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The available therapeutic approaches for cervical cancer can seriously affect the fertility potential of patient; thus, there is a pressing requirement for less toxic and targeted therapies. The membrane proteome is a potential source of therapeutic targets; however, despite the significance of membrane proteins in cancer, proteomic analysis has been a challenging task due to their unique biochemical properties. The aim of the present study was to develop an efficient membrane protein enrichment protocol, and to the best of our knowledge, to compare for the first time the expression pattern of membrane proteins of one normal cell line, HCK1T, and three cervical cancer cell lines, C33A, a human papilloma virus (HPV)-negative cell line, and two HPV-positive cell lines, SiHa (HPV16+) and HeLa (HPV18+). The study aimed to identify the proteins that are involved in cervical carcinogenesis and may constitute novel drug targets. Membrane protein isolation, liquid chromatography coupled with tandem mass spectrometry proteomics and bioinformatics analysis were performed in the membrane fraction of the informative cervical cell lines following a novel enrichment protocol. The percentages of membrane and transmembrane proteins in the enrichment protocol were higher compared with those of the corresponding data derived from total cell extract analysis. Differentially expressed proteins were detected by the comparison of the cervical cancer cell lines with the normal cell line. These proteins constitute molecular features of cancer pathology and participate in biological pathways relevant to malignancy, including 'HIPPO signaling', 'PI3K/Akt signaling', 'cell cycle: G2/M DNA damage checkpoint regulation' and 'EIF2 signaling'. These unique membrane protein identifications offer insights on a previously inaccessible region of the cervical cancer proteome, and may represent putative diagnostic and prognostic markers, and eventually therapeutic targets.

Cervical cancer incidence is tightly linked to HPV infection, and particularly virus types 16 and 18 cause the majority of cases presenting with pre-cancerous stages of cervical intraepithelial neoplasia (CIN). Structural and functional information concerning HPV proteins can offer novel insight into the mechanism(s) of cancer progression in the cervical epithelium. Recently, novel structural determinants of the interactions of viral proteins with their targets in keratinocytes have been elucidated. These exciting findings open the way for the development of targeted anti-oncogenic therapies, and may eventually allow the introduction of novel approaches for a rational cervical cancer treatment.

Background: Oncogenic infection by HPV, eventually leads to cervical carcinogenesis, associated by deregulation of specific pathways and protein expression at the intracellular and secretome level. Thus, secretome analysis can elucidate the biological mechanisms contributing to cervical cancer. In the present study we systematically analyzed its constitution in four cervical cell lines employing a highly sensitive proteomic technology coupled with bioinformatics analysis., Materials and Methods: LC/MS-MS proteomics and bioinformatics analysis were performed in the secretome of four informative cervical cell lines SiHa (HPV16 + ), HeLa (HPV18 + ), C33A (HPV - ) and HCK1T (normal)., Results: The proteomic pattern of each cancer cell line compared to HCK1T was identified and a detailed bioinformatics analysis disclosed inhibition of matrix metalloproteases in cancer cell lines. This prediction was further confirmed via zymography for MMP-2 and MMP-9, western blot analysis for ADAM10 and by MRM for TIMP1. The differential expression of important secreted proteins such as CATD, FUCA1 and SOD2 was also confirmed by western blot analysis. MRM-targeted proteomics analysis confirmed the differential expression of CATD, CATB, SOD2, QPCT and NEU1., Conclusion: High resolution proteomics analysis of cervical cancer secretome revealed significantly deregulated biological processes and proteins implicated in cervical carcinogenesis., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Both HPV-positive and -negative cervical cancers are primarily associated with features of cell cycle and cytoskeletal disruption; however, the actual biological processes affected remain elusive. To this end, we systematically characterized the intracellular proteomic profiles of four distinct and informative cervical cell lines., Materials and Methods: Cell extracts from a normal cervical (HCK1T) and three cervical cancer cell lines, one HPV-negative (C33A), and two HPV-positive, SiHa (HPV16+) and HeLa (HPV18+), were analyzed by 2-dimensional electrophoresis and differentially expressed proteins were identified by MALDI-TOF mass spectrometry, while differential expression was confirmed by western blot analysis., Results: In total, 113 proteins were found differentially expressed between the normal and the cervical cancer lines. Bioinformatics analysis revealed the actin cytoskeleton signaling pathway to be significantly affected, while up-regulation of cofilin-1, an actin depolymerizing factor, was documented and further validated by western blotting. Furthermore, two-way comparisons among the four cell lines, revealed a set of 18 informative differentially expressed proteins., Conclusion: These novel identified proteins provide the impetus for further functional studies to dissect the mechanisms operating in the two distinct pathways of cervical carcinogenesis., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.