Oxidative Stress in HPV-Driven Viral Carcinogenesis: Redox Proteomics Analysis of HPV-16 Dysplastic and Neoplastic Tissues

Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not per se sufficient to induce tumour development. Oxidative stress (OS) represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. To gain insight into the role of OS in cervical cancer, HPV-16 positive tissues were collected from patients with invasive squamous cervical carcinoma, from patients with High Grade dysplastic HPV lesions and from patients with no clinical evidence of HPV lesions. After virological characterization, modulation of proteins involved in the redox status regulation was investigated. ERp57 and GST were sharply elevated in dysplastic and neoplastic tissues. TrxR2 peaked in dysplastic samples while iNOS was progressively reduced in dysplastic and neoplastic samples. By redox proteomic approach, five proteins were found to have increased levels of carbonyls in dysplastic samples respect to controls namely: cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH. In carcinoma samples the peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, Annexin 2 and GAPDH were found less oxidized than in dysplastic tissues. HPV16 neoplastic progression seems associated with increased oxidant environment. In dysplastic tissues the oxidative modification of DNA and proteins involved in cell morphogenesis and terminal differentiation may provide the conditions for the neoplastic progression. Conversely cancer tissues seem to attain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/pro-survival proteins

Oxidative Stress in HPV-Driven Viral Carcinogenesis: Redox Proteomics Analysis of HPV-16 Dysplastic and Neoplastic Tissues

Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not per se sufficient to induce tumour development. Oxidative stress (OS) represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. To gain insight into the role of OS in cervical cancer, HPV-16 positive tissues were collected from patients with invasive squamous cervical carcinoma, from patients with High Grade dysplastic HPV lesions and from patients with no clinical evidence of HPV lesions. After virological characterization, modulation of proteins involved in the redox status regulation was investigated. ERp57 and GST were sharply elevated in dysplastic and neoplastic tissues. TrxR2 peaked in dysplastic samples while iNOS was progressively reduced in dysplastic and neoplastic samples. By redox proteomic approach, five proteins were found to have increased levels of carbonyls in dysplastic samples respect to controls namely: cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH. In carcinoma samples the peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, Annexin 2 and GAPDH were found less oxidized than in dysplastic tissues. HPV16 neoplastic progression seems associated with increased oxidant environment. In dysplastic tissues the oxidative modification of DNA and proteins involved in cell morphogenesis and terminal differentiation may provide the conditions for the neoplastic progression. Conversely cancer tissues seem to attain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/pro-survival proteins

Genital Human Papillomavirus infection and genotype prevalence among Albanian women. A cross sectional study.

International audience“High Risk” HPV types have different geographical distribution and evidence suggests their respective prevalence may vary in different areas and regions. Accurate description of High Risk HPV circulation is a key feature for the rational design of prevention and screening campaigns. A cross-sectional, virological study was conducted on adult Albanian women living either in the Tirana area or in the Duress prefecture. Clinical and gynaecological evaluations were done according to current standard criteria. HPV detection and typing were carried out by a combined MY09/MY11 and GP5+/GP6+ PCR followed by direct sequencing of generated amplicons. Virological data could be obtained from 402 out of 452 patients enrolled between January 2004 and December 2007. Sixtyone patients (15.1% of the cohort) were found to be infected with a genital HPV. As expected viral prevalence was higher among women younger than 30years of age (25.2%) in comparison to those aged 30 or older (13.6%). HPV 16 was found to be the most frequent type (41% of cases), followed by HPV 53, (roughly 7.2%) HPV 31 (5.8%) and HPV 18 (4.3%). HPV 81 and HPV 84, were the most prevalent low risk types detected with prevalences of 11.6 and 5.8 %. No difference was noted in any type specific prevalence between young and mature women. The circulation of HPV types is far more complex than assumed generally. The detailed knowledge of HPV type circulating patterns in specific local geographical areas is essential for appropriate implementation of screening, prevention and surveillance campaigns

Oxidative Stress in HPV-Driven Viral Carcinogenesis: Redox Proteomics Analysis of HPV-16 Dysplastic and Neoplastic Tissues

Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not per se sufficient to induce tumour development. Oxidative stress (OS) represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. To gain insight into the role of OS in cervical cancer, HPV-16 positive tissues were collected from patients with invasive squamous cervical carcinoma, from patients with High Grade dysplastic HPV lesions and from patients with no clinical evidence of HPV lesions. After virological characterization, modulation of proteins involved in the redox status regulation was investigated. ERp57 and GST were sharply elevated in dysplastic and neoplastic tissues. TrxR2 peaked in dysplastic samples while iNOS was progressively reduced in dysplastic and neoplastic samples. By redox proteomic approach, five proteins were found to have increased levels of carbonyls in dysplastic samples respect to controls namely: cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH. In carcinoma samples the peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, Annexin 2 and GAPDH were found less oxidized than in dysplastic tissues. HPV16 neoplastic progression seems associated with increased oxidant environment. In dysplastic tissues the oxidative modification of DNA and proteins involved in cell morphogenesis and terminal differentiation may provide the conditions for the neoplastic progression. Conversely cancer tissues seem to attain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/prosurvival proteins

List of oxidized proteins successfully identified by mass spectrometry along with the peptide hits, sequence coverage, Mw and pI values and the increase/decrease of specific carbonyl levels, indexed as fold oxidation.

<p>List of oxidized proteins successfully identified by mass spectrometry along with the peptide hits, sequence coverage, Mw and pI values and the increase/decrease of specific carbonyl levels, indexed as fold oxidation.</p

Oxidized protein detection by redox proteomics (DYS vs. SCC).

<p>Top: 2D gel maps of DYS (right) and SCC (left) cervical tissues. Protein (300 µg) were separated in first dimension (pH 3–10 linear IPG); second dimension was performed on slab gel (12% gradient SDS-PAGE). Protein detection was achieved using Biosafe Coomassie staining. Bottom: 2D carbonyl immunoblots of DYS (right) and SCC (left) cervical tissues. The spots showing significant increased carbonyl levels are labeled. Relative change in carbonyl immune-reactivity, after normalization of the immunostaining intensities to the protein content, was significant for five spots. The identified proteins are listed in table III.</p

Expression levels, carbonyl levels and enzyme activity of GAPDH in controls (CTR), dysplastic (DYS) and carcinoma tissues (SCC) are reported.

<p>Levels are expressed as fold increase/decrease vs CTR. Enzyme activity is calculated as A₃₄₀/mg protein and also as % vs CTR. Data are expressed as mean ± SEM.</p

Expression levels of stress markers (ERp57, GST, TRX-R2 and iNOS).

<p>Protein expression levels in CTR, DYS and SCC cervical tissues were measured by Western blot analysis using specific antibodies for ERp57 (A), GST (B), TRX-R2 (C) and iNOS (D). Immunoblots were scanned by densitometry and all values were normalized to β-actin levels. Densitometric values shown are given as percentage of the control group, set as 100%. Data are expressed as mean ± SEM. <i>p</i><0.05 versus control (Student's <i>t</i>-test).</p

Total protein oxidation.

<p>Top: Quantification of levels of protein carbonyls in CTR, DYS and SCC cervical tissues. Samples were probed with anti-DNP protein adducts polyclonal antibody as described in <i>Material and Methods</i>. Densitometric values shown are given as percentage of the control group, set as 100%. Data are expressed as mean ± SEM. <i>p</i><0.05 versus control (Student's <i>t</i>-test). Bottom: Protein carbonyl slot- blots from CTR, DYS and SCC samples.</p

Oxidized protein detection by redox proteomics (DYS vs. CTR).

<p>Top: 2D maps of CTR (left) and DYS (right) cervical tissues. Proteins (300 µg) were separated in first dimension (pH 3–10 linear IPG); second dimension was performed on slab gel (12% gradient SDS-PAGE). Protein detection was achieved using Biosafe Coomassie staining. Bottom: 2D carbonyl immunoblots of CTR (left) and DYS (right) cervical tissues. The spots showing significant increased carbonyl levels are labeled. Relative change in carbonyl immune-reactivity, after normalization of the immunostaining intensities to the protein content, was significant for five spots. The identified proteins are listed in table III.</p