The development and clinical applications of proteomics: an Indian perspective.

INTRODUCTION: Proteomic research has been extensively used to identify potential biomarkers or targets for various diseases. Advances in mass spectrometry along with data analytics have led proteomics to become a powerful tool for exploring the critical molecular players associated with diseases, thereby, playing a significant role in the development of proteomic applications for the clinic. AREAS COVERED: This review presents recent advances in the development and clinical applications of proteomics in India toward understanding various diseases including cancer, metabolic diseases, and reproductive diseases. Keywords combined with 'clinical proteomics in India' 'proteomic research in India' and 'mass spectrometry' were used to search PubMed. EXPERT OPINION: The past decade has seen a significant increase in research in clinical proteomics in India. This approach has resulted in the development of proteomics-based marker technologies for disease management in the country. The majority of these investigations are still in the discovery phase and efforts have to be made to address the intended clinical use so that the identified potential biomarkers reach the clinic. To move toward this necessity, there is a pressing need to establish some key infrastructure requirements and meaningful collaborations between the clinicians and scientists which will enable more effective solutions to address health issues specific to India.

Meeting Report: "Proteomics from Discovery to Function:" 6th Annual Meeting of Proteomics Society, India and International Conference-A Milestone for the Indian Proteomics Community.

Proteomics is at the epicenter of post-genomics biotechnologies that are currently driving the next generation system science. Moreover, proteomics is a truly global science. The 6(th) Annual Meeting of Proteomics Society, India (PSI) and International Conference on "Proteomics from Discovery to Function" held from December 7-9, 2014, was a transformative endeavor for global proteomics, bringing together the luminaries in the field of proteomics for the very first time in India. This meeting report presents the lessons learned and the highlights of this international scientific conference that was comprised of nine thematic sessions, pre- and post-conference workshops, and an opportunity to cultivate enduring collaborations for proteomics science to benefit both India and global society. The conference had an unforgettable impression on the participants: for the first time, India hosted past and present President and Council members from the Human Proteome Organization (HUPO), along with eminent scientists and young scholars from India and abroad in the field of proteomics at such a large scale, a major highlight of this international event. In all, the PSI 2014 was a milestone conference that has firmly poised the Indian life sciences community as a leading contributor to post-genomics life sciences, thus cultivating crucial trans-generational capacity and inspiration by recognizing the emerging scholars and omics systems scientists who can think and conduct science from cell to society.

Proteomics research in India: an update.

After a successful completion of the Human Genome Project, deciphering the mystery surrounding the human proteome posed a major challenge. Despite not being largely involved in the Human Genome Project, the Indian scientific community contributed towards proteomic research along with the global community. Currently, more than 76 research/academic institutes and nearly 145 research labs are involved in core proteomic research across India. The Indian researchers have been major contributors in drafting the "human proteome map" along with international efforts. In addition to this, virtual proteomics labs, proteomics courses and remote triggered proteomics labs have helped to overcome the limitations of proteomics education posed due to expensive lab infrastructure. The establishment of Proteomics Society, India (PSI) has created a platform for the Indian proteomic researchers to share ideas, research collaborations and conduct annual conferences and workshops. Indian proteomic research is really moving forward with the global proteomics community in a quest to solve the mysteries of proteomics. A draft map of the human proteome enhances the enthusiasm among intellectuals to promote proteomic research in India to the world.This article is part of a Special Issue entitled: Proteomics in India.

A comparative protein profile of mammalian erythrocyte membranes identified by mass spectrometry.

A comparative analysis of erythrocyte membrane proteins of economically important animals, goat (Capra aegagrus hircus), buffalo (Bubalus bubalis), pig (Sus scrofa), cow (Bos tauras), and human (Homo sapiens) was performed. Solubilized erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE), visualized by staining the gels with Commassie Brilliant Blue (CBB), and identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Emerging results show that all major erythrocyte membrane proteins present in human are also seen in all the animals except for band 4.5 which could not be identified. Band 3 is seen as more intense and compact, band 4.1 appears as a doublet in all the animal erythrocyte membranes, band 4.2 exhibits a slightly higher molecular weight (Mr) in buffalo, and cow and band 4.9 has a higher Mr in all the animals relative to the human protein. In addition, there are two new bands in the goat membrane, band G1, identified as HSP 90α, and band G2 identified as HSP 70. A new band C2 identified as HSP 70 is also seen in cow membranes. Peroxiredoxin II is of lower intensity and/or higher Mr in the animals. The difference in size of the proteins possibly indicates the variations in the composition of the amino acids. The difference in intensity of the proteins among these mammalians highlights the presence of less or more number of copies of that protein per cell. This data complement the earlier observations of differences in the sialoglycoprotein profile and effect of proteases and neuraminidase on agglutination among the mammalian erythrocytes. This study provides a platform to understand the molecular architecture of the individual erythrocytes, and in turn the dependent disorders, their phylogenetic relationship and also generates a database of erythrocyte membrane proteins of mammals. The animals selected for this study are of economic importance as they provide milk for the dairy industry and raw material for leather industry and are routinely sacrificed to obtain non vegetarian food worldwide.

Proteomic profile of keratins in cancer of the gingivo buccal complex: consolidating insights for clinical applications.

Keratins play a major role in several cellular functions. Each tissue type expresses a specific set of keratins. The immense potential of keratins as diagnostic and prognostic markers for different cancers is emerging. Oral cancer is the fifteenth most common cancer worldwide. However, comprehensive information on the profile of keratins in the oral cavity is not available. Several independent reports have identified keratins using antibody based techniques which have pitfalls due to the cross reactivity of the antibodies to this set of very homologous proteins. A few recent proteomic studies have reported the identification of keratins in head and neck cancer. Majority of the studies have used tissues from the head and neck region without specifying subsites. This study reports the analysis of enriched preparations of keratins from cancer of the gingivo buccal complex (GBC) using MS, 2DE, WB, silver staining of 2DE gels and IHC. Our study reveals the absence of K4 and K13 and presence of K14, K16, and K17, in cancers of the GBC and combination of these expression patterns in the cut margins. This report also shows that K13 is glycosylated. This well characterized profile of keratins may have potential to be used in clinics. BIOLOGICAL SIGNIFICANCE: In recent years the immense potential of keratins as diagnostic and prognostic markers for different cancers is emerging. However, comprehensive information on the profile of keratins in the oral cavity is not available. Several independent reports have identified keratins using only antibody based techniques which have pitfalls due to the cross reactivity of the antibodies to this set of very homologous proteins. This study reports the analysis of enriched preparations of keratins from a subsite of the oral cavity, the gingivo buccal complex (GBC) using mass spectrometry, 2DE, western blotting, silver staining of 2DE gels and IHC. The proteomic analysis shows the absence of K4 and K13 and presence of K14, K16, and K17 in cancers of the GBC and combination of these expression patterns in the cut margins. This well characterized profile of keratins from the gingivo buccal complex provides defined markers which may have potential to be used in the clinics.

Identification of human erythrocyte cytosolic proteins associated with plasma membrane during thermal stress.

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis-trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis-trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.

Prognostic utility of autoantibodies to α-enolase and Hsp70 for cancer of the gingivo-buccal complex using immunoproteomics.

PURPOSE: Studies from our laboratory have reported 14 tumor antigens that elicit an autoantibody response in patients with cancer of the gingivobuccal complex (GBC) In this study, utility of the autoantibody response has been evaluated for prognosis of cancer of the GBC. EXPERIMENTAL DESIGN: Autoantibody response was evaluated using immunoproteomics and the prognostic significance was assessed by Kaplan-Meier survival and multivariate analysis. RESULTS: Autoantibody response against α-enolase isoforms a, b, and c and Hsp70 was detected in 27, 53, 64, and 26% of the 78 patients, respectively. Patients positive for autoantibody response to α-ENO and Hsp70 individually and in combination, showed significantly reduced disease-free survival (DFS) compared to those who do not show autoantibody response to either of them. Further the patients, who exhibit autoantibody response to α-ENO and Hsp70 in combination with nodal involvement and/or differentiation status, have significantly lowered DFS. The relative risk of recurrence is 3.41 for patients who exhibit autoantibody response to both the antigens. CONCLUSIONS AND CLINICAL RELEVANCE: Autoantibody response against α-ENO and Hsp70 provides an additional parameter and may be utilized along with nodal involvement and differentiation status for better prognosis of cancer of GBC.

Design and synthesis of (S)- and (R)-enantiomers of [4-(2-hydroxy-1-phenylethylimino)pent-2-ol]dimethyltin(iv) and 2,2-dimethyl-4-phenyl-1,3,2-oxazastannolidine: in vitro antitumor activity against human tumor cell lines and in vivo assay of (S)-enantiomers.

New dimethyltin derived antitumor drug candidates (S)- and (R)-[4-(2-hydroxy-1-phenylethylimino)pent-2-ol]dimethyltin(iv), 1 and (S)- and (R)-[2,2-dimethyl-4-phenyl-1,3,2-oxazastannolidine], 2 derived from (R)- and (S)-enantiomers of [4-(2-hydroxy-1-phenylethylimino)pent-2-ol] and 2-amino-2-phenylethanol, respectively, were synthesized and thoroughly characterized. Preliminary complex-DNA interaction studies employing various optical methods revealed that the (S)-enantiomer displayed a higher propensity towards the drug target DNA double helix. This was quantified by K(b) and K(sv) values of ligands L and L' and (S)-/(R)-1 and (S)-/(R)-2 complexes, which demonstrated a multifold increase in the case of the (S)-enantiomers in comparison to their (R)-enantiomeric forms. This clearly demonstrates the chiral preference of the (S)-enantiomer over the (R)-enantiomer, and its potency to act as a chemotherapeutic agent. Therefore, the in vitro antitumor activity of the (S)-enantiomer of 1 and 2 was evaluated by the sulforhodamine-B (SRB) assay to assess cellular proliferation against five different human cell lines viz., Hop62, DWD, K562, DU145 and MCF-7. The complex (S)-1 displayed a remarkably pronounced and specific activity for K562, while complex (S)-2 exhibited significant activity towards Hop62, DWD, DU145 and MCF-7. The in vivo antitumor activity of (S)-1 and (S)-2 was carried out, which revealed significant regression in human lung tumors.

Reduced PKC α Activity Induces Senescent Phenotype in Erythrocytes.

The molecular mechanism mediating expression of senescent cell antigen-aggregated or cleaved band 3 and externalized phosphatidylserine (PS) on the surface of aged erythrocytes and their premature expression in certain anemias is not completely elucidated. The erythrocytes with these surface modifications undergo macrophage-mediated phagocytosis. In this study, the role of protein kinase C (PKC) isoforms in the expression of these surface modifications was investigated. Inhibition of PKC α by 30 µM rottlerin (R30) and 2.3 nM Gö 6976 caused expression of both the senescent cell marker-externalized PS measured by FACS analysis and aggregated band 3 detected by western blotting. In contrast to this observation, but in keeping with literature, PKC activation by phorbol-12-myristate-13-acetate (PMA) also led to the expression of senescence markers. We explain this antithesis by demonstrating that PMA-treated cells show reduction in the activity of PKC α, thereby simulating inhibition. The reduction in PKC α activity may be attributed to the known downregulation of PMA-activated PKC α, caused by its membrane translocation and proteolysis. We demonstrate membrane translocation of PKC α in PMA-treated cells to substantiate this inference. Thus loss of PKC α activity either by inhibition or downregulation can cause surface modifications which can trigger erythrophagocytosis.

Eryptotic phenotype in chronic myeloid leukemia: contribution of neutrophilic cathepsin g.

In pathological conditions with concurrent neutrophilia, modifications of erythrocyte membrane proteins are reported. In chronic myeloid leukemia (CML), a myeloproliferative disease wherein neutrophilia is accompanied by enhanced erythrophagocytosis, we report for the first time excessive cleavage of erythrocyte band 3. Distinct fragments of band 3 serve as senescent cell antigens leading to erythrophagocytosis. Using immunoproteomics, we report the identification of immunogenic 43 kDa fragment of band 3 in 68% of CML samples compared to their detection in only 38% of healthy individuals. Thus, excessive fragmentation of band 3 in CML, detected in our study, corroborated with the eryptotic phenotype. We demonstrate the role of neutrophilic cathepsin G, detected as an immunogen on erythrocyte membrane, in band 3 cleavage. Cathepsin G from serum adsorbs to the erythrocyte membrane to mediate cleavage of band 3 and therefore contribute to the eryptotic phenotype in CML.

Keratins in oral cancer: necessity of mass spectrometry for validation of antibody based identifications.

Keratins are intermediate filament family proteins which are predominantly expressed in the epithelial cells. Most of the studies which evaluate the status of keratins in clinical samples of the oral cavity are based on the identification of their presence and localization by immunohistochemistry using monoclonal antibodies. It is very well known that many monoclonal/polyclonal antibodies show cross-reactivity with the other closely related or non-related proteins. This cross-reactivity might be the result of epitope similarity, but it is not always necessary. Therefore studies done with only antibody based techniques can mislead interpretation unless they are validated with additional techniques like mass-spectrometry. In this investigation we have evaluated the status of keratin 18 in cancer of buccal mucosa using 1DE, 2DE and western blotting with monoclonal antibody to keratin 18. The patterns emerging showed aberrant as well as differential expression of K18 in adjacent normal versus tumor tissue samples of buccal mucosa. Mass spectrometry analysis of the immunodetected spots however revealed that it is keratin 13. Thus this study emphasizes the necessity of validation of antibody based findings when dealing with proteins of a large family having similarity/homology in amino acid sequence.

Carbohydrate-conjugate heterobimetallic complexes: synthesis, DNA binding studies, artificial nuclease activity and in vitro cytotoxicity.

New carbohydrate-conjugated heterobimetallic complexes [C(32)H(62)N(10)O(8)NiSn(2)Cl(4)]Cl(2)(1) and [C(32)H(62)N(10)O(8)CuSn(2)Cl(4)]Cl(2) (2) were synthesized and characterized by spectroscopic (IR, (1)H, (13)C, and (119)Sn NMR, EPR, UV-vis, ESI-MS) and analytical methods. The interaction studies of 2 with CT DNA were studied by using various biophysical techniques, which showed high binding affinity of 2 toward CT DNA. The extent of interaction was further confirmed by the interaction of 2 with the nucleotides viz.; 5'-AMP, 5'-CMP, 5'-GMP, and 5'-TMP, by absorption titration. (1)H, (31)P, (119)Sn NMR spectroscopy further validated the interaction mode of 2 with 5'-GMP. The electrophoresis pattern observed for 2 with supercoiled pBR322 DNA, exhibited significantly good nuclease activity following oxidative pathway. The preferential selectivity of 2 toward the major groove was observed on interaction of 2 with pBR322 DNA, in the presence of standard groove binders viz.; DAPI and methyl green. Additionally, in vitro antitumor activity of 2 was evaluated on a panel of human cancer cell lines, exhibiting remarkable cytotoxicity activity against Colo205 (colon) and MCF7 (breast) cell lines with GI(50) values <10 µg/mL.

Synthesis of aryl-substituted naphthalene-linked pyrrolobenzodiazepine conjugates as potential anticancer agents with apoptosis-inducing ability.

A library of new aryl-substituted naphthalene C8-linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates with various linker architectures were designed, synthesized, and evaluated for their anticancer activity against a panel of 11 human cancer cell lines. All 32 conjugates show anticancer potential, with some of them exhibiting particularly high activity (0.01-0.19 µM). Thermal denaturation studies showed effective DNA binding capacity relative to DC-81. In assays for biological activity relating to cell-cycle distribution, these PBD conjugates induce G0/G1-phase arrest and also cause an increase in the levels of p53 and caspase-9 proteins, followed by apoptotic cell death. One conjugate in particular is the most promising candidate of the series, with the potential to be selected for further studies, either alone or in combination with existing anticancer therapies.

Database and interaction network of genes involved in oral cancer: Version II.

The oral cancer gene database has been compiled to enable fast retrieval of updated information and role of the genes implicated in oral cancer. The first version of the database with 242 genes was published in Online Journal of Bioinformatics 8(1), 41-44, 2007. In the second version, the database has been enlarged to include 374 genes by adding 132 gene entries. The architecture and format of the database is similar to the earlier version, and includes updated information and external hyperlinks for all the genes. The functional gene interaction network for important biological processes and molecular functions has been rebuilt based on 374 genes using 'String 8.3'. The database is freely available at http://www.actrec.gov.in/OCDB/index.htm and provides the scientist information and external links for the genes involved in oral cancer, interactions between them, and their role in the biology of oral cancer along with clinical relevance.

Synthesis of 4ß-carbamoyl epipodophyllotoxins as potential antitumour agents.

A series of new 4ß-carbamoyl epipodophyllotoxin analogues have been synthesized and evaluated for their anticancer activity against eleven cancer cell lines including Zr-75-1, MCF7, KB, Gurav, DWD, Colo 205, A-549, Hop62, PC3, SiHa and A-2780. Most of the compounds exhibited better growth-inhibition activities against tested cell lines than that of etoposide. Further, compounds 6g and 6i are also evaluated for their DNA topoisomerase-II (topo-II) inhibition activity and they exhibited significant inhibition of topo-II catalytic activity comparable to etoposide.

Synthesis of carbohydrate-conjugate heterobimetallic Cu(II)-Sn(2)(IV) and Zn(II)-Sn(2)(IV) complexes; their interactions with CT DNA and nucleotides; DNA cleavage, in-vitro cytotoxicity.

The new heterobimetallic Ni(II)-Sn(2)(IV) (1), Cu(II)-Sn(2)(IV) (2) and Zn(II)-Sn(2)(IV) (3) complexes, containing D-glucosamine, 1,8-diamino-3,6-diazaoctane and imidazole were isolated and characterized by spectral and analytical methods. The proposed geometry of Ni(II) and Cu(II) in 1 and 2 was square pyramidal, Zn(II) in 3 exhibited tetrahedral while Sn(IV) exhibits hexacoordinate environment, respectively. The X-ray powder diffraction (XRPD) confirmed the amorphous nature of all the complexes. The interaction studies of 2 and 3 with CT DNA were carried out by various biophysical techniques to show the mode of binding. The interaction of 2 and 3 with nucleotides viz 5'-GMP and 5'-TMP, respectively were further confirmed by (1)H, (31)P and (119)Sn NMR spectroscopy. The complex 2 exhibited effective cleavage activity with pBR322 DNA. Furthermore, the cytotoxicity of 2 was examined on a panel of human tumor cell lines of different histological origins and showed good activity against Colo205 and A2780 (GI50 < 10 µg/ml).

Synthesis, anticancer activity and mitochondrial mediated apoptosis inducing ability of 2,5-diaryloxadiazole-pyrrolobenzodiazepine conjugates.

A series of 2,5-diaryloxadiazole linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates have been prepared and evaluated for their anticancer activity. These conjugates have shown promising activity with GI50 values ranging from <0.1 to 0.29 microM. It is observed that some of these conjugates particularly 4a, 4d, 4i and 4l exhibit significant anticancer activity. Some detailed biological assays relating to the cell cycle aspects associated to Bax and caspases have been examined with a view to understand the mechanism of action of these conjugates.

Synthesis, DNA-binding ability and anticancer activity of benzothiazole/benzoxazole-pyrrolo[2,1-c][1,4]benzodiazepine conjugates.

A series of benzothiazole and benzoxazole linked pyrrolobenzodiazepine conjugates attached through different alkane or alkylamide spacers was prepared. Their anticancer activity, DNA thermal denaturation studies, restriction endonuclease digestion assay and flow cytometric analysis in human melanoma cell line (A375) were investigated. One of the compounds of the series 17d showed significant anticancer activity with promising DNA-binding ability and apoptosis caused G0/G1 phase arrest at sub-micromolar concentrations. To ascertain the binding mode and understand the structural requirement of DNA binding interaction, molecular docking studies using GOLD program and more rigorous 2 ns molecular dynamic simulations using Molecular Mechanics-Poisson-Boltzman Surface Area (MM-PBSA) approach including the explicit solvent were carried out. Further, the compound 17d was evaluated for in vivo efficacy studies in human colon cancer HT29 xenograft mice.

Design, synthesis and biological evaluation of 3,5-diaryl-isoxazoline/isoxazole-pyrrolobenzodiazepine conjugates as potential anticancer agents.

A series of 3,5-diaryl-isoxazoline/isoxazole linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates were prepared. These conjugates showed potent anticancer activity with GI(50) values in the range of <0.1-3.6 microM. Some of these PBD conjugates (6a-c) with promising anticancer activity were further investigated on the cell cycle distribution. Moreover, these PBD conjugates exhibited G0/G1 arrest, enhancement in the levels of p53 protein as well as mitochondrial-mediated intrinsic pathway, leading to release of cytochrome c, activation of caspase-3, cleavage of PARP and subsequent apoptotic cell death. Hence these PBD conjugates with 6a being the most potent one could be be taken up for preclinical studies either alone or in combination with existing therapies.