RESUMEN
Cervical cancer is a type of cancer which affects the cervix cells. The conventional treatments for cervical cancer including surgery, chemotherapy, and radiotherapy are only effective in premature stages and less effective in late stages of this tumor. Therefore, the therapeutic strategies based on biologically active substances from plants are needed to develop for the treatment of cervical cancer. The aim of the present study was to assess in vivo toxicity, hematological and biochemical blood parameters in Wistar rats fed Retama sphaerocarpa aqueous leaf extract (RS-AE), as well as to perform in silico molecular docking studies and dynamic simulation of phenolic compounds against HPV16 oncoprotein E6 in order to identify potential inhibitors. RS-AE was found not to induce acute or sub-acute oral toxicity or significant alterations in hematological and biochemical blood parameters in Wistar rats. A total of 11 phenolic compounds were identified in RS-AE, including dihydrodaidzein glucuronide, chrysoperiol pentoside, genistin and vitexin, which turned out to have the highest binding affinity to HPV16 oncoprotein E6. Based on these results, these RS-AE phenolic compounds could be used as natural drugs against the HPV16 E6 oncoprotein.
Asunto(s)
Simulación del Acoplamiento Molecular , Proteínas Oncogénicas Virales , Fenoles , Extractos Vegetales , Hojas de la Planta , Ratas Wistar , Proteínas Represoras , Animales , Extractos Vegetales/farmacología , Extractos Vegetales/química , Ratas , Fenoles/farmacología , Fenoles/aislamiento & purificación , Fenoles/química , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Hojas de la Planta/química , Proteínas Represoras/antagonistas & inhibidores , Femenino , Fitoquímicos/farmacología , Fitoquímicos/aislamiento & purificación , Estructura Molecular , MasculinoRESUMEN
Human Papillomaviruses have co-evolved with their human host, with each of the over 200 known HPV types infecting distinct epithelial niches to cause diverse disease pathologies. Despite the success of prophylactic vaccines in preventing high-risk HPV infection, the development of HPV anti-viral therapies has been hampered by the lack of enzymatic viral functions, and by difficulties in translating the results of in vitro experiments into clinically useful treatment regimes. In this review, we discuss recent advances in anti-HPV drug development, and highlight the importance of understanding persistent HPV infections for future anti-viral design. In the infected epithelial basal layer, HPV genomes are maintained at a very low copy number, with only limited viral gene expression; factors which allow them to hide from the host immune system. However, HPV gene expression confers an elevated proliferative potential, a delayed commitment to differentiation, and preferential persistence of the infected cell in the epithelial basal layer, when compared to their uninfected neighbours. To a large extent, this is driven by the viral E6 protein, which functions in the HPV life cycle as a modulator of epithelial homeostasis. By targeting HPV gene products involved in the maintenance of the viral reservoir, there appears to be new opportunities for the control or elimination of chronic HPV infections.
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Alphapapillomavirus/efectos de los fármacos , Antivirales/uso terapéutico , Infecciones por Papillomavirus/tratamiento farmacológico , Infección Persistente/tratamiento farmacológico , Antivirales/farmacología , Desarrollo de Medicamentos , Epitelio/efectos de los fármacos , Epitelio/patología , Epitelio/virología , Homeostasis/efectos de los fármacos , Humanos , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Infección Persistente/patología , Infección Persistente/virologíaRESUMEN
AIM: High risk Human Papillomavirus (HPV) is an infectious pathogen implicated in a variety of cancers with poor clinical outcome. The mechanism of HPV induced cellular transformation and its intervention remains to be elucidated. Human ADA3 (hADA3), a cellular target of HPV16 E6, is an essential and conserved component of the ADA transcriptional coactivator complex. High risk HPV-E6 binds and functionally inactivates hADA3 to initiate oncogenesis. The aim of this study was to identify the interaction interface between hADA3 and HPV16E6 for designing inhibitory peptides that can potentially disrupt the hADA3-E6 interaction. MATERIAL METHODS: The present investigation employed structure-based in silico tools supported by biochemical validation, in vivo interaction studies and analysis of posttranslational modifications. KEY FINDINGS: First 3D-model of hADA3 was proposed and domains involved in the oncogenic interaction between hADA3 and HPV16E6 were delineated. Rationally designed peptide disrupted hADA3-E6 interaction and impeded malignant properties of cervical cancer cells. SIGNIFICANCE: Intervention of hADA3-E6 interaction thus promises to be a potential strategy to combat HPV induced oncogenic conditions like cervical cancer. The investigation provides mechanistic insights into HPV pathogenesis and shows promise in developing novel therapeutics to treat HPV induced cancers.
Asunto(s)
Proteínas Oncogénicas Virales/antagonistas & inhibidores , Infecciones por Papillomavirus/complicaciones , Fragmentos de Péptidos/farmacología , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Represoras/antagonistas & inhibidores , Sumoilación , Factores de Transcripción/antagonistas & inhibidores , Neoplasias del Cuello Uterino/tratamiento farmacológico , Comunicación Celular , Transformación Celular Neoplásica , Femenino , Humanos , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Conformación Proteica , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
To investigate the antiviral effect of lithium chloride (LiCl) on the replication of Marek's disease virus (MDV) in chicken embryonic fibroblast (CEF) cells, real-time PCR, Western blotting, plaque counting, and indirect immunofluorescence experiments were performed at different time points of LiCl treated CEF cells with virus infection. The results demonstrated that LiCl could affect multiple steps of virus replication and inhibit viral gene expression and protein synthesis in a dose- and time-dependent manner. Moreover, LiCl could directly affect viral infectivity as well. In addition, LiCl significantly affected the gene expression of IFN-ß related genes in virus-infected cells. These results indicate that LiCl significantly inhibits MDV replication and proliferation in CEF cells and it has the potential to be used as an antiviral agent against MDV.
Asunto(s)
Antivirales/farmacología , Herpesvirus Gallináceo 2/efectos de los fármacos , Cloruro de Litio/farmacología , Proteínas Oncogénicas Virales/genética , Replicación Viral/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Línea Celular , Embrión de Pollo , Pollos , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón beta/genética , Interferón beta/metabolismo , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/metabolismo , Carga Viral/efectos de los fármacosRESUMEN
BACKGROUND: Human Papillomavirus (HPV) is a small, non-enveloped, icosahedral and double-stranded DNA virus with a genome of 8 kb, belonging to the papillomaviridae family. HPV has been associated with 99.7% cases of cervical squamous cell carcinoma worldwide. The HPV E6 protein is known as a potent oncogene and is closely allied with the events that result in the malignant transformation of virally infected cells. OBJECTIVE: The present study aims to target plant derived anticancer molecules for HPV driven cancer using a computational approach. METHODS: In this study, E6 oncoprotein was targeted by 101 plant-derived nutraceuticals using the molecular docking method. The multiple sequence analysis and phylogenetic analysis of low risk and high risk 28 HPV E6 proteins were performed. RESULTS: Withanolide D, Ginkgetin, Theaflavin, Hesperidin, and Quercetin-3-gluconide were identified as the potential inhibitors of HPV 16 E6 protein. The zinc finger domain was identified on all variants of HPV E6 oncoprotein while high-risk HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV58, HPV68 and HPV73: probable risk HPV53 and low-risk HPV43 and HPV70 contain PDZ domain. CONCLUSION: The current study using bioinformatics analysis approaches reveals a promising platform for developing anti-cancerous competitive inhibitors targeting HPV.
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Asunto(s)
Biología Computacional/métodos , Proteínas de Unión al ADN/antagonistas & inhibidores , Suplementos Dietéticos/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Simulación del Acoplamiento Molecular , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Humanos , FilogeniaRESUMEN
There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8+ memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade. SIGNIFICANCE: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/732/F1.large.jpg.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de Cabeza y Cuello/terapia , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/terapia , Rimantadina/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Adolescente , Adulto , Anciano , Animales , Presentación de Antígeno/efectos de los fármacos , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Línea Celular Tumoral/trasplante , Quimioradioterapia/métodos , Estudios de Cohortes , Modelos Animales de Enfermedad , Regulación hacia Abajo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/virología , Voluntarios Sanos , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidad , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Células RAW 264.7 , RNA-Seq , Rimantadina/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Adulto JovenRESUMEN
The malignant growth of human papillomavirus (HPV)-positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV-positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV-positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of prosenescent agents, CPX-induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53-independent Caspase-3/7 activation and induction of apoptosis. CPX also eliminates HPV-positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound antiviral and antiproliferative effects in HPV-positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV-inhibitory, prosenescent and proapoptotic agents in both normoxic and hypoxic environments.
Asunto(s)
Ciclopirox/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas E7 de Papillomavirus/antagonistas & inhibidores , Infecciones por Papillomavirus/tratamiento farmacológico , Proteínas Represoras/antagonistas & inhibidores , Neoplasias del Cuello Uterino/tratamiento farmacológico , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Ciclopirox/uso terapéutico , Proteínas de Unión al ADN/metabolismo , Femenino , Células HCT116 , Células HeLa , Humanos , Quelantes del Hierro/farmacología , Quelantes del Hierro/uso terapéutico , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Proteínas Represoras/metabolismo , Esferoides Celulares , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Despite prophylactic vaccination campaigns, human papillomavirus (HPV)-induced cancers still represent a major medical issue for global population, thus specific anti-HPV drugs are needed. Since the ability of HPV E6 oncoprotein to promote p53 degradation is linked to tumor progression, E6 has been proposed as an ideal target for cancer treatment. Using the crystal structure of the E6/E6AP/p53 complex, we performed an in silico screening of small-molecule libraries against a highly conserved alpha-helix in the N-terminal domain of E6 involved in the E6-p53 interaction. We discovered a compound able to inhibit the E6-mediated degradation of p53 through disruption of E6-p53 binding both in vitro and in cells. This compound could restore p53 intracellular levels and transcriptional activity, reduce the viability and proliferation of HPV-positive cancer cells, and block 3D cervospheres formation. Mechanistic studies revealed that the compound anti-tumor activity mainly relies on induction of cell cycle arrest and senescence. Our data demonstrate that the disruption of the direct E6-p53 interaction can be obtained with a small-molecule compound leading to specific antitumoral activity in HPV-positive cancer cells and thus represents a new approach for anti-HPV drug development.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Infecciones por Papillomavirus/tratamiento farmacológico , Proteínas Represoras/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Cristalografía , Ensayos de Selección de Medicamentos Antitumorales/métodos , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidad , Humanos , Simulación de Dinámica Molecular , Estructura Molecular , Neoplasias/patología , Neoplasias/virología , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Esferoides Celulares , Relación Estructura-ActividadRESUMEN
OBJECTIVE: This work aimed to study the mechanism of lncRNATCF7 upregulating DNMT1 mediated by HPV-18 E6 and regulating the biological behavior of cervical cancer cells by inhibiting miR-155. PATIENTS AND METHODS: HPV-16 E6 enhanced DNMT1 expression in cervical cancer cells, which was detected by Western blotting. The expression of miR-155 in cervical cancer was detected by qPCR, the interaction between TCF-7 and miR-155 by Dual-luciferase reporter gene. The changes in invasion ability of cervical cancer cells and the effect of miR-155 on the invasion ability of cervical cancer cells after inhibiting TCF-7 were detected by the transwell invasion assay, while changes in migration ability of cervical cancer cells and the effect of miR-155 on migration ability of cervical cancer cells after inhibiting TCF-7 were observed by the scratch assay. The effect of inhibiting TCF-7 on the tumor size and volume of cervical cancer was detected by the subcutaneous tumor formation in nude mice. RESULTS: E6 expression was significantly inhibited by E6 siRNA. The knockdown of endogenous HPV-16 E6 markedly inhibited the expression of DNMT1; TCF-7 specifically bound to the 3' UTR of miR-155; inhibition of TCF-7 can inhibit invasion and migration of cervical cancer cells; enhanced miR-155 after the inhibition of TCF-7 can promote the invasion and migration of cervical cancer cells; compared with NC group, the tumor volume and weight of TCF-7-siRNA group tumor-bearing was significantly reduced. CONCLUSIONS: TCF-7 plays an important role in the development of cervical cancer. TCF-7 can target miR-155 to regulate the invasion and migration of cervical cancer cells.
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ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , MicroARNs/metabolismo , Proteínas Oncogénicas Virales/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas Represoras/metabolismo , Regiones no Traducidas 3' , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/genética , Femenino , Humanos , Ratones , Ratones Desnudos , MicroARNs/genética , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/genética , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Regulación hacia Arriba , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The recent advancements in CRISPR/Cas9 engineering have resulted in the development of more targeted and potentially safer gene therapies. The challenge in the cancer setting is knowing the driver oncogenes responsible, and the translation of these therapies is hindered by effective and safe delivery methods to target organs with minimal systemic toxicities, on-target specificity of gene editing, and demonstrated lack of long-term adverse events. Using a model system based on cervical cancer, which is driven by the ongoing expression of the human papillomavirus E6 and E7 proteins, we show that CRISPR/Cas9 delivered systemically in vivo using PEGylated liposomes results in tumor elimination and complete survival in treated animals. We compared treatment and editing efficiency of two Cas9 variants, wild-type (WT) Cas9 and the highly specific FokI-dCas9, and showed that the latter was not effective. We also explored high-fidelity repair but found that repair was inefficient, occurring in 6%-8% of cells, whereas non-homologous end joining (NHEJ) was highly efficient, occurring in â¼80% of the cells. Finally, we explored the post gene-editing events in tumors and showed that cell death is induced by apoptosis. Overall, our work demonstrates that in vivo CRISPR/Cas editing treatment of preexisting tumors is completely effective despite the large payloads.
Asunto(s)
Sistemas CRISPR-Cas , Sistemas de Liberación de Medicamentos , Terapia Genética , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/complicaciones , Neoplasias del Cuello Uterino/terapia , Animales , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Edición Génica , Humanos , Liposomas/administración & dosificación , Ratones , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Infecciones por Papillomavirus/virología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virologíaRESUMEN
Cervical cancer is the fourth most common cancer among women worldwide and its development is mainly associated with human papillomavirus infection, a highly sexually transmissible virus. The expression of E6 and E7 viral oncoproteins deregulates cell repairing mechanisms through impairment of tumor suppressor protein functions, such as p53 or retinoblastoma protein. Although the implementation of new preventive vaccines has decreased the infection rate and cervical cancer progression, there are still many women who are affected by this pathology. Nowadays, the main treatment often requires the use of invasive techniques. From well-established strategies, like DNA vaccines and gene therapy, to innovative gene silencing technologies; different methodologies are currently under scrutiny that target the E6 and E7 oncoproteins and/or their modes of action.
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Descubrimiento de Drogas/métodos , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas E7 de Papillomavirus/antagonistas & inhibidores , Infecciones por Papillomavirus/tratamiento farmacológico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/virología , Femenino , Humanos , Infecciones por Papillomavirus/virologíaRESUMEN
The amino (N)-terminal region of human papillomavirus (HPV) minor capsid protein (L2) is a highly conserved region which is essential for establishing viral infection. Despite its importance in viral infectivity, the role of the HPV N-terminal domain has yet to be fully characterized. Using fine mapping analysis, we identified a 36-amino-acid (aa) peptide sequence of the L2 N terminus, termed L2N, that is critical for HPV infection. Ectopic expression of L2N with the transmembrane sequence on the target cell surface conferred resistance to HPV infection. Additionally, L2N peptide with chemical or enzymatic lipidation at the carboxyl (C) terminus efficiently abrogated HPV infection in target cells. Among the synthetic L2N lipopeptides, a stearoylated lipopeptide spanning aa 13 to 46 (13-46st) exhibited the most potent anti-HPV activity, with a half-maximal inhibitory concentration (IC50) of â¼200 pM. Furthermore, we demonstrated that the 13-46st lipopeptide inhibited HPV entry by blocking trans-Golgi network retrograde trafficking of virion particles, leading to rapid degradation. Fundamentally, the inhibitory effect of L2N lipopeptides appeared to be evolutionarily conserved, as they showed cross-type inhibition among various papillomaviruses. In conclusion, our findings provide new insights into the critical role of the L2N sequence in the HPV entry mechanism and identify the therapeutic potential of L2N lipopeptide as an effective anti-HPV agent.IMPORTANCE HPV is a human oncogenic virus that causes a major public health problem worldwide, which is responsible for approximately 5% of total human cancers and almost all cases of cervical cancers. HPV capsid consists of two structure proteins, the major capsid L1 protein and the minor capsid L2 protein. While L2 plays critical roles during the viral life cycle, the molecular mechanism in viral entry remains elusive. Here, we performed fine mapping of the L2 N-terminal region and defined a short 36-amino-acid peptide, called L2N, which is critical for HPV infection. Specifically, L2N peptide with carboxyl-terminal lipidation acted as a potent and cross-type HPV inhibitor. Taken together, data from our study highlight the essential role of the L2N sequence at the early step of HPV entry and suggests the L2N lipopeptide as a new strategy to broadly prevent HPV infection.
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Proteínas de la Cápside/antagonistas & inhibidores , Cápside/metabolismo , Papillomavirus Humano 16/efectos de los fármacos , Lipopéptidos/farmacología , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Infecciones por Papillomavirus/virología , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiología , Humanos , Lipopéptidos/genética , Lipopéptidos/metabolismo , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/tratamiento farmacológico , Internalización del Virus/efectos de los fármacosRESUMEN
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.
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Antivirales/uso terapéutico , Productos Biológicos/uso terapéutico , Infecciones por Papillomavirus/tratamiento farmacológico , Neoplasias del Cuello Uterino/prevención & control , Adulto , Animales , Antivirales/farmacología , Productos Biológicos/farmacología , Modelos Animales de Enfermedad , Femenino , Células HeLa , Papillomavirus Humano 16/efectos de los fármacos , Papillomavirus Humano 16/patogenicidad , Humanos , Ratones , Persona de Mediana Edad , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas E7 de Papillomavirus/antagonistas & inhibidores , Infecciones por Papillomavirus/virología , Proteínas Represoras/antagonistas & inhibidores , Resultado del Tratamiento , Neoplasias del Cuello Uterino/virología , Carga Viral/efectos de los fármacosRESUMEN
High-risk strains of human papillomavirus (HPV) have been identified as the etiologic agent of some anogenital tract, head, and neck cancers. Although prophylactic HPV vaccines have been approved; it is still necessary a drug-based treatment against the infection and its oncogenic effects. The E6 oncoprotein is one of the most studied therapeutic targets of HPV, it has been identified as a key factor in cell immortalization and tumor progression in HPV-positive cells. E6 can promote the degradation of p53, a tumor suppressor protein, through the interaction with the cellular ubiquitin ligase E6AP. Therefore, preventing the formation of the E6-E6AP complex is one of the main strategies to inhibit the viability and proliferation of infected cells. Herein, we propose an in silico pipeline to identify small-molecule inhibitors of the E6-E6AP interaction. Virtual screening was carried out by predicting the ADME properties of the molecules and performing ensemble-based docking simulations to E6 protein followed by binding free energy estimation through MM/PB(GB)SA methods. Finally, the top-three compounds were selected, and their stability in the E6 docked complex and their effect in the inhibition of the E6-E6AP interaction was corroborated by molecular dynamics simulation. Therefore, this pipeline and the identified molecules represent a new starting point in the development of anti-HPV drugs.
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Antivirales/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Antivirales/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Desarrollo de Medicamentos/métodos , Papillomavirus Humano 16/efectos de los fármacos , Papillomavirus Humano 16/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/virología , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/virología , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Aberrant activation of cyclin-dependent kinase 9 (CDK9) is widespread in human cancers. However, the underlying mechanisms of CDK9 activation and the therapeutic potential of CDK9 inhibition in cervical cancer remain largely unknown. Here, we report that CDK9 is gradually upregulated during cervical lesion progression and regulated by HPV16 E6. CDK9 levels are highly correlated with FIGO stage, pathological grade, deep-stromal invasion, tumor size, and lymph nodes metastasis. Knockdown of CDK9 by specific siRNA inhibits cervical cancer cell proliferation in vitro, as well as tumorigenesis in vivo. CDK9 inhibition causes a significant decreased AKT2 and increased p53 protein expression revealing novel CDK9-regulatory mechanisms. Overexpression of AKT2 rescued the suppressive effects caused by CDK9 knockdown, suggesting that AKT2 induction is essential for CDK9-induced transformation. Moreover, CDK9 expression was positively correlated with AKT2 and negatively correlated with p53 in cervical cancer tissues with HPV16 infection. Our findings demonstrate for the first time that CDK9 acts as a proto-oncogene in cervical cancer, modulating cell proliferation and apoptosis through AKT2/p53 pathway. Therefore, our data provide novel mechanistic insights into the role of CDK9 in cervical cancer development. © 2018 IUBMB Life, 71(3):347-356, 2019.
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Quinasa 9 Dependiente de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Represoras/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/genética , Adulto , Animales , Apoptosis/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/metabolismo , Progresión de la Enfermedad , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/crecimiento & desarrollo , Papillomavirus Humano 16/patogenicidad , Humanos , Metástasis Linfática , Ratones , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Transducción de Señal , Carga Tumoral , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Clinical responses with programmed death (PD-1) receptor-directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18, are attractive targets for therapeutic immunization and offer an immune activation strategy that may be complementary to PD-1 inhibition. PATIENTS AND METHODS: We report phase Ib/II safety, tolerability, and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL12 encoding plasmids) delivered by electroporation with CELLECTRA constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457. RESULTS: MEDI0457 was associated with mild injection site reactions, but no treatment-related grade 3-5 adverse events (AE) were noted. Eighteen of 21 evaluable patients showed elevated antigen-specific T-cell activity by IFNγ ELISpot, and persistent cellular responses surpassing 100 spot-forming units (SFUs)/106 peripheral blood mononuclear cells (PBMCs) were noted out to 1 year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4 of 5 post immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all 5 patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow-cytometric analyses revealed induction of HPV16-specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%). CONCLUSIONS: These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.
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Neoplasias de Cabeza y Cuello/terapia , Inmunoterapia , Infecciones por Papillomavirus/terapia , Vacunas contra Papillomavirus/uso terapéutico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Adulto , Anciano , Antígenos Virales de Tumores/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/inmunología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/clasificación , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/virología , Papillomavirus Humano 16/patogenicidad , Papillomavirus Humano 18/patogenicidad , Humanos , Inmunidad Innata/efectos de los fármacos , Interferón gamma/genética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/inmunología , Proteínas E7 de Papillomavirus/antagonistas & inhibidores , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is a malignancy that often involves the oral cavity, pharynx, larynx, or paranasal sinuses. There is a compelling evidence of the human papilloma virus including HPV16 E6 oncogene drives cell transformation and oncogenic processes of HPV positive (HVP+) HNSCC [in particular, Oropharyngeal Squamous Cell Carcinoma (OPSCC)]. In this study, we determined that human OPSCC-derived, HPV16 E6+ cells (UMSCC-104 and UMSCC-47 cell lines) express CD44 and a regulatory transcription factor, c-Jun. Importantly, interaction between matrix hyaluronan (HA) and CD44 (an HA receptor) promotes c-Jun phosphorylation followed by phospho-c-Jun nuclear translocation and co-localization with HPV16 E6 in the nucleus of both UMSCC-104 and UMSCC-47 cells. Further analyses revealed that HPV16 E6 expression is regulated by an upstream promoter containing AP1/c-Jun binding site(s), and chromatin immunoprecipitation (ChIP) assays demonstrated that stimulation of HPV16 E6 expression by HA-CD44 interaction is phospho-c-Jun dependent in these HPV16+ UMSCC-104 and UMSCC-47 cells. This process results in an upregulation of survival proteins, inhibitors of the apoptosis family of proteins (IAPs) and chemoresistance in these HPV16+ cells. Treatment of UMSCC-104 or UMSCC-47 cells with c-Jun-specific or HPV16 E6-specific small interfering RNAs effectively blocks HA/CD44-mediated c-Jun signaling and abrogates HPV16 E6 expression as well as causes downregulation of survival proteins (cIAP-1 and cIAP-2) expression and enhancement of chemosensitivity. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in HPV16 E6 upregulation leading to survival protein (cIAP-1/cIAP-2) production and chemoresistance in HPV16+ UMSCC-104 and UMSCC-47 cells. Most importantly, using a mouse xenograft model, we have observed that Cisplatin chemotherapy combined with the suppression of CD44, c-Jun and HPV16 E6 (by treating both UMSCC-104 cells and UMSCC-47 cells with CD44shRNA or c-Jun shRNA or HPV16 E6 shRNA) appears to be more effective in tumor size reduction than chemotherapy alone. Thus, these newly-discovered HA/CD44-c-Jun/HPV16E6 signaling pathways may provide new drug targets for overcoming cisplatin chemoresistance in HPV16E6-positive OPSCC cells.
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Carcinoma de Células Escamosas/virología , Resistencia a Antineoplásicos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Neoplasias Orofaríngeas/virología , Infecciones por Papillomavirus/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Represoras/metabolismo , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidad , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/genética , Ratones , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/genética , Neoplasias Orofaríngeas/tratamiento farmacológico , Neoplasias Orofaríngeas/metabolismo , Infecciones por Papillomavirus/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This review highlights current interventional clinical trials for HIV-associated malignancies (HIVAMs), with emphasis on 4 mechanistic areas: immunomodulatory therapies and gene therapies, including immune checkpoint inhibitors; cytotoxic therapies; novel tumor-targeted and virally targeted therapies in both AIDS-defining and non-AIDS-defining cancers (NADC); and other screening or topical/ablative interventions. A search on ClinicalTrials.gov located 35 trials, including 12 immunomodulatory or gene therapy trials, 6 cytotoxic therapy trials, 10 trials of therapies with tumor or viral molecular targets, and 7 trials evaluating screening interventions or topical or ablative therapies. Study drugs, mechanisms, and outcomes of interest, including future directions, are discussed. Targeted therapies and immunotherapies address not only the tumor but underlying viral oncogens, including possible benefits on HIV-specific immunologic control. The resulting science from the trials listed in this review will provide important translational breakthroughs for people living with HIV (PLWH) and cancer. We highlight disease-specific challenges that could be addressed in future studies, including testing the safety and efficacy of cutting-edge immunotherapy and targeted treatments used in the general cancer population, and improving gaps in knowledge and practice for cancer screening and its treatment, especially in low-resource regions. Additional important considerations include identification of novel therapies for virally mediated tumors that disproportionally present in PLWH, how to treat persons with HIVAM and advanced immunosuppression, and how to comanage both diseases in antiretroviral therapy-naïve persons and those receiving care in settings where supportive therapies for hematologic toxicities and infections are limited. Current and future clinical trials should address needs of both resource-replete and -limited regions, as well as cancers that are uncommon in or respond differently to HIV-negative populations (eg, Kaposi sarcoma or anal cancer), in addition to an increased focus on NADCs not traditionally linked with HIV, such as lung or gastrointestinal tumors.
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Infecciones por VIH/complicaciones , VIH/patogenicidad , Inmunoterapia/métodos , Neoplasias/terapia , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Ensayos Clínicos como Asunto , Terapia Genética/métodos , VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Infecciones por VIH/virología , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Terapia Molecular Dirigida/métodos , Neoplasias/inmunología , Neoplasias/mortalidad , Neoplasias/virología , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/inmunología , Factores de Riesgo , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Despite the availability of prophylactic vaccines the burden of human papillomavirus (HPV) associated malignancy remains high and there is a need to develop additional therapeutic strategies to complement vaccination. We have previously shown that the poorly characterised E5 oncoprotein forms a virus-coded ion channel or viroporin that was sensitive to the amantadine derivative rimantadine. We now demonstrate that alkylated imino sugars, which have antiviral activity against a number of viruses, inhibit E5 channel activity in vitro. Using molecular modelling we predict that imino sugars intercalate between E5 protomers to prevent channel oligomerisation. We explored the ability of these viroporin inhibitors to block E5-mediated activation of mitogenic signalling in keratinocytes. Treatment with either rimantadine or imino sugars prevented ERK-MAPK phosphorylation and reduced cyclin B1 expression in cells expressing E5 from a number of high-risk HPV types. Moreover, viroporin inhibitors also reduced ERK-MAPK activation and cyclin B1 expression in differentiating primary human keratinocytes containing high-risk HPV18. These observations provide evidence of a key role for E5 viroporin function during the HPV life cycle. Viroporin inhibitors could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation.
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Antivirales/farmacología , Iminoazúcares/farmacología , Canales Iónicos/antagonistas & inhibidores , Papillomaviridae/efectos de los fármacos , Línea Celular , Ciclina B1/metabolismo , Humanos , Queratinocitos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Infecciones por Papillomavirus/virología , Fosforilación , Rimantadina/farmacología , Transducción de Señal , Proteínas Virales/antagonistas & inhibidoresRESUMEN
In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells in vitro and was even able to reduce tumor development in experimental HPV-related cancer models. Nevertheless, for the development of therapeutic tools to be employed in humans, it is important to achieve maximum safety guarantee, which can be provided by the protein format. In the current study, two anti-16E6 scFvs derived from I7 were expressed in E. coli and purified in soluble form by affinity chromatography. Specificity, sensitivity and stability in physiologic environment of the purified scFvs were demonstrated by binding studies using recombinant 16E6 as an antigen. The scFvs functionality was confirmed by immunofluorescence in cervical cancer cells, where the scFvs were able to recognize the nuclear E6. Furthermore, an antiproliferative activity of the scFvI7nuc delivered in protein format to HPV16-positive cell lines was observed. Our results demonstrate that functional anti-16E6 scFvs can be produced in E. coli, suggesting that such purified antibodies could be used in the diagnosis and treatment of HPV-induced malignancies.