Chondroitin Sulfate Proteoglycans Are De Facto Cellular Receptors for Human Papillomavirus 16 under High Serum Conditions.

Human papillomaviruses (HPVs) are nonenveloped double-stranded DNA viruses that utilize heparan sulfate proteoglycans (HSPGs) as initial attachment factors prior to cell entry and infection. While extensively characterized, the selective interaction between HPV and HSPGs is generally studied using standard in vitro conditions, which fail to account for the effects that media additives, such as fetal bovine serum (FBS), can have on viral binding. As environmental conditions and growth factors associated with wound healing are thought to play a role in natural HPV infection, we sought to investigate the effects that serum or platelet extracts could have on the binding and infectivity of HPV. Here, we demonstrate that high concentrations of FBS and human serum greatly inhibit HPV16 binding, and that for FBS, this effect results from the obstruction of cell surface HSPGs by serum-derived heparin-binding proteins (HBPs). Surprisingly, we found that under these conditions, HPV particles utilize 6O-sulfated chondroitin sulfate proteoglycans (CSPGs) as initial binding receptors prior to infection. These findings were corroborated by small interfering RNA (siRNA)-mediated knockdown experiments, as well as through a cancer cell line screen, where we identified a strong association between viral binding in high serum and the expression of chondroitin sulfate biosynthesis genes. Furthermore, HPV binding in the presence of human platelet lysate also demonstrated an increased dependance on CSPGs, suggesting a possible role for these receptor proteoglycans in active wound healing environments. Overall, this work highlights the significant influence that serum/platelet factors can have on virus binding and identifies CSPGs as alternative cell attachment receptors for HPV. IMPORTANCE Heparan sulfate proteoglycans (HSPGs) have previously been identified as primary attachment factors for the initial binding of human papillomaviruses (HPVs) prior to infection. Here, we demonstrate that in vitro, HPV binding to HSPGs is strongly dependent on the surrounding experimental conditions, including the concentration of fetal bovine serum (FBS). We found that high concentrations of FBS can block HSPG-binding sites and cause a dependence on 6O-sulfated chondroitin sulfate proteoglycans (CSPGs) as alternative initial viral receptors. Further, we demonstrate that use of a human-derived alternative to FBS, human platelet lysate, also occludes HSPG-dependent binding, causing a shift toward CSPGs for viral attachment. As HPV infection of basal epithelial cells is thought to occur at sites of microtrauma with exposure to high serum levels and platelet factors, these unexpected findings highlight a possible role for CSPGs as important cellular receptors for the binding and infectivity of HPV in vivo.

Cell-penetrating peptide inhibits retromer-mediated human papillomavirus trafficking during virus entry.

Virus replication requires critical interactions between viral proteins and cellular proteins that mediate many aspects of infection, including the transport of viral genomes to the site of replication. In human papillomavirus (HPV) infection, the cellular protein complex known as retromer binds to the L2 capsid protein and sorts incoming virions into the retrograde transport pathway for trafficking to the nucleus. Here, we show that short synthetic peptides containing the HPV16 L2 retromer-binding site and a cell-penetrating sequence enter cells, sequester retromer from the incoming HPV pseudovirus, and inhibit HPV exit from the endosome, resulting in loss of viral components from cells and in a profound, dose-dependent block to infection. The peptide also inhibits cervicovaginal HPV16 pseudovirus infection in a mouse model. These results confirm the retromer-mediated model of retrograde HPV entry and validate intracellular virus trafficking as an antiviral target. More generally, inhibiting virus replication with agents that can enter cells and disrupt essential protein-protein interactions may be applicable in broad outline to many viruses.

Low-Dose Aspirin Has Antiproliferative and Apoptosis Effects in HPV16 Tumor Cells and Delays Tumor Development and Growth in an Experimental Model.

OBJECTIVES: The purpose of this study was to investigate the effect of aspirin on epithelial HPV16-transformed cells and its antitumor effects, in an experimental HPV16-positive tumor model. DESIGN: The design of the study is experimental (in vitro and in vivo). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: SiHa and BMK-16/myc cells were treated with aspirin and cell proliferation was determined by MTT; Caspase-Glo 3/7 Assay was used to determine apoptosis. The tumor-bearing mice group was treated with 50 mg/gr/day of aspirin (orally) during 30 days and the antitumor effect was determined. RESULTS: Here, we provide evidence that aspirin has a negative effect on proliferation and induces apoptosis in the human (SiHa) and murine (BMK-16/myc) HPV16 cells. Furthermore, aspirin showed inhibition of tumor growth, and in mice treated with aspirin prior to implantation of tumor cells, the tumor growth was delayed. Also, the effect of aspirin increased survival in tumor-bearing mice and in mice pre-treated with aspirin. LIMITATIONS: It is necessary to carry out in vitro and in vivo studies of the molecular mechanisms involved in the effects of aspirin on tumor cells. CONCLUSION: Aspirin showed antiproliferative effects in tumor cells and inhibited tumor progression and could be effective as a chemopreventive agent. Thus, aspirin deserves further exploration for the treatment of cervical cancer and other neoplasms.

The study protocol of the evaluation for the preventive efficacy of the HPV vaccine for persistent HPV16/18 infection in Japanese adult women: the HAKUOH study.

BACKGROUND: In general, human papillomavirus (HPV) vaccines have demonstrated efficacy in young women worldwide, but there is limited evidence on the efficacy of the quadrivalent HPV6/11/16/18 vaccine in adult women and no evidence of its effectiveness in Japanese adult women in particular. This study aims to evaluate the efficacy of the quadrivalent HPV6/11/16/18 vaccine for persistent HPV16/18 infection in Japanese women aged 27-45 years. METHODS: This is an interventional, nonrandomized, non-double-blind prospective cohort study designed to compare the rates of persistent HPV16/18 infection between the vaccinated arm and unvaccinated arm. The subjects will consist of all women aged 27-45 years who have normal cytology results confirmed by cervical cancer screening from May 2019 to March 2021. The follow-up time is two years. The subjects will be divided into two groups: the vaccinated group and the unvaccinated group. The study will need to enroll 600 vaccinated participants (experimental arm) and 2200 unvaccinated participants (control arm). DISCUSSION: The findings of this trial (HAKUOH study) might provide the first local evidence on the subject and be significantly useful not only to medical academia but also to the Japanese Ministry of Health, Labour and Welfare. The findings could contribute to public health improvement by providing local supportive knowledge on the prevention of HPV infection through HPV vaccination in young adult women in Japan, where active recommendations have been suspended for a long time due to adverse effects. TRIAL REGISTRATION: Trial registration number: NCT04022148 . Registration began on December 1, 2019.

The DNA damage response activates HPV16 late gene expression at the level of RNA processing.

We show that the alkylating cancer drug melphalan activated the DNA damage response and induced human papillomavirus type 16 (HPV16) late gene expression in an ATM- and Chk1/2-dependent manner. Activation of HPV16 late gene expression included inhibition of the HPV16 early polyadenylation signal that resulted in read-through into the late region of HPV16. This was followed by activation of the exclusively late, HPV16 splice sites SD3632 and SA5639 and production of spliced late L1 mRNAs. Altered HPV16 mRNA processing was paralleled by increased association of phosphorylated BRCA1, BARD1, BCLAF1 and TRAP150 with HPV16 DNA, and increased association of RNA processing factors U2AF65 and hnRNP C with HPV16 mRNAs. These RNA processing factors inhibited HPV16 early polyadenylation and enhanced HPV16 late mRNA splicing, thereby activating HPV16 late gene expression.

REBACIN® as a noninvasive clinical intervention for high-risk human papillomavirus persistent infection.

The development of highly sensitive HPV-genotyping tests has opened the possibility of treating HPV-infected women before high-grade lesions appear. The lack of efficient intervention for persistent high-risk HPV infection necessitates the need for development of novel therapeutic strategy. Here we demonstrate that REBACIN®, a proprietary antiviral biologics, has shown potent efficacy in the clearance of persistent HPV infections. Two independent parallel clinical studies were investigated, which a total of 199 patients were enrolled and randomly divided into a REBACIN®-test group and a control group without treatment. The viral clearance rates for the REBACIN® groups were 61.5% (24/39) and 62.5% (35/56), respectively, for the two independent parallel studies. In contrast, the nontreatment groups showed self-clearance rates at 20.0% (8/40) and 12.5% (8/64). We further found that REBACIN® was able to significantly repress the expression of HPV E6 and E7 oncogenes in TC-1 and Hela cells. The two viral genes are well known for the development of high-grade premalignancy lesion and cervical cancer. In a mouse model, REBACIN® was indicated to notably suppress E6/E7-induced tumor growth, suggesting E6 and E7 oncogenes as a potential target of REBACIN®. Taken together, our studies shed light into the development of a novel noninvasive therapeutic intervention for clearance of persistent HPV infection with significant efficacy.

Potential effectiveness of prophylactic HPV immunization for men who have sex with men in the Netherlands: A multi-model approach.

BACKGROUND: Men who have sex with men (MSM) are at high risk for anal cancer, primarily related to human papillomavirus genotype 16 (HPV16) infections. At 8.5 per 100,000 per year, the incidence rate of anal cancer among MSM is similar to that of cervical cancer among adult women in the Netherlands. However, MSM are not included in most HPV vaccination programs. We explored the potential effectiveness of prophylactic immunization in reducing anogenital HPV16 transmission among MSM in the Netherlands. METHODS AND FINDINGS: We developed a range of mathematical models for penile-anal HPV16 transmission, varying in sexual contact structure and natural history of infection, to provide robust and plausible predictions about the effectiveness of targeted vaccination. Models were informed by an observational cohort study among MSM in Amsterdam, 2010-2013. Parameters on sexual behavior and HPV16 infections were obtained by fitting the models to data from 461 HIV-negative study participants, considered representative of the local MSM population. We assumed 85% efficacy of vaccination against future HPV16 infections as reported for HIV-negative MSM, and age-specific uptake rates similar to those for hepatitis B vaccination among MSM in the Netherlands. Targeted vaccination was contrasted with vaccination of 12-year-old boys at 40% uptake in base-case scenarios, and we also considered the effectiveness of a combined strategy. Offering vaccine to MSM without age restrictions resulted in a model-averaged 27.3% reduction (90% prediction interval [PI] 11.9%-37.5%) in prevalence of anal HPV16 infections, assuming similar uptake among MSM as achieved for hepatitis B vaccination. The predicted reduction improved to 46.1% (90% PI 21.8%-62.4%) if uptake rates among MSM were doubled. The reductions in HPV16 infection prevalence were mostly achieved within 30 years of a targeted immunization campaign, during which they exceeded those induced by vaccinating 40% of preadolescent boys, if started simultaneously. The reduction in anal HPV16 prevalence amounted to 74.8% (90% PI 59.8%-93.0%) under a combined vaccination strategy. HPV16 prevalence reductions mostly exceeded vaccine coverage projections among MSM, illustrating the efficiency of prophylactic immunization even when the HPV vaccine is given after sexual debut. Mode of protection was identified as the key limitation to potential effectiveness of targeted vaccination, as the projected reductions were strongly reduced if we assumed no protection against future infections in recipients with prevalent infection or infection-derived immunity at the time of immunization. Unverified limitations of our study include the sparsity of data to inform the models, the omission of oral sex in transmission to the penile or anal site, and the restriction that our modeling results apply primarily to HIV-negative MSM. CONCLUSIONS: Our findings suggest that targeted vaccination may generate considerable reductions in anogenital HPV16 infections among MSM, and has the potential to accelerate anal cancer prevention, especially when combined with sex-neutral vaccination in preadolescence.

A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin.

BACKGROUND: Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development. METHODS: Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed. RESULTS: After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 µmol/L. The cell line was inhibited to a maximum of 18% by cetuximab. CONCLUSIONS: We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.

Enhancement of therapeutic DNA vaccine potency by melatonin through inhibiting VEGF expression and induction of antitumor immunity mediated by CD8+ T cells.

To be effective, therapeutic cancer vaccines should stimulate both an effective cell-mediated and a robust cytotoxic CD8+ T-cell response against human papillomavirus (HPV)-infected cells to treat the pre-existing tumors and prevent potential future tumors. In this study, the therapeutic experiments were designed in order to evaluate antitumor effect against the syngeneic TC-1 tumor model. The anti-tumor efficacy of a HPV-16 E7 DNA vaccine adjuvanted with melatonin (MLT) was evaluated in a C57BL/6 mouse tumor model by measuring tumor growth post vaccination and the survival rate of tumor-bearing mice, analyzing the specific lymphocyte proliferation responses in control and vaccinated mice by MTT assay. The E7-specific cytotoxic T cells (CTL) were analyzed by lymphocyte proliferation and lactate dehydrogenates (LDH) release assays. IFN-γ, IL-4 and TNF-α secretion in splenocyte cultures as well as vascular endothelial growth factor (VEGF) and IL-10 in the tumor microenvironment were assayed by ELISA. Our results demonstrated that subcutaneous administration of C57BL/6 mice with a DNA vaccine adjuvanted with MLT dose-dependently and significantly induced strong HPV16 E7-specific CD8+ cytotoxicity and IFN-γ and TNF-α responses capable of reducing HPV-16 E7-expressing tumor volume. A significantly higher level of E7-specific T-cell proliferation was also found in the adjuvanted vaccine group. Furthermore, tumor growth was significantly inhibited when the DNA vaccine was combined with MLT and the survival time of TC-1 tumor bearing mice was also significantly prolonged. In vivo studies further demonstrated that MLT decreased the accumulation of IL-10 and VEGF in the tumor microenvironment of vaccinated mice. These data indicate that melatonin as an adjuvant augmented the cancer vaccine efficiency against HPV-associated tumors in a dose dependent manner.

Inhibition of Tetraspanin Functions Impairs Human Papillomavirus and Cytomegalovirus Infections.

Tetraspanins are suggested to regulate the composition of cell membrane components and control intracellular transport, which leaves them vulnerable to utilization by pathogens such as human papillomaviruses (HPV) and cytomegaloviruses (HCMV) to facilitate host cell entry and subsequent infection. In this study, by means of cellular depletion, the cluster of differentiation (CD) tetraspanins CD9, CD63, and CD151 were found to reduce HPV16 infection in HeLa cells by 50 to 80%. Moreover, we tested recombinant proteins or peptides of specific tetraspanin domains on their effect on the most oncogenic HPV type, HPV16, and HCMV. We found that the C-terminal tails of CD63 and CD151 significantly inhibited infections of both HPV16 and HCMV. Although CD9 was newly identified as a key cellular factor for HPV16 infection, the recombinant CD9 C-terminal peptide had no effect on infection. Based on the determined half-maximal inhibitory concentration (IC50), we classified CD63 and CD151 C-terminal peptides as moderate to potent inhibitors of HPV16 infection in HeLa and HaCaT cells, and in EA.hy926, HFF (human foreskin fibroblast) cells, and HEC-LTT (human endothelial cell-large T antigen and telomerase) cells for HCMV, respectively. These results indicate that HPV16 and HCMV share similar cellular requirements for their entry into host cells and reveal the necessity of the cytoplasmic CD151 and CD63 C-termini in virus infections. Furthermore, this highlights the suitability of these peptides for functional investigation of tetraspanin domains and as inhibitors of pathogen infections.

3-(2-Chloropropyl amide)-4-methoxy-N-phenylbenzamide inhibits expression of HPV oncogenes in human cervical cancer cell.

BACKGROUND: Human papillomaviruses (HPVs) are the primary causative agents for cervical cancer, and HPV oncoproteins E6 and E7 are known to be the main reason for the onset and maintenance of the malignancies. Therefore, inhibition of viral E6 and E7 oncoproteins expression represents a viable strategy to cervical cancer therapies. This study is to evaluate the antiviral effect of a novel N-Phenylbenzamide derivative, 3-(2-Chloropropyl amide)-4-methoxy-N-phenylbenzamide (L17), against HPV16 in vitro and identify its associated mechanism of action in cervical cancer cells. METHODS: The cytotoxic effect of L17 was assessed by MTT assay. The mRNA and protein levels of E6 and E7 oncogenes were analyzed by quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot, respectively. p53 and Rb protein levels were also detected by Western blot. The effect of L17 on cell cycle was analyzed by flow cytometry. RESULTS: The cytotoxic effect of L17 was greater in cervical carcinoma cells than in normal cells. L17 significantly reduced the expression of HPV16 E6 and E7 mRNA and protein, at least partly by enhancing degradation of HPV16 E6 and E7 mRNA. Moreover, reduced expression of E6 and E7 induced by L17 resulted in the up-regulation of p53 and Rb expression, which subsequently induced CaSki cells arrest at G0/G1 phase. CONCLUSIONS: L17 has antiviral activity through suppressing E6 and E7 oncogene expression and could inhibit CaSki cell proliferating by inducing cells arrest at G0/G1 phase at nontoxic concentration, implying that L17 might be exploited as a candidate agent for HPV-associated cervical cancer prevention and treatment.

Synergistic effect of programmed cell death protein 1 blockade and secondary lymphoid tissue chemokine in the induction of anti-tumor immunity by a therapeutic cancer vaccine.

The use of DNA vaccines has become an attractive approach for generating antigen-specific cytotoxic CD8+ T lymphocytes (CTLs), which can mediate protective antitumor immunity. The potency of DNA vaccines encoding weakly immunogenic tumor-associated antigens (TAAs) can be improved by using an adjuvant injected together with checkpoint antibodies. In the current study, we evaluated whether the therapeutic effects of a DNA vaccine encoding human papilloma virus type 16 (HPV-16) E7 can be enhanced by combined application of an immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway and secondary lymphoid tissue chemokine (SLC) also known as CCL21 adjuvant, in a mouse cervical cancer model. The therapeutic effects of the DNA vaccine in combination with CCL21 adjuvant plus PD-1 blockade was evaluated using a tumor growth curve. To further investigate the mechanism underlying the antitumor response, cytolytic and lymphocyte proliferation responses in splenocytes were measured using non-radioactive cytotoxicity and MTT assays, respectively. Vascular endothelial growth factor (VEGF) and IL-10 expression in the tumor and the levels of IFN-γ and IL-4 in supernatants of spleno-lymphocyte cultures were measured using ELISA. The immune efficacy was evaluated by in vivo tumor regression assay. The results showed that vaccination with a DNA vaccine in combination with the CCL21 adjuvant plus PD-1 blockade greatly enhanced cytotoxic T lymphocyte production and lymphocyte proliferation rates and greatly inhibited tumor progression. Moreover, the vaccine in combination with adjuvant and blockade significantly reduced intratumoral VEGF, IL-10 and splenic IL-4 but induced the expression of splenic IFN-γ. This formulation could be an effective candidate for a vaccine against cervical cancers and merits further investigation.

Human papillomavirus type 16 viral load is decreased following a therapeutic vaccination.

In the dose-escalation phase of a Phase I clinical trial in which six subjects each were vaccinated with PepCan at the 50, 100, 250, and 500 µg per peptide dose, the 50 µg dose showed the best histological regression rate. Ten additional subjects were vaccinated at this dose in the final dose phase. As with the dose-escalation phase, no dose-limiting toxicities were observed. Overall, the histological regression rates were 50% at the 50 µg dose (7 of 14) and 100 µg dose (3 of 6), and 45 % overall (14 of 31). Of subjects in whom HPV type 16 (HPV 16) was detected at entry, it became undetectable in three subjects after vaccination, and the viral loads significantly decreased in nine subjects in whom HPV 16 infection was detected at entry and exit (p = 0.008). Immune profiling revealed increased T-helper type 1 cells after vaccinations (p = 0.02 and 0.0004 after 2 and 4 vaccinations, respectively). T-helper type 2 cells initially increased after two vaccinations (p = 0.01), but decreased below the baseline level after four vaccinations although not significantly. Pre-vaccination regulatory T cell levels were significantly lower in histological responders compared to non-responders (p = 0.03). Feasibility of testing plasma for multiplex cytokine/chemokine analysis and of performing proteomic analysis of PBMCs was examined for potentially identifying biomarkers in the future. While these analyses are feasible to perform, attention needs to be given to how soon the blood samples would be processed after phlebotomy. As sufficient safety of PepCan has been demonstrated, enrollment for the Phase II clinical trial has been opened.

Susceptibility of HPV16 and 18 to high level disinfectants indicated for semi-critical ultrasound probes.

Ultrasound probes used in endocavitary procedures have been shown to be contaminated with high-risk HPV after routine use and HPV is also known to be resistant to some high level disinfectants (HLDs). This study compared efficacy of two leading ultrasound probe HLD methods; liquid ortho-phthalaldehyde (Cidex® OPA) and an automated device using sonicated hydrogen peroxide (trophon® EPR) against HPV16 and HPV18 in a hard-surface carrier test. Native HPV16 and HPV18 virions were generated in organotypic epithelial raft cultures. Viral lysates were dried onto carriers with a 5% (v/v) protein soil. Efficacy tests were performed against the automated device at 35% and 31.5% H2 O2 and 0.55% OPA in quadruplicate with matched input, neutralization, and cytotoxicity controls. Hypochlorite was included as a positive control. Infectivity was determined by the abundance (qRT-PCR) of the spliced E1^E4 transcript in infected recipient cells. The automated HLD device showed excellent efficacy against HPV16 and HPV18 (>5 log10 reductions in infectivity) whereas OPA showed minimal efficacy (<0.6 log10 reductions). While HPV is highly resistant to OPA, sonicated hydrogen peroxide offers an effective disinfection solution for ultrasound probes. Disinfection methods that are effective against HPV should be adopted where possible.

A1E reduces stemness and self-renewal in HPV 16-positive cervical cancer stem cells.

BACKGROUND: Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. METHODS: A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. RESULTS: A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. CONCLUSIONS: Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.

Griffithsin and Carrageenan Combination To Target Herpes Simplex Virus 2 and Human Papillomavirus.

Extensive preclinical evaluation of griffithsin (GRFT) has identified this lectin to be a promising broad-spectrum microbicide. We set out to explore the antiviral properties of a GRFT and carrageenan (CG) combination product against herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) as well as determine the mechanism of action (MOA) of GRFT against both viruses. We performed the experiments in different cell lines, using time-of-addition and temperature dependence experiments to differentiate inhibition of viral attachment from entry and viral receptor internalization. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins, and immunoprecipitation and immunohistochemistry were used to identify the specific glycoprotein involved. We determined the antiviral activity of GRFT against HSV-2 to be a 50% effective concentration (EC50) of 230 nM and provide the first evidence that GRFT has moderate anti-HPV activity (EC50 = 0.429 to 1.39 µM). GRFT blocks the entry of HSV-2 and HPV into target cells but not the adsorption of HSV-2 and HPV onto target cells. The results of the SPR, immunoprecipitation, and immunohistochemistry analyses of HSV-2 combined suggest that GRFT may block viral entry by binding to HSV-2 glycoprotein D. Cell-based assays suggest anti-HPV activity through α6 integrin internalization. The GRFT-CG combination product but not GRFT or CG alone reduced HSV-2 vaginal infection in mice when given an hour before challenge (P = 0.0352). While GRFT significantly protected mice against vaginal HPV infection when dosed during and after HPV16 pseudovirus challenge (P < 0.026), greater CG-mediated protection was afforded by the GRFT-CG combination for up to 8 h (P < 0.0022). These findings support the development of the GRFT-CG combination as a broad-spectrum microbicide.

HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4.

Minichoromosome maintenance (MCM) proteins play key role in cell cycle progression by licensing DNA replication only once per cell cycle. These proteins are found to be overexpressed in cervical cancer cells. In this study, we depleted MCM4, one of the MCM 2-7 complex components by RNA interference (RNAi) in four cervical cancer cell lines. The four cell lines were selected on the basis of their human papillomavirus (HPV) infection: HPV16-positive SiHa, HPV18-positive ME-180, HPV16- and HPV18-positive CaSki, and HPV-negative C-33A. The MCM4-deficient cells irrespective of their HPV status grow for several generations and maintain regular cell cycle. We did not find any evidence of augmented response to a short-term (48 h) cisplatin treatment in these MCM4-deficient cells. However, MCM4-/HPV16+ SiHa cells cannot withstand a prolonged treatment (up to 5 days) of even a sublethal dosage of cisplatin. They show increased chromosomal instability compared to their control counterparts. On the other hand, MCM4-deficient CaSki cells (both HPV16+ and 18+) remain resistant to a prolonged exposure to cisplatin. Our study indicates that cervical cancer cells may be using excess MCMs as a backup for replicative stress; however, its regulatory mechanism is dependent on the HPV status of the cells.

Update on Head and Neck Cancer: Current Knowledge on Epidemiology, Risk Factors, Molecular Features and Novel Therapies.

Tobacco use and alcohol consumption are the main risk factors associated with head and neck squamous cell carcinoma (SCC) development due to their cytotoxic and mutagenic effects on the exposed epithelia of the upper aerodigestive tract. Epstein-Barr virus (EBV) and high-risk human papillomaviruses (HPVs), both encoding viral oncoproteins able to interfere with cell cycle control, have been recognized as the etiological agents of nasopharynx carcinoma and a fraction of oropharyngeal carcinoma, respectively. Head and neck SCC is a deadly disease and despite innovative treatments represents a major challenge for patients. Recently, a number of genomic studies have highlighted the molecular heterogeneity of head and neck SCC based on methylation profiles, microRNA expression, mutated genes and new druggable pathways which may represent new targets for cancer-tailored therapies. To date, cetuximab is the only FDA-approved anti-epidermal growth factor receptor therapy for the treatment of head and neck SCC. In addition, a number of monoclonal antibodies targeting AKT, mTOR and PI3K pathways are under evaluation. Several therapeutic vaccines against HPV16 and EBV proteins are also under study. The purpose of this article is to review the epidemiology, pathogenesis and molecular features of head and neck SCC, with an emphasis on new therapies.

Activity of Andrographolide and Its Derivatives on HPV16 Pseudovirus Infection and Viral Oncogene Expression in Cervical Carcinoma Cells.

Andrographolide (Androg) has been reported to contain antiviral and antitumor activities, but the effects of Androg on human papillomavirus (HPV) infection and cervical cancer have not been elucidated. This study investigated the effects of Androg and its derivatives, namely, 14-deoxy-11,12-didehydroandrographolide (14-DDA) and 3,19-isopropylidene andrographolide (IPAD), on HPV16 pseudovirus (HPV16PsV) infectivity, HPV16 E6 oncogene expression and cervical cancer cell apoptosis. The result demonstrated that all compounds inhibited HPV16PsV infection and that 14-DDA showed the highest potency. Only Androg suppressed long control region (LCR) transcription activity of HPV16 in transiently transfected C33A cells and significantly inhibited E6 oncogene expression in SiHa cells in a dose-dependent manner. A twofold subcytotoxic concentration of IPAD exhibited an inhibitory effect on E6 oncogene expression at 48-h posttreatment. Interestingly, p53 protein was restored in a downstream process and was detected earlier by IPAD treatment than by Androg treatment. This result corresponded to the level of cell apoptosis and cell cycle arrest at the G2/M phase. E6 oncogene expression was also suppressed in CaSki cells treated with Androg and IPAD leading to cell apoptosis. These findings imply that Androg and its derivatives have different activities and may be effective agents for HPV prevention and cervical cancer treatment.

Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo.

The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated ß-galactosidase (SA-ß-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.