Ayyappan Nair, Ph.D.
Head, Business Development (Technologies, Discovery Biology), Anthem Biosciences & DavosPharma, New Jersey, USA
Inhibition of NF-κB regulated gene expression by chrysoeriol suppresses tumorigenesis in breast cancer cells
Amrutha K1, Pandurangan Nanjan1, Sanu K Shaji1, Damu Sunilkumar1, Subhalakshmi K1, Rashmi U Nair1, Lakshmi Rajakrishna2, Asoke Banerji1, Ayyappan Ramesh Nair1*,2
- School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri Campus, Clappana P.O., Kollam – 690 525, Kerala, India
- Anthem Biosciences, No 49, Canara Bank Road, Bommasandra Industrial Area, Phase 1, Hosur Road, Bangalore – 560 099, Karnataka, India
Abstract: A large number of effective cancer-preventing compounds inhibit the activation of nuclear factor-κ B (NF-κB). It has been previously demonstrated that some flavonoids that are a vital component of our diet inhibits this pathway. As a consequence, many flavonoids inhibit genes involved in various aspects of tumorigenesis and have thus emerged as potential chemopreventive candidates for cancer treatment. We studied the effect of 17 different flavonoids, including the highly evaluated quercetin on the NF-κB pathway, and on the expression of MMP-9 and COX-2 (two NF-κB regulated genes involved in metastasis) in the highly invasive human breast cancer cell line MDA-MB-231. The findings suggest that not all the quercetin like flavone backbone compounds inhibit the NF-κB pathway, and that the highly hydoxylated flavonols quercetagetin and gossypetin did not inhibit this pathway, nor did it inhibit the expression of MMP-9 and COX-2. This indicates a correlation between inhibition of NF-κB and subsequent suppression of these NF-κB regulated genes. Here, we also report the novel observation that the not so well characterized methoxylated flavone chrysoeriol inhibited the NF-κB pathway, and was most potent in reducing the expression of MMP-9 and COX-2. Based on these observations, the cellular effects of chrysoeriol were evaluated in MDA-MB-231. Chrysoeriol caused cell cycle arrest at G2/M, inhibited migration and invasion, and caused cell death of macrophages that contributed to migration of these cancer cells. These effects of chrysoeriol make it a potential therapeutic candidate for breast cancer metastasis.
Sanjeeva Srivastava, Ph.D.
Assistant Professor, Proteomics Lab, IIT-Bombay, India
Identification of Potential Early Diagnostic Biomarkers for Gliomas and Various Infectious Diseases using Proteomic Technologies
The spectacular advancements achieved in the field of proteomics research during the last decade have propelled the growth of proteomics for clinical research. Recently, comprehensive proteomic analyses of different biological samples such as serum or plasma, tissue, CSF, urine, saliva etc. have attracted considerable attention for the identification of protein biomarkers as early detection surrogates for diseases (Ray et al., 2011). Biomarkers are biomolecules that can be used for early disease detection, differentiation between closely related diseases with similar clinical manifestations as well as aid in scrutinizing disease progression. Our research group is performing in-depth analysis of alteration in human proteome in different types of brain tumors and various pathogenic infections to obtain mechanistic insight about the disease pathogenesis and host immune responses, and identification of surrogate protein markers for these fatal human diseases.
Applying 2D-DIGE in combination with MALDI-TOF/TOF MS we have analyzed the serum and tissue proteome profiles of glioblastoma multiforme; the most common and lethal adult malignant brain tumor (Gollapalli et al., 2012) (Figure 1). Results obtained were validated by employing different immunoassay-based approaches. In serum proteomic analysis we have identified some interesting proteins like haptoglobin, ceruloplasmin, vitamin-D binding protein etc. Moreover, proteomic analysis of different grades (grade-I to IV) of gliomas and normal brain tissue was performed and differential expressions of quite a few proteins such as SIRT2, GFAP, SOD, CDC42 have been identified, which have significant correlation with the tumor growth. While proteomic analysis of cerebrospinal fluid from low grade (grade I & II) vs. high grade (grade III & IV) gliomas revealed modulation of CSF levels of apolipoprotein E, dickkopf related protein 3, vitamin D binding protein and albumin in high grade gliomas. The prospective candidates identified in our studies provide a mechanistic insight of glioma pathogenesis and identification of potential biomarkers. We are also studying the role of JAK/STAT interactome and therapeutic potential of STAT3 inhibitors in gliomas using proteomics approach. Several candidates of the JAK/STAT interactome were identified with altered expression and a significant correlation was observed between STAT3 and PDK1 transcript expression level.
We have also investigated the changes in human serum proteome in different infectious diseases including falciparum and vivax malaria (Ray et al., 2012a; Ray et al., 2012b), dengue (Ray et al., 2012c) and leptospirosis (Srivastava et al., 2012). Although, quite a few serum proteins were found to be commonly altered in different infectious diseases and might be a consequence of inflammation mediated acute phase response signaling, uniquely modulated candidates were identified in each pathogenic infection indicating the some inimitable responses. Further, a panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models employing PLSDA and other classification methods to predict the clinical phenotypic classes and 91.37% overall prediction accuracy was achieved (Figure 2). ROC curve analysis was carried out to evaluate the individual performance of classifier proteins. The excellent discrimination among the different disease groups on the basis of differentially expressed proteins demonstrates the potential diagnostic implications of this analytical approach.
Keywords: Diagnostic biomarkers, Gliomas, Infectious Diseases, Proteomics, Serum proteome
Acknowledgments: This disease biomarker discovery research was supported by Department of Biotechnology, India grant (No. BT/PR14359/MED/30/916/2010), Board of Research in Nuclear Sciences (BRNS) DAE young scientist award (2009/20/37/4/BRNS) and a startup grant 09IRCC007 from the IIT Bombay. The active support from Advanced Center for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Hospital (TMH), and Seth GS Medical College and KEM Hospital Mumbai, India in clinical sample collection process is gratefully acknowledged.
References :
- Ray S, Reddy PJ, Jain R, Gollapalli K. Moiyadi A, Srivastava S. Proteomic technologies for the identification of disease biomarkers in serum: advances and challenges ahead. Proteomics 11: 2139-61, 2011.
- Gollapalli K, Ray S, Srivastava R, Renu D, Singh P, Dhali S, Dikshit JB, Srikanth R, Moiyadi A, Srivastava S. Investigation of serum proteome alterations in human glioblastoma multiforme. Proteomics 12(14): 2378-90, 2012.
- Ray S, Renu D, Srivastava R, Gollapalli K, Taur S, Jhaveri T, Dhali S, Chennareddy S, Potla A, Dikshit JB, Srikanth R, Gogtay N, Thatte U, Patankar S, Srivastava S. Proteomic investigation of falciparum and vivax malaria for identification of surrogate protein markers. PLoS One 7(8): e41751, 2012a.
- Ray S, Kamath KS, Srivastava R, Raghu D, Gollapalli K, Jain R, Gupta SV, Ray S, Taur S, Dhali S, Gogtay N, Thatte U, Srikanth R, Patankar S, Srivastava S. Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response. J Proteomics 75(10): 3063-80, 2012b.
- Srivastava R, Ray S, Vaibhav V, Gollapalli K, Jhaveri T, Taur S, Dhali S, Gogtay N, Thatte U, Srikanth R, Srivastava S. Serum profiling of leptospirosis patients to investigate proteomic alterations. J Proteomics 76: 56-68, 2012.
- Ray S, Srivastava R, Tripathi K, Vaibhav V, Srivastava S. Serum proteome changes in dengue virus-infected patients from a dengue-endemic area of India: towards new molecular targets? OMICS 16(10): 527-36, 2012c.
* Correspondence: Dr. Sanjeeva Srivastava, Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400 076, India: E-mail: sanjeeva@iitb.ac.in; Phone: +91-22-2576-7779, Fax: +91-22-2572-3480
![Figure 1 (a) Differentially expressed proteins in GBM identified using 2D-DIGE. Representative 2D- DIGE image to compare serum proteome of HC and GBM patients. GBM and HC samples were labeled with Cy3 and Cy5 respectively, while the protein reference pool (internal standard) was labeled with Cy2. Graphical and 3D fluorescence intensity representations of few selected statistically significant (p < 0.05) differentially expressed proteins in GBM patients identified in biological variation analysis (BVA) using DeCyder 2D software. (b) Involvement of different essential physiological pathways with differentially expressed proteins in GBM. Members of multiple essential physiological processes including cell growth and proliferation, vitamin D metabolism, lipoprotein metabolism and transport, oxidative stress regulation, complement cascade, and platelet activation found to be modulated in the GBM patients (Gollapalli et al., Proteomics 2012).](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/sanjeeva.jpg)
![Figure 2 (a) Western blot analysis of haptoglobin (HP), serum amyloid A (SAA), and clusterin (CLU) from serum samples of healthy control (HC) [n = 12], falciparum malaria (FM) [n = 12], vivax malaria (VM) [n = 12], Leptospirosis (Lep) [n = 6], dengue fever [DF] [n = 6] and non infectious disease control (NIDC:GBM) [n = 12]. Representative blots of the target proteins are depicted along with their respective relative abundance volumes (volume X 104). All the data are represented as mean ± SE. (b) Discrimination of malaria from dengue, leptospirosis and GBM using PLS-DA analysis. PLS-DA scores Plot for FM (blue spheres, n = 8), VM (green spheres, n = 8), DF (red spheres, n = 6), Lep (grey spheres, n = 6) and GBM (brown spheres, n = 8) samples based on 6 differentially expressed proteins (serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I) identified using DIGE. The axes of the plot indicate PLSDA latent variables t0-t2.](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/sanjeeva-2.jpg)
![Delegate Talk: AIB1 Mediated Modulation of CXCR4-SDF1 Signaling in Breast Cancer @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Binu K Aa, Jem Prabhakarb, Thara Sc and Lakshmi Sd,∗
aDepartment of Clinical Diagnostics Services and Translational Research, Malabar Cancer Centre, Thalassery, Kerala, India.
bDivision of Surgical Oncology, Division of Pathology
dDivision of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India.
Introduction
AIB1, a member of the nuclear co activators, promotes the transcriptional activity of multiple nuclear receptors such as the ER and other transcription factors. Chemokines produced by stromal cells have potential to influence ERα-positive breast cancer progression to metastasis. CXCR4 is the physiological receptor for SDF1, together shown to stimulate the chemotactic and invasive behavior of breast cancer cells to serve as a homing mechanism to sites of metastasis. We propose that over expression of AIB1 in breast cancer cells leads to increased SDF1 and CXCR4 expression, which induces invasion and metastasis of cancer cells.
Materials and Methods
Breast tumor and normal breast tissues from patients in Regional Cancer Centre, Thiruvananthapuram were used for study. The modulatory effect of AIB1 was studied in MCF-7 cells with AIB1 siRNA transfection along with treatment of 17β-Estradiol (E2), 4-hydroxytamoxifen (4OHT), combinations of E2 and 4OHT. The gene expression pattern and protein localization were assessed by RT-PCR and immunofluorescence microscopy respectively. The metastatic and invasive properties were assessed by wound healing assay. Quantitative colocalization analyses were done to assess the association of proteins using Pearson’s correlation coefficient.
Result and Conclusion
The mRNA and protein level expression of AIB1, CXCR4 and SDF1 were higher in tumor samples than in normal samples. AIB1 was localized to the nuclei whereas CXCR4 and SDF1 immunoreactivity were observed in the cytoplasm and to a lesser extent in the nuclei of tumor epithelial cells. In tumor samples the gene level expressions of AIB1 showed significant positive correlations with SDF1(r = 0.213, p = 0.018). CXCR4 showed significant positive correlation with SDF1 in gene (r = 0.498, p = 0.000) and protein levels(r = 0.375, p = 0.002). Quantitative colocalization analyses showed a marked reduction in expression of CXCR4 and SDF1 in siAIB1MCF-7 cells than MCF-7 cells with different treatment groups. Wound healing assay shows reduced wound healing in siAIB1 treated MCF-7 cells.
In recent years, targeting specific cancer pathways and key molecules to arrest tumor growth and achieve tumor eradication have proven a challenge; due to acquired resistance and homing of cancer cells to various metastatic sites. The present study revealed that silencing AIB1 can prevent the over expression of SDF1 and CXCR4. Co activator levels determine the basal and estrogen-inducible expression of SDF1, a secreted protein that controls breast cancer cell proliferation and invasion through autocrine and paracrine mechanisms (Hall et al. 2003). The effects of CXCR4 overexpression has been correlated with SDF1 mediated activation of downstream signaling via ERK1/2 and p38 MAPK and with an enhancement of ER-mediated gene expression (Rhodes et al. 2011). It is possible that over expression of AIB1 as a stimulant involved in the expression of CXCR4 might up-regulate the expression of prometastatic and angiogenic genes. Thus based on these observations it can be concluded that SDF1/CXCR4 overexpression, with significant association with AIB1 expression, itself contribute to the development of mammary cancer and metastatic progression.
![Delegate Talk: Insilico Analysis of hypothetical proteins from Leishmania donovani: A Case study of a membrane protein of the MFS class reveals their plausible roles in drug resistance @ Sathyam Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/bicb.jpg)
Nitish Sathyanrayanan, Sandesh Ganji and Holenarsipur Gundurao Nagendra.
Insilico Analysis of hypothetical proteins from Leishmania donovani: A Case study of a membrane protein of the MFS class reveals their plausible roles in drug resistance
Kala-azar or visceral leishmaniais (VL), caused by protozoan parasite Leishmania donovani, is one of the leading causes of morbidity and mortality in Bihar, India (Guerin et al. 2002; Mubayi et al. 2010). The disease is transmitted to the humans mainly by the vector, Phlebotmus argentipes, commonly known as Sand fly. The majority of VL (> 90%) occurs in only six countries: Bangladesh, India, Nepal, Sudan, Ethiopia and Brazil (Chappuis et al. 2007). In the Indian subcontinent, about 200 million people are estimated to be at risk of developing VL and this region harbors an estimated 67% of the global VL disease burden. The Bihar state only has captured almost 50% cases out of total cases in Indian sub-continent (Bhunia et al. 2013). ‘Conserved hypothetical’ proteins pose a challenge not just to functional genomics, but also to biology in general (Galperin and Koonin 2004). Leishmania donovani (strain BPK282A1) genome consists of a staggering ∼65% of hypothetical proteins. These uncharacterized proteins may enable better appreciation of signalling pathways, general metabolism, stress response and even drug resistance.
![Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi
Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.
Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.
Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.
Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.
![Delegate Talk: A proteomic approach to decipher the mechanism of action and molecular targets of curcumin in Bacillus subtilis @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Jaipal Panga Reddy, Dulal Panda and Sanjeeva Srivastava
The rapid emergence of microbial resistance against the ever increasing number of infectious diseases has been a major hurdle in hunt for novel antibiotics and inventive targets required to combat these dreaded pathogens. Despite the discovery of synthetic and semi-synthetic drugs, infectious diseases remain a major cause of concern. The process of drug discovery started from natural products as an antibacterial drug, an old art which was widely adopted in ancient civilizations in India and China. Most of the existing antibiotics are derived from the backbone of natural compounds (Butler M S 2005). Drug discovery process from natural products to synthetic medicine has continued for thousands of years to battle with pathogenic organisms. Although synthetic drugs played a vital role as antimicrobial drugs during the last decade, the over-use of antibiotics has led to the unanticipated changes at the genomic level, resulting into emergence of antibiotic-resistant strains (Singh P et al. 2010). To combat drug-resistant strains there is a need to develop and characterize new drugs by screening natural and synthetic compounds. Curcumin is a well known natural product, with a potential to cure wide range of cancers. Apart from antitumor activity, it also has anti-inflammatory, anti-viral, anti-genotoxic, phototoxic and antimicrobial activities. The exact mechanism of action of curcumin is still obscure and further investigations are required to identify its molecular/cellular targets. Recently, it was observed that curcumin is able to perturb the FtsZ assembly dynamics and elongate the bacterial cell length by inhibiting bacterial cell division (Rai D et al. 2008).
In the present study, we aimed at deciphering the mechanism of action and molecular targets of curcumin in B. subtilis AH75 using classical two dimensional electrophoresis, 2D-difference gel electrophoresis (2DDIGE) in combination with MALDI-TOF/TOFMS. Comparative proteome analysis of control and IC50 curcumin treated B. subtilis has revealed differential expression of 48 proteins (p ≤ 0.05) in response to curcumin treatment. Further, in silico analysis has revealed the involvement of the differential expressed proteins in protein synthesis, transcription/nucleotide synthesis, stress regulation, central metabolism and amino acid metabolic process. Additionally, suppression in major central metabolic dehydrogenase such as glyceraldehyde-3-phosphate dehydrogenase 2, dihydrolipoyl dehydrogenase, malate dehydrogenase, pyruvate dehydrogenase E1 component subunit beta, 2-oxoglutarate dehydrogenase E1 component, ATP synthase epsilon chain, ATP synthase subunit beta and probable NADdependent malic enzyme 1 has been identified (Figure 1). Reduction in expression levels of major dehydrogenases indicates the disturbance in cell membrane integrity to transfer the electrons from NADH to molecular oxygen or maintain the PMF or maintain the membrane potential. Selected potential proteins obtained from proteomic analysis were validated with real-time expression analysis and metabolic assays such as resazurin assay for metabolic activity, CTC assay for respiratory activity and propidium iodide staining for membrane integrity. Findings from this study have revealed that curcumin has potential effects on multiple physiological pathways in bacteria and inhibition of the cell division machinery is one of the prime targets of this drug. Multiple proteins involved in cell division process such as GroEL, ClpC, MetK, Tuf and major dehydrogenases are significantly modulated as a consequence of the drug treatment.
Akhilesh Pandey, Ph.D.
Professor, Johns Hopkins University School of Medicine, Baltimore, USA
A draft map of the human proteome
We have generated a draft map of the human proteome through a systematic and comprehensive analysis of normal human adult tissues, fetal tissues and hematopoietic cells as an India-US initiative. This unique dataset was generated from 30 histologically normal adult tissues, fetal tissues and purified primary hematopoietic cells that were analyzed at high resolution in the MS mode and by HCD fragmentation in the MS/MS mode on LTQ-Orbitrap Velos/Elite mass spectrometers. This dataset was searched against a 6-frame translation of the human genome and RNA-Seq transcripts in addition to standard protein databases. In addition to confirming a large majority (>16,000) of the annotated protein-coding genes in humans, we obtained novel information at multiple levels: novel protein-coding genes, unannotated exons, novel splice sites, proof of translation of pseudogenes (i.e. genes incorrectly annotated as pseudogenes), fused genes, SNPs encoded in proteins and novel N-termini to name a few. Many proteins identified in this study were identified by proteomic methods for the first time (e.g. hypothetical proteins or proteins annotated based solely on their chromosomal location). We have generated a catalog of proteins that show a more tissue-restricted pattern of expression, which should serve as the basis for pursuing biomarkers for diseases pertaining to specific organs. This study also provides one of the largest sets of proteotypic peptides for use in developing MRM assays for human proteins. Identification of several novel protein-coding regions in the human genome underscores the importance of systematic characterization of the human proteome and accurate annotation of protein-coding genes. This comprehensive dataset will complement other global HUPO initiatives using antibody-based as well as MRM mass spectrometry-based strategies. Finally, we believe that this dataset will become a reference set for use as a spectral library as well as for interesting interrogations pertaining to biomedical as well as bioinformatics questions.
![Delegate Talk: Proteomic profiling of gallbladder cancer secretome – a source for circulatory biomarker discovery @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/bioanalt.jpg)
Tejaswini Subbannayya, Nandini A. Sahasrabuddhe, Arivusudar Marimuthu, Santosh Renuse, Gajanan Sathe, Srinivas M. Srikanth, Mustafa A. Barbhuiya, Bipin Nair, Juan Carlos Roa, Rafael Guerrero-Preston, H. C. Harsha, David Sidransky, Akhilesh Pandey, T. S. Keshava Prasad and Aditi Chatterjee
Proteomic profiling of gallbladder cancer secretome – a source for circulatory biomarker discovery
Gallbladder cancer (GBC) is the fifth most common cancer of the gastrointestinal tract and one of the common malignancies that occur in the biliary tract (Misra et al. 2006; Lazcano-Ponce et al. 2001). It has a poor prognosis with survival of less than 5 years in 90% of the cases (Misra et al. 2003). The etiology is ill-defined. Several risk factors have been reported including cholelithiasis, obesity, female gender and exposure to carcinogens (Eslick 2010; Kumar et al. 2006). Poor prognosis in GBC is mainly due to late presentation of the disease and lack of reliable biomarkers for early diagnosis. This emphasizes the need to identify and characterize cancer biomarkers to aid in the diagnosis and prognosis of GBC. Secreted proteins are an important class of molecules which can be detected in body fluids and has been targeted for biomarker discovery. There are challenges faced in the proteomic interrogation of body fluids especially plasma such as low abundance of tumor secreted proteins, high complexity and high abundance of other proteins that are not released by the tumor cells (Tonack et al. 2009). Profiling of conditioned media from the cancer cell lines can be used as an alternate means to identify secreted proteins from tumor cells (Kashyap et al. 2010; Marimuthu et al. 2012). We analyzed the invasive property of 7 GBC cell lines (SNU-308, G-415, GB-d1, TGBC2TKB, TGBC24TKB, OCUG-1 and NOZ). Four cell lines were selected for analysis of the cancer secretome based on the invasive property of the cells. We employed isobaric tags for relative and absolute quantitation (iTRAQ) labeling technology coupled with high resolution mass spectrometry to identify and characterize secretome from the panel of 4GBC cancer cells mentioned above. In total, we have identified around 2,000 proteins of which 175 were secreted at differential abundance across all the four cell lines. This secretome analysis will act as a reservoir of candidate biomarkers. Currently, we are investigating and validating these candidate markers from GBC cell secretome. Through this study, we have shown mass spectrometry-based quantitative proteomic analysis as a robust approach to investigate secreted proteins in cancer cells.