Conceived and designed the experiments: FDM MP. Performed the experiments: FDD EB AF. Analyzed the data: MES AG CF. Contributed reagents/materials/analysis tools: CB KF DAB. Wrote the paper: MP RC.
The authors have declared that no competing interests exist.
Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not
Human Papillomavirus type 16 (HPV16) is the most prevalent High Risk (HR) type in premalignant and malignant cervical lesions
The present poor knowledge about the neoplastic progression mechanisms has dramatic consequences on the clinical side. As a matter of fact based on the current screening methods, it is not possible to predict the clinical outcome of a single lesion. Thus while just a very minor part of them tend to progress all of them have to be regarded as a potentially progressive. Consequently a large number of patients have to be treated as potentially progressive patients and are therefore submitted to ultimately unnecessary surgical treatments. In the search for molecular markers able to predict the clinical outcome of dysplastic lesions many of viral, host-related and environmental factors have been taken into account and examined. Nevertheless HPV related carcinogenesis remains poorly understood and current screening protocols still wait improvements. Among environmental factors Oxidative Stress (OS), although appearing a good candidate as cancer promoting factor has been comparatively neglected so far. OS is a condition arising when the production of reactive oxygen species (ROS) is not matched by the antioxidant/repairing pathways of the cell. ROS are constantly generated in aerobic cells by the incomplete reduction of molecular O2 to H2O during mitochondrial oxidative phosphorylation, as well as during a number of processes such as inflammation, infections, mechanical and chemical stresses, exposure to UV and to ionising irradiation
The study design and enrolment criteria were approved by the Regina Elena's local Ethical Committee. All participants provided full written informed consent
Fresh or frozen histological samples were cut in small pieces and incubated for 20 min in ice with lysis buffer (10 mM Hepes pH 7.9, 10 mM K2EDTA, 5 mM NaCl, 1% TritonX-100, 10 mM â-mercapto-ethanol, aprotinine 5 mg/L). Lysates were sequentially disrupted by a mechanical mincer (Ultra-Turrax IKA T10) and by a potter device, until a fine, cloudy suspension was obtained. The suspension was then clarified in a JA-21 Beckman Super-centrifuge at 16,000 g for 20 min at +4°C. Protein concentration in the supernatant was determined by the Coomassie Plus Pierce Protein Assay (Pierce Inc., Rockford, IL, USA). Samples were then divided into aliquots and stored at −80°C until use.
DNA was extracted from a small piece of tissue by the QIAamp DNA Mini Kit (QIAGEN Gmbh, Hilden, Germany) used according to the manufacturer's instructions.
For HPV detection the samples were amplified using the MY09/MY11 primer couple
Viral typing was assessed by direct sequencing of amplified products by the BigDye Terminator 1.1 Cycle Sequencing Kit (Sanger method). Sequences were aligned to prototype viral sequence through the BLAST resource at the NCBI(
The viral load of HPV-16 positive samples was determined by a SYBR Green quantitative PCR (qPCR) procedure based on the work of Roberts et al.
PRIMERS | 5′-3′ SEQUENCES | Bp | Tm(C°) | reference |
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Manos et al 1989 |
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Saiki et al 1988 |
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Nagao et al 2002 |
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Nagao et al 2002 |
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Yoshinouchi et al 1999 |
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Names of HPV16 primers include sense of primer extension followed by the position of the 5′ nt on prototype sequence for rapid primer location.
To obtain information about the physical status of viral genome the Rolling Circle Amplification (RCA) was used
Endoplasmic Reticulum protein 57 (ERp-57), glutathione S-transferase (GST), inducible nitric oxide synthase (i-NOS) and thioredoxin reductase 2 (TrxR2) levels were evaluated by Western blot analyses. Sample aliquots (40 µg of protein) were subjected to 12.5% SDS-PAGE and electroblotted (1 h at 100 V) to nitrocellulose membranes (Bio-Rad) using 25 mM Tris, 192 mM glycine and 20% (v/v) methanol. Equal protein loading was confirmed by staining with 0.2% v/v Ponceau S in 7% acetic acid. Blotted membranes were blocked with 3% albumin in T-TBS and challenged with appropriate primary antibodies, namely anti-Erp-57 rabbit polyclonal antibody, anti-GST mouse monoclonal antibody, anti-iNOS rabbit polyclonal antibody (Upstate, Millipore S.p.a, MI, IT) and TrxR2 goat polyclonal antibody (Santa Cruz Biotech. Inc., Santa Cruz, CA,USA) for 1 h at room temperature. Unbound antibodies were removed by washing twice with Tris-buffered saline containing 0.1% Tween 20, for 5 minutes. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Sigma–Aldrich Inc. St. Louis, MO) diluted 1∶5000. Protein bands were visualized with ECL Plus™ (Amersham) according to the manufacturer's protocol. Blots were scanned on a GS880 densitometer (Biorad) and quantified by QuantityOne image software.
Protein oxidation was measured according to Butterfield et al.
For the first-dimension electrophoresis, proteins (200 µg in 200 µL of rehydration buffer) were applied to a ReadyStrip™ IPG strip pH 3–10 (Bio-Rad). The strips were soaked in the sample solution for 1 h to allow uptake of the proteins. The strips were then actively rehydrated in Protean IEF Cell Apparatus (Bio-Rad) for 16 h at 50 V. The isoelectric focusing was performed at 300 V for 2 h linearly; 500 V for 2 h linearly; 1000 V for 2 h linearly, 8000 V for 8 h linearly and 8000 V for 10 h rapidly. All the processes above were carried out at room temperature. The focused IEF strips were stored at −80°C until second dimension electrophoresis was performed.
For second dimension electrophoresis, thawed strips were equilibrated for 10 min in 50 mM Tris-HCl (pH 6.8) containing 6 M urea, 1% (w/v) sodium dodecyl sulfate (SDS), 30% (v/v) glycerol, and 0.5% dithiothreitol, and then re-equilibrated for 15 min in the same buffer containing 4.5% iodacetamide in place of dithiothreitol. 12% Precast criterion gels (Bio-Rad) were used to perform second dimension electrophoresis. Precision Protein™ Standards (Bio-Rad) were run along with the sample at 200 V for 65 min. After electrophoresis, the gels were fixed (7% acetic acid, 10% methanol) and stained with Bio-Safe Coomassie Gel Stain (Bio-Rad).
To identify carbonylated proteins, samples (200 µg proteins) were derivatized as above described, subjected to 2-DE and transferred to nitrocellulose membrane using Criterion Blotter apparatus (Bio-Rad) at 100 V for 1 h. The carbonylated proteins were detected as above reported.
The 20 gels (n = 7 controls, n = 6 dysplasia and n = 7 carcinoma) and 20 nitrocellulose blots were scanned and saved in TIF format using a GS-800 densitometer (Bio-Rad). PDQuest 2D Analysis software (version 7.2.0, Bio-Rad) was used for matching and analysis of visualized protein spots among differential gels and membranes. The anti-DNP immune-reactivity of individual proteins was normalized to protein content evaluated by the intensity of Coomassie blue stained spots. After completion of spot matching, the normalized intensity of each protein spot from individual gels was compared among the groups using statistical analysis. Statistical significance was assessed by a two-tailed Student's
Selected spots were manually excised from gel and submitted to trypsin proteolysis
GAPDH activity was measured by a colorimetric assay kit (ScienCell, Research Laboratories Co, Carlsbad, CA). The method is based on the oxidization of â-NADH to â-NAD in the presence of 3-phosphoglyceric acid (3-PGA), adenosine 5′-triphosphate (ATP) and GAPDH. The GAPDH activity is determined by assaying the rate of NADH oxidation, which is proportional to the reduction in absorbance at 340 nm over time (A340 nm/min). Briefly, 5 µl of each sample or standard is added to each well, in the 96-well plate, containing 145 µl of GAPDH assay mixture, and the A340 nm kinetic was measured. Enzyme activity is calculated as U.A./mg protein.
DNA oxidation was evaluated by the 8-OH-2deoxy Guanosine EIA kit (StressMarq Biosciences Inc, Victoria BC CANADA) used according to the manufacturer's instructions.
All other materials used unless otherwise specified were analytical grade products purchased from the current laboratory suppliers either Sigma–Aldrich (St. Louis, MO, USA) or Bio-Rad (Bio-Rad Laboratories, Milan, Italy).
Two-sided, Student's
During the period from January 2008 to December 2009 a total of 87 patients yielded their consent to participate to the study. Among them 35 had an invasive squamous cell carcinoma (SCC), 1 an adeno-carcinoma, 12 were affected by a cervical dysplastic lesion and 23 were suffering for a uterine fibroleiomyoma. The remaining 15 patients turned out to be affected by other inflammatory or chronic/degenerative pelvic diseases and were excluded from further analyses. Viral typing showed that HPV16 was present in 25/35 patients with invasive SCC, in 6/12 patients with dysplastic lesion and in 7/23 patients with uterine fibroleiomyoma. These latter, for the sole purpose of this work are here considered as control patients. All the HPV16 patients, listed in
ID | Age | DNAviral load | RCA | Cytology | Kolposcopy | Histology | Stage (FIGO) | Hist Grade | LymphNode Met | DistantMet |
Z A | 51 | 3,17×10−3 | ND | Normal | ND | Negative | - | - | - | - |
E L | 44 | 3,00×10−4 | ND | Normal | ND | Negative | - | – | – | - |
V M | 32 | 2,19×10−2 | ND | Normal | ND | Negative | - | - | - | - |
Q M | 50 | 3,50×10−2 | Positive | Normal | ND | Negative | - | - | - | - |
H C | 55 | 1,98×10−3 | ND | Normal | ND | Negative | - | - | - | - |
H S | 51 | 2,30×10−3 | ND | Normal | ND | Negative | - | - | - | - |
B Z | 51 | 3,11×10−3 | ND | Normal | ND | Negative | - | - | - | - |
D H | 48 | 1,52×10−2 | ND | HSIL | ANTZ G1 | CIN-II | - | - | - | - |
E M | 35 | 7,60×10−3 | ND | HSIL | ANTZ G2 | CIN-III | - | - | - | - |
M B | 40 | 3,30×10−3 | ND | HSIL | ANTZ G2 | CIN-III | - | - | - | - |
M T | 31 | 9,2×10−3 | ND | HSIL | ANTZ G2 | CIN-II | - | - | - | - |
L B | 43 | 6,68×10−2 | ND | HSIL | ANTZ G1 | CIN-III | - | - | - | - |
T S | 39 | 4,27×10−3 | ND | HSIL | ANTZ G2 | CIN-III | - | - | - | - |
M R | 55 | 1,1×102 | Positive | HSIL/worse | ANTZ G2 | Invasive SCC | IIA | G3 | No | No |
D A | 48 | 5,60×101 | Positive | HSIL/worse | ANTZ G2 | Invasive SCC | IIB | G3 | No | No |
M S | 64 | 2,00×101 | ND | HSIL/worse | ANTZ G1 | Invasive SCC | IB1 | G2 | No | No |
C G | 44 | 1,81×101 | ND | HSIL/worse | ANTZ G2 | Invasive SCC | IIB | G3 | No | No |
D′A A | 68 | 9,20×10−4 | ND | HSIL/worse | ANTZ G1 | Invasive SCC, with basaloid aspects | IIA | G2 | No | No |
G I | 40 | 5,88×100 | ND | HSIL/worse | ANTZ G2 | Invasive EC | IIB | G3 | No | No |
N B | 59 | 3,00×102 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIB | G3 | No | No |
F R | 52 | 6,23×10−1 | ND | HSIL/worse | ANTZ G2 | Invasive EC | IIB | G3 | Yes | No |
S C | 47 | 2,35×102 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIB | G3 | No | No |
A M | 43 | 1,22×101 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IA1 | G3 | No | No |
P B | 45 | 3,00×100 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIA | G2 | No | No |
C C | 46 | 4,60×102 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIA | G3 | No | No |
A M | 52 | 6,50×102 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIB | G2 | No | No |
P MC | 55 | 5,4×101 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIA | G3 | No | No |
G.R | 73 | 3,60×10−3 | ND | HSIL/worse | ANTZ G2 | Invasive EC | IIA | G2 | Yes | No |
A.LE | 70 | 7,80×100 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IB2 | G3 | No | No |
M P | 58 | 1,25×103 | ND | HSIL/worse | ANTZ G2 | Invasive SCC | IB2 | G2 | No | No |
M M | 75 | 7,30×10−2 | ND | HSIL/worse | ANTZ G1 | Invasive SCC, | IB2 | G2 | No | No |
P E | 53 | 1,7×100 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIB | G3 | No | No |
M M | 56 | 7,43×102 | ND | HSIL/worse | ANTZ G2 | Invasive EC | IB2 | G2 | No | No |
V A | 49 | 4,91×10−2 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIA | G3 | Yes | No |
M GM | 51 | 6,90×101 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIB | G2 | Yes | No |
D′A L | 47 | 1,3×10−1 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IIA | G3 | No | No |
T E | 59 | 8,3×102 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IA2 | G3 | No | No |
M R | 56 | 7,70×100 | ND | HSIL/worse | ANTZ G2 | Invasive SCC, | IB1 | G2 | No | No |
HK-168 | - | 2.8×100 | ND | ND | - | HPV-16 |
- | - | - | - |
SiHa | - | 1.09×100 | ND | ND | - | Invasive SCC cell line | - | - | - | - |
Caski | - | 655×102 | ND | ND | - | Invasive SCC cell line | - | - | - | - |
HaCaT | - | ND | ND | ND | - | Spontaneously Immortalized human keratinocytes cell line | - | - | - | - |
ID: patient's identification code; DNA viral load was calculated as the E6/beta globin ratio and expressed in copy per haploid cellular genome CHCG; HSIL: High grade Squamous Intraepithelial Lesion; ANTZ: Abnormal Transformation Zone; Histological Grade: G1 Well differentiated; G2 Moderately differentiated; G3 Poorly differentiated. Met: metastases. ND: none detected; SCC samples selected for redox-proteomics analysis are boxed.
Viral load and viral genome physical status have been claimed to be relevant determinants in HPV infection outcome and in clinical evaluation of dysplastic and neoplastic lesions. Various methodologies are described for the evaluation of viral load. We worked with the SYBR Green method following the recommendation of Roberts et al.
Viral loads in CTR, DYS and SCC cervical tissues are expressed as E6 copies/β-globin copies/cell.
The physical status of viral genomes in the samples is reported in
Expression levels of selected stress response proteins, including Endoplasmic Reticulum protein 57 (ERp57), Glutathione S-Transferase (GST), inducible Nitric Oxide Synthase (iNOS) and mitochondrial Thioredoxin Reductase (TrxR2) were evaluated in control, dysplastic and neoplastic tissues (
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.
Erp57 is an ER stress marker and our previous work showed that it is a selective target of OS in epithelial cells
GST is a detoxifying enzymes found to be overexpressed in different tumors, though no data are available in cervical cancer. Further, growing studies proposed that GST polymorphism is a candidate risk factor for developing cervical cancer. GST was sharply increased in dysplastic and in neoplastic cells compared with controls (up to 1.8 and 6 fold respectively) (
The TrxR2, participates in mitochondrial redox signaling events and it has been recently regarded as a cancer development
i-NOS, the inducible isoform of Nitric Oxide Synthase, is a well-established marker of nitrosative stress and inflammation. Its specific role in tumor biology is still under debate. iNOS was found to be progressively decreased in dysplastic (65% of control) or neoplastic (25% of control) samples as compared with control tissues (
To assess the extent of total protein oxidation, protein carbonyl levels were evaluated by slot-blot analysis (
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
For the redox proteomics analysis, a set of 7 SCC samples were selected from those reported in
Two representative 2D gels, and the corresponding blots, from control and dysplastic samples are pictured in
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.
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.
Protein | Swiss Proteincode | TheoricalMw/pI | SequenceCoverage % | Score | Foldoxidation |
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Keratin, type II cytoskeletal 6AKeratin, type II cytoskeletal 6BKeratin, type II cytoskeletal 6C | P02538P48668P04259 | 60293/8.0960273/8.0960315/8.09 | 535049 | 327283311 | +9.08 |
Glyceraldehyde-3-phosphate dehydrogenase | P04406 | 36201/8.57 | 46 | 131 | +8.62 |
Cornulin | Q9UBG3 | 53730/5.73 | 48 | 229 | +4.26 |
Retinal Dehydrogenase 1 | P00352 | 55454/6.30 | 27 | 125 | +31.68 |
Actin, cytoplasmic 1Actin, cytoplasmic 2 | Q96HG5P63261 | 42052/5.2942108/5.31 | 7979 | 2727 | +9.06 |
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Glyceraldehyde-3-phosphate dehydrogenase | P04406 | 36201/8.57 | 41 | 112 | −10.57 |
Peptidyl-prolyl cis-trans isomerase A | P62937 | 18229/7.68 | 41 | 79 | −313.34 |
Erp 57 | P30101 | 57146/5.98 | 22 | 96 | −16.55 |
Serpin B3 | Q8IXI3 | 44594/6.35 | 187 | 37 | −5.18 |
Annexin A2 | P07355 | 38808/7.57 | 45 | 168 | −106.75 |
Two proteins were identified in the same spot: the fragments/isoforms A,B & C of cytoskeletal Keratin 6 (with a score of 327, 283 and 311 respectively) and the pyruvate kinase with a score of 66 (around the threshold limit).
Redox proteomics analysis was also performed to compare dysplastic and neoplastic samples. Results indicated that five proteins, namely Serpin B3, Annexin 2 (Anx2), ERp57 and peptidyl-prolyl cis/trans isomerase (Pin1) and GAPDH were less oxidized in neoplastic samples compared with dysplastic ones (
In order to see the effect of oxidative modification on protein function, the enzymatic activity of GAPDH has been measured in dysplastic and neoplastic tissues compared with healthy controls. We found that dysplastic tissue had a lower enzymatic activity compared with controls (normalized to protein expression levels) while a recovery of the activity was evidenced in SCC samples (
CTR | DYS | SCC | |
1±0.1 | 1.6±0.3 | 2.4±0.2 | |
1±0.3 | 9.0±0.4 | 1.2±0.3 | |
45±5.4(100±12%) | 26.8±6.3(59.5±14%) | 52.8±4.9(117.3±11%) |
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.
In order to support data on protein oxidation with results obtained from alternative approaches the extent of DNA oxidation was assessed by the levels of 8-OH-dG. This is an established marker of DNA oxidative damage in response to increased OS and ROS production. As it can be seen in
8-OH-dG | CTR | DYS | SCC |
|
168±14 | 213±18 | 60±8 |
Data are expressed as mean ± SEM.
Aiming to identify new candidate markers able to predict the clinical outcome of lesions it was necessary to compare lesions with highly homogeneous clinical and biological features. Thus considering that viral load and viral genome physical status have been assumed to be most relevant determinants in cervical infection outcome
Remarkable differences were shown in stress response markers among the three groups of lesions. The ERp57 was found to be significantly up-regulated only in neoplastic tissue compared with both dysplastic and controls tissues. The ERp57 is a ER resident member of the protein-disulphide isomerase family, which assists the maturation and transport of unfolded secretory proteins by facilitating disulphide bond formation and rearrangement reactions. ERp57 expression is induced during neoplastic transformation
The TrxR2 is part of an important mechanism for maintaining the reduced intracellular environment. In addition to its possible implication in many aspects of cancer biology
The inducible form of NOS has been commonly associated with malignant diseases, however its role in carcinogenesis and tumor biology is far from being clarified. Our results indicated that compared with the level found in control tissues, iNOS expression was gradually reduced in dysplastic and neoplastic lesions. Though divergent from what observed in most cancer types, this finding is an agreement with the results by Mazibrada
Taken together the above data support the view that highly active detoxifying systems (ERp57; TrxR2; GST) and reduced iNOS might be part of a complex adaptive metabolic profile allowing cell survival in an increasingly oxidant environment.
In order to better understand the role of OS in cervical cancer, we measured the extent of total protein oxidation. We found that protein carbonyls were significantly increased in dysplastic tissues, while levels detected in neoplastic tissues were not significantly different to control ones. This unexpected trend was also paralleled by the extent of oxidative DNA damage. Indeed we found that 8-OH–dG levels were clearly increased respect to both SCC and controls. These findings support the view that dysplastic state is highly vulnerable to oxidative damage, a major factor of genetic instability, providing the conditions for the neoplastic evolution of transformed cells. Conversely established tumours seem to fit well with stress conditions.
Since it has been demonstrated that protein oxidation results in diminished, complete loss of, or a toxic gain in protein function
Cytokeratins (CKs) contribute to cytoskeleton organization and are well known markers of cell differentiation both in normal and neoplastic epithelia
Cornulin is a calcium binding protein member of the fused-gene family
The RDH is an enzyme involved in the synthesis of retinoic acid, a fundamental regulator of cell differentiation, embryogenesis, tissue homeostasis and renewal
Taken together the above data indicate that in dysplastic lesions a selective oxidation of CK6, Actin, Cornulin and RDH, concur to impair their functions contributing to cytoskeleton derangement, suppression of terminal differentiation and reduced control on viral oncogenes activity.
Comparative analysis between SCC and CTR tissues did not reveal any significant increase of carbonylated protein. This result is consistent with total protein oxidation levels. However, further larger studies are needed to clarify such an apparent enigma.
Comparing the redox proteomics pattern of dysplastic and neoplastic tissues a number of proteins exhibited a lower oxidation in neoplastic tissues namely the ERp57; Anx2; Serpin B3; Pin1 and GAPDH.
Interestingly, the increased expression of ERp57 in neoplastic tissues is also associated with its reduced oxidation possibly resulting in a much greater increase of activity than expected based on purely quantitative data. This result underscores the relevance that elevated protein folding/unfolding activity may have for cell survival of cancer cells and suggests that the achievement of the neoplastic phenotype is accompanied by the activation of compensatory mechanisms able to counteract oxidative damage of selected targets and allowing the cell to fit to the hostile environment.
A similar lower level of carbonylation in tumor tissues compared with dysplastic lesions was also showed for Anx2. Recent studies suggest that Anx2 might be linked to carcinogenesis through its implication in the invasion and neovascularisation processes
Serpin B3, also known as Squamous Cell Carcinoma Antigen 1, is a serine protease inhibitor involved in regulation of plasminogen activation, inhibition of inflammation and promotion of epithelial proliferation
Pin1 [named after the acronym: Protein Interacting with NIMA ( = Never In A Mithosis)] is indeed a Peptidyl prolyl cis-trans isomerase that isomerizes phospho-Serine/Threonine-Proline [p(S/T)-P] motifs causing them to twist between two completely distinct conformations. Pin1 is required for cell division, regulates the cell cycle and, once over-expressed, can promote oncogenesis through a number of signaling pathways
GAPDH has long been considered a “simple” glycolitic enzyme and has been widely (and erroneously) used as an internal standard reference for RNA expression. Indeed GAPDH is tightly regulated at both transcriptional and post-translational level
This protection may once again represent a pro-survival mechanism which makes tumors/adapted cell more resistant to stress stimuli and therefore able to proliferate
In order to identify putative molecular marker(s) able to correlate with clinical and biological evolution of cervical dysplastic lesions, we analyzed the expression of stress response proteins and the pattern of oxidative adducts on DNA and proteins in a set of normal, dysplastic and neoplastic cervical tissues infected with HPV-16 mostly in an integrated status.
The up-regulation of stress protein markers indicated that an increased oxidative environment occur both in dysplastic and neoplastic tissues. However, in dysplastic tissues this condition resulted in oxidative modification of DNA and of proteins involved in cell morphogenesis and terminal differentiation such as CK6, actin, cornulin, RDH and GAPDH, providing the conditions for the neoplastic progression. Conversely cancer tissues seem to gain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/pro-survival proteins such as ERp57, Anx2, Serpin B3, Pin1 and GAPDH (
Function of the proteins showing altered oxidation in both DYS and SCC are listed. DYS is characterized by an increased oxidative environment, compared with both control and SCC tissues.
Further studies are needed to better understand the effects of protein oxidation on cell transformation and cancer promotion. The comprehension of these phenomena may also concur to improve current clinical protocols for screening and for prognostic evaluation of cervical lesions.