RESUMEN
Here we show that treatment of the HPV16-positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan-induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down-regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1-negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half-life of the HPV16 E6 and E7 mRNAs renders HPV16-driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Melfalán/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Neoplasias Tonsilares/virología , Animales , Línea Celular Tumoral , Semivida , Papillomavirus Humano 16 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas E7 de Papillomavirus/biosíntesis , Proteínas E7 de Papillomavirus/efectos de los fármacos , Infecciones por Papillomavirus/complicaciones , Estabilidad del ARN/efectos de los fármacos , Proteínas Represoras/biosíntesis , Proteínas Represoras/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The scope of this work was to study synergism between human papillomavirus (HPV) infection and tobacco in vitro, both known to be independent risk factors for oral cancer. METHODS: HPV-positive and HPV-negative oral keratinocytes and oral HPV-negative fibroblasts were exposed to smokeless tobacco extract (STE) prepared from the Scandinavian (STE1) and US-type (STE2) snuff. Cell cycle profiles were determined with flow cytometry, and HPV E6/E7 mRNA expression in HPV-positive cells was assayed using RT-qPCR. RESULTS: The exposure of HPV-positive keratinocytes with STE2 increased the number of aneuploid cells from 27% to 80% of which 44% were in S-phase, while none of the diploid cells were in S-phase. The changes after STE1 exposure were less than seen after STE2: from 27% to 31% of which 34% were in S-phase. STE had no effect on HPV16 E6/E7 expression in HPV-positive keratinocytes. In oral spontaneously transformed, HPV-negative keratinocytes, the number of aneuploid cells at G2-M stage increased after STE1 and STE2 exposure from 3% to 9% and 7%, respectively. In HPV-negative oral fibroblasts, the number of cells at G2-M phase increased from 11% to 21% after STE1 and 29% after STE2 exposure. CONCLUSIONS: The effect of STE varied in the cell lines studied. STE2 increased significantly the proportion of aneuploid cells in HPV-positive oral keratinocytes, but not HPV16 E6/E7 expression. This indicates that tobacco products may enhance the effects of HPV 16 and the risk of DNA aneuploidy increasing risk to malignant transformation.
Asunto(s)
Aneugénicos/efectos adversos , Aneuploidia , Transformación Celular Viral/fisiología , Papillomavirus Humano 16/fisiología , Queratinocitos/efectos de los fármacos , Mucosa Bucal/efectos de los fármacos , Extractos Vegetales/efectos adversos , Tabaco sin Humo/efectos adversos , División Celular/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Diploidia , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Fase G2/efectos de los fármacos , Encía/citología , Encía/virología , Humanos , Queratinocitos/virología , Mucosa Bucal/citología , Mucosa Bucal/virología , Nicotina/efectos adversos , Proteínas Oncogénicas Virales/análisis , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas E7 de Papillomavirus/análisis , Proteínas E7 de Papillomavirus/efectos de los fármacos , Proteínas Represoras/análisis , Proteínas Represoras/efectos de los fármacos , Fase S/efectos de los fármacos , Piel/citología , Piel/virología , TetraploidíaRESUMEN
INTRODUCTION: Human Papillomaviruses (HPVs) are the main etiological agents for the development of most ano-genital cancers and for a subset of head and neck neoplasias. The oncogenic capacity of HPV is due to the combined activity of the viral oncoproteins E6 and E7. A defining feature of all HPV associated cancers is the continued retention and expression of these two viral oncoproteins throughout the development of the disease, and this highlights their value as potential targets for therapeutic intervention, in HPV-induced malignancies. AREAS COVERED: In this review, the authors focus on the HPV E6 oncoprotein functions and its interactions with cellular targets containing either LxxLL motifs or PDZ domains. New approaches leading to the prevention such interactions are described, showing the advantage of E6 as a target for therapeutic intervention against malignant transformation and cancer. EXPERT OPINION: The high degree of conservation in E6-LxxLL interactions across multiple HPV types makes this a compelling therapeutic target for pathologies caused by diverse HPV types. Combining this with therapeutics directed against E6-PDZ interactions offers great promise for the treatment of malignancies caused by high-risk HPV types.
Asunto(s)
Alphapapillomavirus/aislamiento & purificación , Neoplasias/virología , Proteínas Oncogénicas Virales/efectos de los fármacos , Secuencia de Aminoácidos , Humanos , Proteínas Oncogénicas Virales/química , Homología de Secuencia de AminoácidoRESUMEN
The present study further investigated the mode of action of methyl jasmonate (MJ) in different cervical cancer cell lines. We show that in addition to the short term cytotoxicity, MJ effectively reduced the survival of cervical cancer cells (clonogenicity assays). MJ induced apoptosis in all cervical cancer cells. In some cell lines, MJ caused elevation of the mitochondrial superoxide anion, notably, in HeLa and CaSki. Changes in the expression of p53 and bax were variable, yet, downregulation of survivin was common to all cervical cancer cells. MJ significantly reduced the levels of the human papillomavirus (HPV) E6 and E7 proteins without alteration of the mRNA levels. Moreover, ectopic expression of E6, E7 or both in cervical cancer cells that lack HPV (C33A), did not alter significantly their response to MJ. Our studies point to MJ as an effective anticancer agent against a variety of cervical cancer cells acting through shared and different pathways to induce cell death regardless of the presence of HPV.
Asunto(s)
Acetatos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclopentanos/farmacología , Proteínas de Unión al ADN/efectos de los fármacos , Regulación hacia Abajo , Proteínas Inhibidoras de la Apoptosis/efectos de los fármacos , Proteínas Oncogénicas Virales/efectos de los fármacos , Oxilipinas/farmacología , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Represoras/efectos de los fármacos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Represoras/metabolismo , Survivin , Neoplasias del Cuello Uterino/metabolismoRESUMEN
Standard treatment of locally advanced cervical cancer currently consists of concurrent chemoradiation, leading to a 5-year disease-free survival of 66-79%, indicating that there is still ample room for improvement. Characteristic of cervical cancer is the presence of high risk (HR) human papillomavirus (HPV) DNA in more than 99% of these tumors. When the HR HPV genome integrates into the host genome, oncogenic E6 and E7 proteins become constitutively expressed. These oncogenes are also active earlier in the infection cycle and hence are available as therapeutic targets at the preneoplastic stages as well. E7 plays an important role in the early stage of carcinogenesis by stimulating proliferation. HR HPV E6-induced proteasomal degradation of p53 hampers p53 functionality in cell cycle arrest and apoptosis. As p53 plays a key role in the intrinsic apoptotic pathway, current chemoradiation cannot optimally activate this pathway. In this review, we focus on targeted anticancer drugs to eliminate the consequences of HR HPV E6 and E7 activity. Strategies for direct and indirect targeting of HR HPV E6 and E7, including RNA interference, small molecules, proteasome inhibitors, and histone deacetylase inhibitors, are described. In addition, the extrinsic apoptotic pathway as possible alternative therapeutic target for apoptosis induction is reviewed. The rational for implementing recombinant human TRAIL and death receptor agonists and the latest developments on combining these drugs with standard treatment in preclinical settings as well as clinical trials are discussed.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Oncogénicas Virales/efectos de los fármacos , Papillomaviridae/aislamiento & purificación , Neoplasias del Cuello Uterino/prevención & control , Apoptosis , Cisplatino/farmacología , ADN Viral/análisis , Femenino , Humanos , Papillomaviridae/genética , Radiación Ionizante , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Several mucosotropic human papillomaviruses (HPV), including HPV type 16 (HPV-16), are etiologic agents of a subset of anogenital cancers and head and neck squamous cell carcinomas. In mice, HPV-16 E7 is the most potent of the papillomaviral oncogenes in the development of cervical disease. Furthermore, interfering specifically with the expression of E7 in HPV-positive cell lines derived from human cervical cancers inhibits their ability to proliferate, indicating that the expression of E7 is important in maintaining the transformed phenotype in vitro. To assess the temporal role of E7 in maintaining HPV-associated tumors and precancerous lesions in vivo, we generated Bi-L E7 transgenic mice that harbor a tetracycline-inducible transgene that expresses both HPV-16 E7 and firefly luciferase. When we crossed Bi-L E7 mice to a K5-tTA transgene-inducing line of mice, which expresses a tetracycline-responsive transactivator selectively in the stratified squamous epithelia, the resulting Bi-L E7/K5-tTA bitransgenic mice expressed E7 and luciferase in the skin and cervical epithelium, and doxycycline repressed this expression. Bitransgenic mice displayed several overt and acute epithelial phenotypes previously shown to be associated with the expression of E7, and these phenotypes were reversed on treatment with doxycycline. Repressing the expression of E7 caused the regression of high-grade cervical dysplasia and established cervical tumors, indicating that they depend on the continuous expression of E7 for their persistence. These results suggest that E7 is a relevant target not only for anticancer therapy but also for the treatment of HPV-positive dysplastic cervical lesions.
Asunto(s)
Papillomavirus Humano 16 , Proteínas Oncogénicas Virales/genética , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virología , Cuello del Útero/efectos de los fármacos , Cuello del Útero/patología , Cartilla de ADN , Replicación del ADN/efectos de los fármacos , Doxiciclina/farmacología , Femenino , Luciérnagas/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica , Genes Reporteros , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/virología , Humanos , Luciferasas/efectos de los fármacos , Luciferasas/genética , Ratones , Ratones Transgénicos , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas E7 de Papillomavirus , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Cidofovir is a nucleoside analogue that is used off-license to treat recurrent respiratory papillomatosis (RRP) caused by HPV6/11. However, the effect of this drug upon low-risk HPV 6/11 gene expression is unknown. METHODS: The expression of E6 was evaluated by RT-PCR in HPV-ve C33A cervical carcinoma cells stably transfected with both low- and high-risk HPV E6 cDNA's and in SiHa (HPV16+ve) cervical carcinoma cells after treatment with 2 doses and durations of exposure to cidofovir. RESULTS: Compared to the vector only transcript, E6 RNA levels showed an 8-fold increase in low-risk and 20-fold increase in high-risk E6-expressing cells. High-risk E6 protein levels were also detected by Western blot in cidofovir-treated C33A Type16 E6-transfected cells. CONCLUSION: These data may indicate a potential rationale for increased risk of genetic instability and thus transformation due to drug-induced increase in the level of E6.
Asunto(s)
Antivirales/farmacología , Citosina/análogos & derivados , Papillomavirus Humano 6/efectos de los fármacos , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas Virales/metabolismo , Organofosfonatos/farmacología , Neoplasias del Cuello Uterino/virología , Técnicas de Cultivo de Célula , Cidofovir , Citosina/farmacología , Femenino , Papillomavirus Humano 6/genética , Papillomavirus Humano 6/metabolismo , Humanos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Viral/efectos de los fármacos , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Neoplasias del Cuello Uterino/patologíaRESUMEN
OSU03012 is a non-COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21(WAF1/CIP1) were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21(WAF1/CIP1) were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53-p21(WAF1/CIP1) checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21(WAF1/CIP1)-/- mouse embryo fibroblasts were more sensitive to OSU03012-induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21(WAF1/CIP1) is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Mucosa Bucal/efectos de los fármacos , Pirazoles/farmacología , Sulfonamidas/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Bromodesoxiuridina/metabolismo , Línea Celular , Ciclina A/efectos de los fármacos , Ciclina A/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Embrión de Mamíferos , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Fase G1/efectos de los fármacos , Silenciador del Gen , Humanos , Inmunoprecipitación , Ratones , Mucosa Bucal/citología , Mucosa Bucal/virología , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/metabolismo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Infecciones Tumorales por Virus/metabolismoRESUMEN
Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K(D)=24.1microM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/efectos de los fármacos , Podofilotoxina/farmacología , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Ratones , Conformación Molecular , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Biblioteca de Péptidos , Podofilotoxina/síntesis química , Podofilotoxina/química , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Estereoisomerismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Eukaryotic cells continuously produce reactive oxygen species (ROS) and have mechanisms to control ROS levels. ROS have been shown to mediate cell proliferation and transformation. We studied the effect of CuZn-superoxide dismutase (CuZnSOD) on the focus-forming ability of bovine papillomavirus (BPV-1) wtDNA and hypertransforming mutant of its major oncoprotein E5, E5-17S. We found that CuZnSOD suppresses the focus-forming ability of BPV-1 wtDNA and E5 oncoprotein. Significantly fewer foci were detected in pCGCuZnSOD- and BPV-1 DNA-cotransfected cell culture compare to BPV-1 DNA-transfected cell culture (p<0.001). CuZnSOD decreases the rate of cell proliferation in both non-transformed C127 and BPV-1- and E5-transformed cell lines. CuZnSOD decelerates cell entry into the S phase of the cell cycle and has a suppressing effect on the actively dividing cells. As the transformed cells proliferate faster than normal cells when confluent, CuZnSOD inhibits the growth of foci. These results indicate that superoxide radicals may be involved in signaling for cell proliferation and that SOD suppresses cell proliferation.
Asunto(s)
Papillomavirus Bovino 1/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Viral/efectos de los fármacos , Fibroblastos/virología , Proteínas Oncogénicas Virales/efectos de los fármacos , Superóxido Dismutasa/farmacología , Animales , Papillomavirus Bovino 1/fisiología , Bovinos , Células Cultivadas , ADN Viral , Fibroblastos/efectos de los fármacos , Ratones , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Superóxidos/metabolismo , TransfecciónRESUMEN
AIMS AND BACKGROUND: It was documented that nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) and that EBV-encoded latent membrane protein-1 expression (LMP1) plays an important role in the pathogenesis of NPC. In preclinical studies, arsenic trioxide (As2O3) has been identified as a promising anticancer agent for treatment of NPC. The purpose of this study is to investigate if this agent can inhibit the expression of LMP1 and therefore lead to growth inhibition of NPC cells in vitro. METHODS: LMP1-positive NPC cells, HNE1-LMP1, were treated with 3 micromol/L of As2O3 for 96 hours. The LMP1 protein expression and mRNA level in HNE1-LMP1 cells were determined by western blot, confocal immunofluorescence staining and semiquantitative reverse transcriptase reaction (RT-PCR). Apoptosis was determined by light microscopy and the TUNEL method. Alterations in the cell cycle distribution were also investigated by flow cytometry. MTT assay and colony formation assay were used to detect the proliferation of the cells. The LMP1-negative parental cell lines HNE1 and HNE2 were used as control in an attempt to elucidate the role of LMP1 in the anticancer effect of As2O3 on NPC cells. RESULTS: The expression of LMP1 at the protein and mRNA level was reduced after exposure to 3 micromol/L As2O3. This dose of As2O3 significantly induced apoptosis and growth retardation of HNE1-LMP1 cells. In addition, more HNE1-LMP1 cells were induced to G0/G1 and G2/M arrest. The same dose of As2O3 had a moderate effect on HNE1 and HNE2 cells. CONCLUSION: Arsenic trioxide can inhibit LMP1 expression and dictate apoptosis and alterations of cell cycle distribution as well as growth retardation. LMP1-positive NPC cells are more sensitive to As2O3 treatment than LMP1-negative NPC cells.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Óxidos/farmacología , Proteínas de la Matriz Viral/efectos de los fármacos , Proteínas de la Matriz Viral/metabolismo , Apoptosis , Trióxido de Arsénico , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Humanos , Etiquetado Corte-Fin in Situ , Neoplasias Nasofaríngeas/patología , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas Virales/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayo de Tumor de Célula Madre , Proteínas de la Matriz Viral/genéticaRESUMEN
Adeno-associated virus (AAV) Rep 78 protein is known to inhibit the promoter site of several oncogenes and viral genes, including the human papillomavirus (HPV) type 16 E6 transforming genes. The biochemical studies of Rep 78 have been reported, but the effects of Rep 78 gene-mediated inhibition of HPV 16 E6 promoter activity on the various human cervical carcinoma cells have not been characterized. pEGFP-N1 vector, cloned by AAV-mediated Rep 78, is transfected into cervical carcinoma cells. Transfection efficiency of Rep 78 was approximately 30-60% different. Messenger RNA (mRNA) and protein expression of Rep 78 gene was significantly higher on day 1 of the transfection of Rep 78 DNA in CaSki cells, and DNA level of HPV 16 E6 was decreased on day 1 of the transfection. The growth of CaSki cervical cancer cells was only 10-15% inhibited by Rep 78, and the other cervical cells, HeLa, HeLaS3, HT3, and QGU, were unaffected by Rep 78 transfection. In spite of the high efficiency of Rep 78 gene transformation and expression rate, we could not show the significant growth inhibition in various cervical cancer cell lines. Taken together, long-term expression of Rep 78 strategy might be needed for cervical carcinoma gene therapy using AAV vector.
Asunto(s)
Vacunas contra el Cáncer/farmacología , Transformación Celular Viral/genética , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/genética , Vacunas contra Papillomavirus , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Virales , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/efectos de los fármacos , Papillomaviridae/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Probabilidad , Sensibilidad y Especificidad , Transfección , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Although HIV protease inhibitor (PI) drugs predominantly target HIV proteases 1 and 2, it is also known that part of their efficacy is due to selective inhibition of the proteasome. The pathogenicity of high-risk human papilloma virus (HPV) is dependent on expression of viral E6 proteins which inappropriately activate the 26S proteasome to degrade p53 and other cellular proteins that are detrimental to viral replication. Comparison of the ability of the PIs indinavir, ritonavir, amprenavir, lopinavir, atazanavir, nelfinavir and saquinavir to inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells showed that 15 microM lopinavir, 1 mM indinavir or 125 microM ritonavir treatment for 24 h produced a stable increase in the level of nuclear p53 in these cells with minimal cell death. After 4 h exposure of HPV16+ve SiHa cells to 15 microM lopinavir, a transient increase in wild-type p53 expression was observed associated with a 7% reduction in the chymotryptic activity of the 205 proteasome and apoptosis after 24h. Comparison of growth rates of PI treated SiHa, CaSki, C33A, C33A-E6 and non-transformed NIH/3T3 cells showed that SiHa were the most sensitive, whereas NIH/3T3 were least affected. In conclusion, these data show that specific HIV PIs such as lopinavir and possibly indinavir, can induce selective toxicity of HPV-transformed cervical carcinoma cells expressing wild-type p53 and may form the basis of a topically applied alternative to surgery for the treatment of HPV-related premalignant lesions of the cervix.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Proteínas Oncogénicas Virales/metabolismo , Pirimidinonas/farmacología , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Carcinoma/tratamiento farmacológico , Carcinoma/virología , Línea Celular Tumoral/efectos de los fármacos , Femenino , Humanos , Lopinavir , Ratones , Células 3T3 NIH , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas Virales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Represoras/efectos de los fármacos , Proteínas Represoras/genética , Transfección , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/virologíaRESUMEN
Jaceosidin (4',5,7-trihydroxy-3',6-dimethoxyflavone) was isolated from Artemisia argyi as a putative oncogene inhibitor. Jaceosidin inhibited binding between oncoprotein E6 of the human papillomavirus and the p53 tumor suppressor protein. In addition, jaceosidin inhibited binding between the E7 oncoprotein and the Rb tumor suppressor protein, and also inhibited the function of HPV-16 harboring cervical cancer cells, including SiHa and CaSki. Collectively, jaceosidin inhibited the functions of the E6 and E7 oncoproteins of the human papillomavirus, suggesting that this compound might be used as a potential drug for the treatment of cervical cancers associated with the human papillomavirus.
Asunto(s)
Artemisia , Flavonoides/farmacología , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Represoras/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Neoplasias del Cuello Uterino , Supervivencia Celular/efectos de los fármacos , Femenino , Flavonoides/aislamiento & purificación , Humanos , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus , Hojas de la Planta , Proteínas Represoras/metabolismoRESUMEN
Here we show that human papillomavirus (HPV) E6 and E7 oncoproteins induce hWAPL expression. In addition, small interfering RNA (siRNA) of hWAPL suppressed the growth of tumours derived from SiHa cells in nude mice. Thus, hWAPL may be one of the effective targets of uterine cervical cancer therapy.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas/genética , Papillomaviridae/genética , ARN Interferente Pequeño/uso terapéutico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Animales , Femenino , Humanos , Immunoblotting , Ratones , Proteínas Nucleares , Proteínas Proto-OncogénicasRESUMEN
High-risk human papillomaviruses (HR-HPVs) are one of the most devastating oncogenic viruses worldwide and have been causally linked with the development of human cervical cancer. Several prophylactic and therapeutic clinical HPV vaccine trials are in progress. Although prophylactic vaccines are useful in preventing the incidence of cervical cancer, the elimination of existing HPV infections needs to be addressed, because cervical cancer is the leading female cancer in developing countries. Several different and encouraging strategies have been investigated in a preclinical and clinical setting for the treatment and elimination of existing HPV-induced infection. This review summarizes the therapeutic clinical trials and the different preclinical research strategies that are under investigation whereby HR-HPV E6 and E7 oncogenes are delivered in a nucleic acid form, in viral and bacterial vectors, or as peptide- and protein-based vaccines.
Asunto(s)
Proteínas Oncogénicas Virales/efectos de los fármacos , Papillomaviridae/efectos de los fármacos , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/uso terapéutico , Vacunas de ADN , Humanos , Papillomaviridae/inmunología , Infecciones por Papillomavirus/tratamiento farmacológico , Vacunas ViralesRESUMEN
The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6. This ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. These findings show that RNAi provides a powerful molecular strategy to inactivate intracellular E6 function efficiently. Moreover, they define E6 as a most promising therapeutic target to eliminate HPV-positive tumour cells specifically by RNAi. Thus, by sequence-specific targeting of antiapoptotic genes, siRNAs may be developed into novel therapeutics that can efficiently correct the apoptosis deficiency of cancer cells.
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Proteínas de Unión al ADN , Proteínas Oncogénicas Virales/genética , ARN Interferente Pequeño/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Terapia Genética/métodos , Células HeLa/efectos de los fármacos , Células HeLa/virología , Humanos , Proteínas Oncogénicas Virales/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Transfección , Células Tumorales Cultivadas , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Our previous work with primary bovine fibroblasts demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 and G2/M, in correlation with p53 activation. The expression of bovine papillomavirus type 4 (BPV-4) E7 overcame this arrest and lead to the development of tumorigenic cells lines (Beniston et al., 2001). Given the possible link between papillomavirus infection, bracken fern in the diet and cancer of the upper gastrointestinal (GI) tract in humans, we investigated whether a similar situation would occur in human cells transformed by human papillomavirus type 16 (HPV-16) oncoproteins. Quercetin arrested primary human foreskin keratinocytes in G1. Arrest was linked to an elevation of the cyclin-dependent kinase inhibitor (cdki) p27(Kip1). Expression of the HPV16 E6 and E7 oncoproteins in transformed cells failed to abrogate cell cycle arrest. G1 arrest in the transformed cells was also linked to an increase of p27(Kip1) with a concomitant reduction of cyclin E-associated kinase activity. This elevation of p27(Kip1) was due not only to increased protein half-life, but also to increased mRNA transcription.
Asunto(s)
Proteínas de Ciclo Celular/efectos de los fármacos , Fase G1/efectos de los fármacos , Mutágenos/farmacología , Proteínas Oncogénicas Virales/efectos de los fármacos , Quercetina/farmacología , Proteínas Represoras , Proteínas Supresoras de Tumor/efectos de los fármacos , Línea Celular Transformada , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Humanos , Queratinocitos/efectos de los fármacos , Proteínas E7 de Papillomavirus , Pteridium/metabolismoRESUMEN
Human papillomaviruses (HPV) are causative agents of human cancers including those of the cervix and also of the head and neck; HPV16 is the most commonly found type in these diseases. The viral E2 protein regulates transcription from the viral genome by interacting with DNA-binding sequences in the HPV transcriptional control region; it also regulates replication by interacting with and recruiting the HPV replication factor E1 to the viral origin. Therefore, E2 is essential for the viral life cycle. The E2 protein interacts with several proteins involved in the cellular response to DNA damage including p53, TopBP1, and PARP. We therefore set out to establish whether DNA-damaging agents can regulate E2 activity. Here we show that UVB irradiation downregulates transcriptional activity of both HPV16 and HPV8 E2, while hydroxyurea and etoposide do not. This downregulation of E2 activity is independent of p53 function as it occurs in p53 wild type and null cell types as well as in the presence of functional HPV16 E6 that degrades p53. Using stable cell lines expressing E2 we show that this downregulation of E2 function by UVB is due to a reduction of the E2 protein half-life. The identification of the pathway(s) through which UVB downregulates E2 transcriptional activity and protein levels will present a novel target for the treatment of HPV-related diseases.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Oncogénicas Virales/metabolismo , Activación Transcripcional/efectos de la radiación , Rayos Ultravioleta , Antineoplásicos/farmacología , Daño del ADN , Regulación hacia Abajo/efectos de la radiación , Semivida , Células HeLa , Humanos , Leupeptinas/farmacología , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/efectos de la radiación , Transactivadores/efectos de los fármacos , Transactivadores/metabolismo , Transactivadores/efectos de la radiación , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/efectos de la radiaciónRESUMEN
We have recently shown that staurosporine (ST) can trigger apoptosis of CaSki and HeLa cervical tumor cells from G2/M checkpoint, though the mechanism remains elusive. In this study, we reported that ST induced the inhibition of E6 and E7 viral oncogene and MDM2 expression, while it led to increased levels of p53, which was transiently located to mitochondria. Additionally, the proteins of the p53-regulated genes, p21(WAF1) and Bax, were increased with a similar time, while Bcl-2 and Bcl-X(L) expression was lowered. Upon ST treatment, the cytochrome c was released into the cytosol and, then, activation of caspases-9 and -3 led to Poly(ADP)RibosePolymerase (PARP) cleavage. Finally, characteristic morphological signs confirmed the apoptosis execution. Thus, taken together, all these observations suggest that apoptosis can be reactivated in HPV-positive human carcinoma cells and highlight that ST could be used as a potently chemotherapy agent to enhance the sensitivity of tumor cells to apoptosis.