Elucidating Molecular Interactions of Ten Natural Compounds Targeting E6 HPV High Risk Oncoproteins Using Microsecond Molecular Dynamics Simulations.

BACKGROUND: Cervical cancer is a major public health issue worldwide, occurring in the vast majority of cases (85%) in low-income countries. Human papillomavirus (HPV) mainly infects the mucosal epithelium, and a small portion causes over 600,000 cases every year worldwide at various anatomical spots, mainly leading to anogenital and head and neck. INTRODUCTION: The E6 oncoprotein encoded by cancer-associated alpha HPV can transform epithelial cells into tumorigenic tissue. Therapy for this infection and blocking of the HPV E6 oncoprotein could be provided with cost-effective and abundant natural products which are an exponentially growing topic in the literature. Finding an active natural compound that readily blocks HPV E6 oncoprotein which could be available for developing countries without expensive extraction processes or costly synthetic pathways is of major interest. METHODS: Molecular dynamics simulation was performed using the most up-to-date AMBER protein force field ff14SB and a GPU enabled high performance computing cluster. RESULTS: In this research, we present a study of the binding properties between 10 selected natural compounds that are readily available with two variants of the E6 oncoprotein types (HPV-16 and HPV-18) using 10+ microsecond molecular dynamics simulations. CONCLUSION: Our results suggest that crocetin, ergosterol peroxide and κ-carrageenan natural products bind strongly to both HPV-16 and HPV-18 and could potentially serve as a scaffolding for further drug development.

Mesenchymal Stromal Cells Derived from Normal Cervix and Cervical Cancer Tumors Increase CD73 Expression in Cervical Cancer Cells Through TGF-ß1 Production.

Mesenchymal stromal cells (MSCs) in the tumor microenvironment (TME) participate together with tumor cells to suppress antitumor effector cells through the production of immunosuppressive factors, such as transforming growth factor-beta 1 (TGF-ß1). Furthermore, TGF-ß1 can induce 5'-nucleotidase (CD73) expression in various cell types; this functional activity is associated with the production of adenosine (Ado), which is an immunosuppressive nucleoside. In this study, we provide evidence that coculture of MSCs derived from cervical tumors (CeCa-MSC) with CeCa tumor cells increases CD73 expression in tumor cells and the capacity of these cells to generate Ado in a MSC ratio-dependent manner. Interestingly, the increase in CD73 in the CeCa cell membrane corresponded to an increase in the TGF-ß1 expression level in the tumor cells and the TGF-ß1 content in the supernatants of the CeCa/CeCa-MSC cocultures. The addition of anti-hTGF-ß neutralizing antibodies strongly reversed CD73 expression in the tumor cells. This phenomenon was not exclusive to CeCa-MSCs; coculture of MSCs derived from the normal cervix with CeCa cells produced similar results. These results suggest that the interaction of MSCs with CeCa tumor cells in the TME may condition higher TGF-ß1 production to maintain an immunosuppressive status not only through the activity of this cytokine per se but also through its ability to induce CD73 expression in tumor cells and generate an immunosuppressive microenvironment rich in Ado.

High-risk human papillomavirus (HPV) DNA sequences in metaplastic breast carcinomas of Mexican women.

BACKGROUND: Metaplastic carcinoma, an uncommon subtype of breast cancer, is part of the spectrum of basal-like, triple receptor-negative breast carcinomas. The present study examined 20 surgical specimens of metaplastic breast carcinomas, for the presence of high-risk Human papillomavirus (HPV), which is suspected to be a potential carcinogenic agent for breast carcinoma. METHODS: Mastectomy specimens from patients harboring metaplastic breast carcinoma, as defined by the World Health Organization (WHO), and who attended the Instituto Nacional de Cancerologia in Mexico City, were retrieved from the files of the Department of Pathology accumulated during a 16-year period (1995-2008). Demographic and clinical information was obtained from patients' medical records. DNA was extracted from formalin-fixed, paraffin-embedded tumors and HPV type-specific amplification was performed by means of Polymerase chain reaction (PCR). Quantitative Real-time (RT) PCR was conducted in HPV positive cases. Statistically, the association of continuous or categorical variables with HPV status was tested by the Student t, the Chi square, or Fisher's exact tests, as appropriate. RESULTS: High-risk HPV DNA was detected in eight (40%) of 20 metaplastic breast carcinomas: seven (87.5%) HPV-16 and one (12.5%) HPV-18. Mean age of patients with HPV-positive cases was 49 years (range 24-72 years), the same as for HPV-negative cases (range, 30-73 years). There were not striking differences between HPV + and HPV- metaplastic carcinomas regarding clinical findings. Nearly all cases were negative for estrogen, progesterone and Human epidermal growth factor receptor 2 (HER2), but positive for Epidermal growth factor receptor (EGFR). CONCLUSIONS: High-risk HPV has been strongly associated with conventional breast carcinomas, although the subtle mechanism of neoplastic transformation is poorly understood. In Mexican patients, the prevalence of HPV infection among metaplastic breast carcinomas is higher than in non-metaplastic ones, as so the HPV viral loads; notwithstanding, HPV viral loads show wide variation and remain even lower than cervical and other non-cervical carcinomas, making it difficult to assume that HPV could play a key role in breast carcinogenesis. Further studies are warranted to elucidate the meaning of the presence of high-risk HPVDNA in breast carcinomas.

Regulation of p14ARF expression by HPV-18 E6 variants.

A common causative agent for uterine cervical cancer is the human papillomavirus type 18 (HPV-18) which has three phylogenic variants: Asian-Amerindian, European, and African. Each variant shows significant molecular differences in the E6 gene. E6 oncoprotein is a negative regulator of tumor suppressor protein p53, hence, this oncoprotein indirectly regulates the expression of tumor-suppressor p14(ARF) . p14(ARF) and p16(INK4A) genes are overexpressed in--and have been proposed as markers for--HPV-related cervical cancer. In order to dissect the role of E6 on the regulation of p14(ARF) expression, separating it from that of other intervening factors, transfection of E6 variants to MCF-7 cells was performed, assessing cDNA transcript levels by RT-PCR, whereas p14(ARF) and p53 expression were evaluated by immunocytochemistry and Western blot. E6 transfected cells differentially expressed transcripts of two molecular forms: E6 and E6*. The ratio of these two forms varied with the transfected E6 variant. With the Asian-Amerindian variant, the ratio was E6 > E6*, whereas with the European and the African the ratio was E6* > E6. As expected with the E6* construct, E6* transcripts were solely observed. In addition, when E6 > E6* and p53 expression was low, p14(ARF) was high and when E6* > E6 and p53 expression was high, p14(ARF) was low. In conclusion, each E6 variant distinctively affects p53 levels and consequently p14(ARF) expression, finding that could be related with the differences in oncogenic effect of infection with the diverse high-risk HPV variants.

Gene expression profiles induced by E6 from non-European HPV18 variants reveals a differential activation on cellular processes driving to carcinogenesis.

Cervical cancer in developed countries remains as a major concern on public health policies due to incidence and mortality rates. Persistent infection with high risk human papillomavirus is a necessary etiological agent in the progression to invasive cervical carcinoma. A proposed hypothesis is the association between more aggressive HPV variants and the risk to develop cervical cancer. In order to have a global perspective in terms of cellular transcripts and molecular pathways affected by HPV18 E6 intratype variants; we conducted a genome wide analysis of gene expression. Our results show that E6 derived from non-European variants are able to up-regulate cellular transcripts associated to the hallmarks of cancer; such as cell cycle, migration, Wnt pathway and mTor signaling. Moreover, we were able to show that HPV18 E6 from African variant had a major effect on cellular processes such as cell cycle and migration as confirmed by functional studies.

[RNA interference (RNAi) and its therapeutic potential in cancer]. / RNA de interferencia y su potencial terapéutico en cáncer.

Small RNAs belong to a newly discovered strain of molecules. These molecules are composed of double strand RNA comprised by just about 19-31 nucleotides. They have two main characteristics that make them unique. Firstly, they are noncoding for proteins and second they interfere post-transcriptional with mRNA. This interfering action is the distinguishing hallmark, therefore known as interfering RNA or RNAi. There are three main subclasses of which micro-RNA and siRNA are the most widely studied. Interference RNAs participate in a myriad of cellular functions mainly through modulation of genetic expression. Due to these capabilities it has been used as therapeutic weapon in a number of diseases including cancer. It is known that both miRNA and siRNA participate in carcinogenesis, either inhibiting suppressor genes, or stimulating oncogenes. It has been demonstrated that manipulating small interfering RNAs in cell lines and animal models, the malignant and metastatic phenotype can be reversed. Up to now a few clinical trials using RNAi as a therapeutic agent have demonstrated some success and feasibility. It is forseeable that in the near future cancer treatment with small RNAs will be widely applicable, once the many constrains for its systemic application are surpassed.

Nuclear co-expression of p14ARF and p16INK4A in uterine cervical cancer-derived cell lines containing HPV.

The Papanicolaou test (Pap) has been responsible for a significant reduction of cervical cancer-related morbimortality. In order to increase its sensitivity and specificity new markers have been studied and incorporated to cytological and histological methods for diagnosis for cervical cancer, such as p16INK4A that has been considered the immunocytochemical marker of choice for detection of HPV related cancers. We considered that p14ARF could be a complementary marker in order to improve the accuracy of cytological diagnosis because its genetic proximity to p16INK4A. We performed a systematic analysis of several putative cervical cancer markers in order to evaluate their performance in the detection of malignancy, in comparison with p16INK4A and p14ARF, using immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses. Most markers were non-specific and could not discriminate HPV infected cancer cell lines from other non HPV malignant. In contrast, nuclear co-expression of p16INK4A and p14ARF was observed only in HPV-transformed cancer cell lines. Notably, in C-33A cervical cancer cells (HPV negative), p14ARF was present in the nucleoli, but p16INK4A was conspicuously absent from the nuclei of these cells. We conclude that both markers; p16INK4A and p14ARF are complementary and should be evaluated jointly in order to improve the accuracy of cytological diagnosis of cervical cancer.

Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid.

BACKGROUND: DNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells. METHODS: Cell lines C33A (HPV-), CaSki (HPV-16+) and MS751 (HPV-18+) were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 microM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid. RESULTS: Valproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma. CONCLUSION: These results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines is used.

[Toward cervical cancer prevention: strategies employed in the development of HPV vaccines]. / Hacia la prevención del cáncer cérvico-uterino: estrategias empleadas en el desarrollo de vacunas contra el VPH.

An HPV persistent infection is doubtless the main factor involved in cervical cancer development. It is clear that the majority of HPV infections are self-limited and not all high-risk HPV infections are destined to progress to a higher grade lesion. Due to viral mechanisms that evade the immune system, in some cases the immune response against HPV is not as effective, allowing the establishment of persistent infections. The promise of a vaccine that can avoid HPV infections and therefore decrease cervical cancer incidence, has been motive of great interest and enthusiasm on the search of different strategies for obtaining an effective vaccine. At present, several prophylactic vaccines have been developed based in virus like particles (VLPs) produced with L1 viral proteins. Results of these vaccines applied to women between 16 and 23 years old show high tolerability and immunogenicity with higher antibody titers than those seen in an HPV natural infection. Even these vaccines can not wholly prevent infections caused by HPV types included in the vaccine design; its efficacy has been demonstrated for their ability to eliminate HPV persistent infections and to prevent the development of cervical intraepithelial neoplasia. These vaccines are currently in phase III of clinical trials, whose results will determine its impact in the general population. Therapeutic vaccines are focused in the elimination of established cervical lesions; nevertheless their efficacy has not been proved for clinical use.

Differential splicing of E6 within human papillomavirus type 18 variants and functional consequences.

Persistent infections of the uterine cervix with 'high-risk' human papillomavirus (HPV) are now recognized as necessary for the development of cervical cancer. Among them, HPV types 16 and 18 exhibit numerous variants associated with different risks for cervical cancer development. In this study, the questions of whether different HPV type 18 variants exhibit changes in early gene transcription and the molecular mechanisms underlying these differences were investigated. It was shown that, indeed, type 18 variants exhibited singular differences in E6 transcripts in vivo. Higher levels of the E6*I transcript were detected regularly in clones harbouring the African variant, as opposed to low levels of this transcript detected in clones containing the reference clone (Asian-Amerindian), where significantly higher levels of full-length E6 transcript were usually observed. As a direct consequence, higher levels of p53 protein were found in the presence of African E6, as opposed to the low levels of p53 observed with the Asian-Amerindian E6. These variations in consequence affected the levels of cellular proteins regulated by p53, such as Bax. Similar changes in the relative levels of E6 transcripts were observed when tumours containing type 18 E6 variants were analysed. The different ability of cells containing variant E6 genes to form tumours in nude mice was suggested by the fact that tumour volumes were considerably higher when cells expressed the Asian-Amerindian E6. Mutagenesis analysis of the reference clone showed that a C491A change reverts the phenotype. These results suggest that different splicing patterns of E6 within HPV type 18 variants may possibly have biological implications in viral tumorigenesis.

Verruciform xanthoma of the esophagus.

Verruciform xanthoma is a distinctive lesion of oral mucosa and genital skin. It can be solitary or multifocal, as well as sporadic or associated with inflammatory, autoimmune, immunodeficient, metabolic, neoplastic, or congenital diseases. To our knowledge, it has not yet been described in the esophagus. The case of a 61-year-old man suffering from primary non-Hodgkin lymphoma of the testis is presented. Two years after initial diagnosis, mediastinal adenopathies were disclosed. Fractioned radiotherapy was administered; 3 years later, verruciform xanthoma of middle third of the esophagus was endoscopically resected. Histologically, the lesion showed acanthotic squamous mucosa infiltrated by neutrophils. Papillae were packed with foam cells that were positive for CD68 and vimentin antibodies. Verruciform xanthoma is a condition observed exclusively in squamous epithelia. From our viewpoint, physical agents play a preponderant role in the etiology, although viral agents may occasionally be involved in the development of this enigmatic lesion.

Circulating nucleosomes and response to chemotherapy: an in vitro, in vivo and clinical study on cervical cancer patients.

It is known that cell-free DNA circulates in plasma/serum of patients with cancer and that part of this DNA circulates as nucleosomes that can be quantified by ELISA. We analyzed the effect of tumor and chemotherapy upon the levels of nucleosomes in vitro, in vivo and in cervical cancer patients. The levels of nucleosomes pre- and post-treatment were correlated with response in 11 patients receiving chemotherapy. Nucleosomes were determined in nude mice treated with or without cisplatin and carrying tumors generated with HeLa cells, and in the cell lysate and supernatant of HeLa cells exposed to cisplatin in culture. In addition, nucleosomes were determined at different time points in patients and in rats receiving chemotherapy. Nucleosomes were higher in patients that controls (1,760 vs. 601, p = 0.0001). After 24 hr of treatment with oxaliplatin and gemcitabine, the levels decreased in 6 patients of whom 5 had response. Nucleosome levels differed between mice xenografted and not xenografted (765 vs. 378, p = 0.001) and between xenografted treated with or without cisplatin (650 vs. 765, p = 0.010), but not in tumor-free animals treated and untreated with cisplatin (378 vs. 379, p = 0.99). In vitro, nucleosomes reached at peak 8 hr in cell lysates to decrease thereafter, whereas in supernatant, levels continued to increase up to 24 hr. Serial determination of nucleosomes in patients showed a rise within 6-12 hr and then a reduction to below the basal at 24 hr. In rats, nucleosomes had no major changes in those receiving oxaliplatin or the triple combination of cisplatin, gemcitabine and paclitaxel as compared to untreated controls. An overdose of this triple combination produced a transient elevation of almost 1,000 AU over the basal. Our results demonstrate that most of circulating nucleosomes originate from the tumor and that chemotherapy produces an early rise most likely due to tumor apoptosis and that nucleosomes are rapidly cleared from circulation. On the contrary, chemotherapy within the therapeutic range of doses has no effect on nucleosome levels in healthy mice and rats. This data suggests that the determination of circulating nucleosomes pre- and post-treatment could be a useful test to predict response to chemotherapy in cancer patients.

Sondas de DNA para el diagnóstico de tuberculosis: ¿utopía o realidad? / DNA probes for the diagnosis of tuberculosis: utopia or reality?

La tuberculosis sigue siendo un serio problema de salud pública. Tan sólo durante 1989/90 la taza de infección en el Continente Americano (exceptuando los Estados los Estados Unidos t Canadá) fue de 117 millones de personas. Esta traduce la dificultad de mantener un control efectivo sobre la población potencialmente infectada, debido entre otras razones, a la falta de técnica de diagnóstico que permitan una detección rápida y sensible del bacilo. Las técnicas comúnmente utilizadas implican el análisis microscópico de muestras de expectoración y/o el cultivo in vitro de los bacilos. A pesar de que ambas técnicas presentan una sensibilidad aceptable, requieren mucho tiempo para la obtención de los resultados. Es por esta razón que muchos investigadores se han abocado a la tarea de deseñar nuevas estrategias que permiten una detección rápida y sensible de este patógeno. En este contexto la utilización de sondas de este patógeno. En este contexto la utilización de sondas de DNA y la amplificación por PCR ha abierto nuevas esperanzas en la obtención de una técnica rápida y sensible, que puede ser utilizada como ayuda en el diagnóstico de la tuberculosis. Sin embargo, a pesar de las múltiples ventajas que conlleva su aplicación, en algunas ocaciones cabe ponderar su utulización como técnica diagnóstica.