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Identification of Apoptosis Inducing Signaling Pathways and Characterization of Sclerotium rolfsii Lectin (SRL) Binding Proteins from Human Colon Epithelial Cancer HT29 cells
Author(s) -
Guha Nilanjan,
Gudihal Ravindra,
Barkeer Sreekanth,
Deepak SA,
Padmanaban Arunkumar,
Inamdar Shashikala
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.831.2
Subject(s) - apoptosis , biology , signal transduction , microbiology and biotechnology , cell cycle , mapk/erk pathway , sclerotium , biochemistry , horticulture
Sclerotium rolfsii lectin (SRL) is isolated from fungus Sclerotium rolfsii and exhibits strong receptor mediated anti‐proliferative activity towards human colon, breast and ovarian cancer cells by inducing apoptosis . In this study, we investigated the signaling pathways that lead to apoptosis by combining transcriptomics data from gene expression and miRNA microarray analysis data and performed pathway analysis using features of GeneSpring 13.1 (GS 13.1). We also identified SRL binding membrane proteins from HT29 cells using proteomics experiments. It was found that SRL treatment results in altered expression of several hundred molecules including MAPK and c‐JUN‐associated, apoptosis‐associated, cell cycle and DNA replication‐associated signaling molecules. Pathway analysis using GS 13.1 revealed that SRL treatment induces changes of MAPK and c‐JUN associated signaling pathways as early as 2h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected after 24h. A significant change of miRNA expression was also observed after 12h SRL treatment. These changes were further validated by qRT‐PCR and immunoblotting. The study suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c‐JUN, followed by miRNA‐associated cell activity and apoptosis. Membrane proteins from HT29 cells were isolated by phase separation and purified by affinity chromatography using SRL‐Sepharose‐4B matrix. Affinity purified proteins were subjected to in‐gel and in‐solution trypsin digestion, analyzed by ESI‐Q‐TOF LC‐MS and Agilent Spectrum Mill software. Considering the specificity of SRL towards O‐glycans, the presence of O‐GalNAc sites in SRL interacting proteins were tested using NetOGlyc software. Western blotting was performed to validate the MS identified proteins. A major protein band around 25kDa following in‐gel trypsin digestion was identified as Keratin 1 by MS. In‐solution trypsin digestion followed by MS identified 25 SRL interacting proteins namely, keratins, heat shock proteins, tubulins, pyruvate kinase M2, peroxiredoxin‐1, ATP synthase subunit alpha, actin, annexin‐A2, prohibitin, ADP/ATP translocase‐2 and alpha enolase. NetOGlyc software analysis revealed 21 proteins positive for O‐glycosylation sites including keratins containing 27 to 50 O‐GalNAc sites. Keratin 1 identified and validated by western blotting as major SRL interacting protein showed 49 O‐GalNAc sites. Proteins identified in our proteomics studies contain O‐GalNAc sites and are known to be involved in cell survival, apoptosis and tumorigenesis. These findings demonstrate the signature profiles of SRL ‐induced gene expression which helps in understanding the lectin's mechanism of action.