided into groupings based on levels of viral activity and that this may represent additional clinical information necessary to develop appropriate treatment plans. The most frequently applied assay to identify HPV activity in a patient sample is to detect the over-expression of the surrogate protein marker p16 by Immunohistochemisty (IHC), which has been shown to increase expression in response to HPV active infections in oropharyngeal cancer. An alternative technique to quantitatively detect the presence and level of transcriptional activity of HPV in tissue samples is to measure the expression of oncogenes E6 and E7. We have analyzed the fresh frozen tissue samples corresponding to matched tumor, normal and metastatic patient tissues using a quantitative PCR (qPCR) approach to determine the expression of HPV oncogenes and p16 gene expression. We observed that there are three groups of oropharyngeal tumors when grouped by HPV16 E6 and E7 gene expression. The first are the most rare and are tumors that contain no HPV sequences or expression of the E6 and E7 ocogenes. The second are tumors that contain HPV DNA sequences but very low expression of E6 or E7 and the third are tumors that contain HPV DNA sequence and robust expression of E6 and E7. Additionally, we found variable correlation between HPV16 E6 and E7 expression and p16 gene expression, suggesting that factors other than HPV oncogene expression may influence the expression of p16. Our results would indicate that the best assays for quantifying the activity of an HPV infection in oropharyngeal cancers would be those optimized to accurately measure either E6 and E7 transcripts or their protein products directly out of paraffin-embedded tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3859. doi:10.1158/1538-7445.AM2011-3859

Estrogen and progesterone receptor expression in HPV-positive and HPV-negative cervical carcinomas

Human papillomavirus (HPV) is widely accepted as the main cause of cervical cancer. However, the presence of HPV DNA does not inescapably lead to the development of the cancerous phenotype of the infected cell. Therefore, it is considered that the induction of full cancerous expression of HPV requires additional cofactors. The aim of this study was to assess the expression of estrogen receptor α (ERα) and progesterone receptor (PR) in archived tissue blocks of squamous cell carcinoma and adenocarcinoma of the uterine cervix and to ascertain whether expression of these receptors is associated with the presence of HPV DNA. The investigation was performed using formalin-fixed, paraffin-embedded cervical cancer specimens obtained from 250 women who underwent surgery for histologically confirmed neoplastic lesions. The control group consisted of normal cervical tissues obtained from 50 patients who underwent myomectomy. The results of this study revealed that the expression of ER and PR in planoepithelial cancers and adenocarcinomas of the cervix were decreased to undetectable levels. Only in singular cases in the pattern of staining the expression of ER and PR was noted. In stromal cells of the tested neoplasms, higher expression of both types of receptors was found. Comparison of the expression of ER and PR in the staining pattern and stroma of both squamous cell carcinoma and adenocarcioma of the cervix, showed statistically higher expression in the stromal cells. Strong expression (+1, +2, +3) of ER and PR was noted in the stromal cells irrespective of HPV infection, histopathological type of cancer, and clinical and histopathological grade.

Exogenous and endogenous virus interactions in head and neck cancer immunity (105.34)

Abstract We report that two common tumor viruses etiologically associated with head and neck cancer, Epstein-Barr virus (EBV) with undifferentiated nasopharyngeal carcinoma, and human papillomavirus (HPV) with oropharyngeal squamous cell carcinoma, transactivate an endogenous retrovirus (HERV-K18), which encodes a superantigen that activates TCRBV13 T cells. EBV LMP-2 transactivated the HERV-K18 superantigen in nasopharyngeal epithelial cells, and EBV-associated nasopharyngeal carcinomas expressed the superantigen. HPV type-16 E6 and E7 proteins also transactivated the HERV-K18 superantigen in oropharyngeal epithelial cells, and superantigen expression was significantly increased in HPV16-associated carcinomas. Serum cytokine levels in head and neck carcinoma patients indicated that there were distinct inflammatory profiles associated with exogenous and endogenous viruses. HPV-associated patients had increased levels of serum IL-8, while HPV-negative patients had higher serum IL-6 and G-CSF levels. Interestingly, HERV-K18 superantigen levels correlated independently with serum levels of the Th2 cytokine IL-13. Among HPV+ tumors, gene expression array analyses indicated that B cell specific genes were significantly upregulated in the superantigen high group. Our results suggest that endogenous superantigen expression in HPV+ tumors was associated with a Th2 immune response, demonstrating that chronic and endogenous viruses comprising the virome affect both host and tumor immunity.

Human papillomavirus 16 E7 protein inhibits interferon‐γ‐mediated enhancement of keratinocyte antigen processing and T‐cell lysis

Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ineffective virus-specific immune response, and prolonged infection promotes anogenital cancer. To determine whether poor antigen presentation by HPV-infected keratinocytes (KCs) contributes to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein (E7-KCs) were compared for susceptibility to T-cell-mediated lysis directed to ovalbumin (OVA) processed for presentation by the KCs. Interferon (IFN)-γ efficiently enhanced susceptibility to lysis of KCs presenting OVA, but not of E7-KCs similarly presenting OVA. E7-KCs also exhibited impaired IFN-γ-induced upregulation of transcription of major histocompatibility complex class I antigen processing and presentation-associated genes, and of membrane SIINFEKL-H-2K(b) complexes. Thus, expression of HPV 16 E7 protein in KCs may inhibit enhancement by IFN-γ of KC sensitivity to T-cell lysis, by impairing antigen presentation.

Curcumin suppresses human papillomavirus oncoproteins, restores p53, rb, and ptpn13 proteins and inhibits benzo[a]pyrene‐induced upregulation of HPV E7

Curcumin has great potential as a chemopreventive and chemotherapeutic agent; however, its effects on human papillomavirus (HPV)-associated molecular events are inadequately explored. This study examined the effects of curcumin on HPV-associated pathways involved in developing cervical cancer. We demonstrate for the first time that curcumin treatment suppresses cervical cancer cell growth in a three-dimensional raft culture system. Curcumin also inhibits tumorigenic characteristics as shown by decreases in both clonogenic potential and cell motility. Additionally, our findings show that curcumin treatment inhibits the transcription of HPV16 E6/E7 as early as 6 h posttreatment and restores the expression of tumor suppressor proteins p53, retinoblastoma protein, and PTPN13. While smoking is a recognized risk factor for cervical cancer, the molecular effects of smoke carcinogens on the expression of HPV E6/E7 oncogenes are not well known. We show for the first time that exposure to benzo[a]pyrene (BaP), a tobacco carcinogen, increases the expression of HPV E7 oncoprotein suggesting a molecular link between smoking and cervical cancer. Importantly, curcumin decreases the BaP induced increase in the expression of HPV E7 oncoprotein. The results of this study clearly demonstrate that curcumin alters HPV-associated molecular pathways in cervical cancer cells. These novel findings imply that curcumin may be an effective chemopreventive and therapeutic agent for cervical cancer prevention and treatment.

Detection of Human Papillomavirus in Cervical Gradings by Immunohistochemistry and Typing of HPV 16 and 18 in High-Grades by Polymerase Chain Reaction.

Background:Cervical cancer has a major impact on developing countries, where screening programs are not well established or effective.Aim:This study aims to investigate Human papillomavirus (HPV) 6, 11 and 18 expression in cervical biopsies by immunohistochemistry, (IHC) followed by typing of high-risk HPV 16 and 18 in high-grades by polymerase chain reaction (PCR).Settings and Design:During the study period of six months, 30 biopsy samples were obtained from patients attending various gynecology clinics in and around Trichy District, Tamil Nadu, between January and June 2009.Materials and Methods:The ecto- and endoscopic biopsy specimens of the cervix were fixed in 10% buffered formalin; routine paraffin sections were taken for processing and stained with hematoxylin and eosin. The samples were graded as Normal cervicitis, Cervical intraepithelial neoplasia (CIN) I, II, III, and squamous cell carcinoma (SCC), for original diagnosis by pathologists. The extra sections were studied for the expression of HPV 6, 11 and 18 by immunohistochemistry and HPV DNA 16 and18 by PCR.Results:Out of thirty samples, 15 expressed positive and 15 negative for HPV marker. Twenty-seven cases of cervical gradings have been categorized into high grade CIN II/III, SCC (23) and low grade CIN I (4). The high grades were subjected to PCR for high-risk typing. The results revealed that 15 cases were positive for HPV genotype 16 and eight cases for HPV genotype 18. The prevalence of HPV infection was found to be higher in women aged between 50 and 59.Conclusion:This study reveals a significant detection of HPV in the South Indian suspected individuals, by the use of advanced techniques such as IHC and PCR.

Anti-Human Papillomavirus (HPV) 16 E6 Activity of Ling Zhi or Reishi Medicinal Mushroom, Ganoderma lucidum (W. Curt.: Fr.) P. Karst. (Aphyllophoromycetideae) Extracts

Cancer of the cervix ranks second in the demographics of cancers affecting women worldwide. Human papillomavirus (HPV) type 16 is one of the major causative agents for cervical cancer. The E6 region of the HPV 16 genome encodes for an oncoprotein responsible for the pathogenesis of the disease. The E6 oncoprotein is abundant in cervical cancer and cervical cancer-derived cell lines. Mushrooms of the genus Ganoderma have long been used traditionally as an immunomodulating and antitumor agent, with recent studies validating its use in chemotherapeutics. Due to the importance of the E6 oncoprotein in the carcinogenesis of cervical cancer, this study was undertaken to screen the crude dichloromethane, ethanol, water, and polysaccharide extracts of Ganoderma lucidum for the ability to suppress the expression of HPV 16 E6 and to evaluate secondary metabolite classes present in the active crude extract. CaSki cells were treated with the crude extracts and were then subjected to immunocytochemistry protocols. All crude extracts of G. lucidum presented HPV 16 E6 suppression, with the dichloromethane extract being the most active compared to the other crude extracts. The crude dichloromethane extract was then subjected to thin-layer chromatography to evaluate the secondary metabolite classes present. The present study shows that the crude dichloromethane extract of G. lucidum possesses flavonoids, terpenoids, phenolics, and alkaloids. Further isolation work on the dichloromethane extract of G. lucidum could direct us to lead compounds with promising anti-HPV 16 E6 activity.

Human papillomavirus-associated induction of human β-defensins in anal intraepithelial neoplasia

Antimicrobial peptides and proteins (AMPs) are widely distributed effector molecules of the innate immune system with well-known antibacterial activity. However, there is a paucity of information regarding antiviral effects of AMPs. The present study was performed to analyse expression of AMPs in human papillomavirus (HPV)-associated anal skin lesions of human immunodeficiency virus (HIV)-positive men who have sex with men (MSM), a special high-risk group for persistent HPV infections and anal dysplasia. Skin lesions were analysed for the presence of LL-37, RNase 7, and human beta-defensin (hBD)-1, hBD-2 and hBD-3. Moreover, HPV typing and HPV DNA load determination for HPV types 6, 11, 16, 18, 31 and 33 were performed to evaluate possible correlations between expression of AMPs and lesional HPV types. Skin biopsies of 45 HIV-positive MSM with anal intraepithelial neoplasia (AIN), anal condylomata acuminata or unaffected anal mucosa, as well as condylomata acuminata of eight HIV-negative MSM, were analysed for AMP mRNA expression. Additionally, immunohistochemical analysis for hBD-2 and hBD-3 was performed in a total of 45 samples. hBD-2 and hBD-3 gene and protein expression was significantly increased in both AIN and condyloma, whereas LL-37, RNase 7 and hBD-1 gene expression did not differ significantly from unaffected anal mucosa. AMP expression correlated neither with the number of HPV types nor with the high-risk and low-risk HPV DNA loads of the quantified types. No significant differences in AMP expression were observed in condylomata of HIV-positive and HIV-negative MSM. hBD-2 and hBD-3 expression was shown to be significantly upregulated in HPV-associated anal skin lesions of both HIV-positive and HIV-negative MSM. Their biological significance in the innate immunity against these lesions needs further research.

The anti‐condyloma acuminatum effects of interferon‐inducible protein 10 in vitro

Interferon-inducible protein 10 (IP-10) has been reported to show an effective antiviral and antineoplastic role in various murine models. To investigate the anti-condyloma acuminatum effect and human papillomavirus (HPV)-inhibiting efficacy of a recombinant plasmid encoding IP-10 in vitro. A recombinant plasmid DNA carrying IP-10 cDNA was constructed. Condyloma acuminatum tissue particles were transfected with IP-10 and examined for apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry techniques. Relative real-time polymerase chain reaction (PCR) was used to validate the HPV inhibited level of the treatment groups. Immunohistochemistry was used to determine the IP-10 expression on condyloma acuminatum tissues. Transfected IP-10 was expressed mainly in the cytoplasm of the condyloma acuminatum tissues. Plentiful apoptosis was observed in condyloma acuminatum tissues transfected with IP-10. In addition, HPV expression was lower in IP-10-treated tissues than in control tissues. These observations suggest that IP-10 has strong anti-condyloma acuminatum effects, inducing apoptosis and inhibiting HPV, and therefore may be a novel and potentially effective therapy for condyloma acuminatum.