A study on effect of concanavalin on differential gene expression in human cell lines.

1. A study on effect of concanavalin on differential gene expression in human cell lines. (Funding agency- DST, Extramural research) Dr. Bhagya B. Sharma, Assistant Professor, Genetics unit, Dept of Anatomy, Yenepoya Medical College, YenepoyaUniversity Presented by: GeethaSuvarna A PhD scholar, Reg no- 123/ Jan 2014 Yenepoya University Priniciple investigator: Co-investigator: • Dr. Arun A.B., • Dydirector, Yenepoya Research center, Yenepoya University

2. Project summary The lectins are carbohydrate binding proteins that agglutinate cells or precipitate glycoconjugates. Lectins exhibit diverse functions like mitogenicity, immunomodulation, antimicrobial activity, antiviral and antiproliferative properties. In cancer research lectins are extensively used as diagnostic tool and also as antiproliferative agent. In the present study, Con A lectin is used to study the genes involved in mitogenicity and antiproliferative effect induced by this lectin. Microarray technique is used to study the gene expression induced by Con A in normal human normal lymphocytes and acute myeloid leukaemia cell line (HL 60). Key words:Concanavalin, microarray, mitogenicity, antiproliferation

3. Introduction Origin of the proposal • Binding of mitogenic lectins -involving different signal transduction pathways which ultimately results in cell proliferation. • Lectins have been found to possess anti-cancer properties • Concanavalin A is a mannose/glucose binding lectin isolated from Canavaliaensiformis. • Con A act as a T-cell mitogen and in cancer cells it induces cell death through apoptotic and autophagicpathway different molecular processes involved in mitogenicityand cytotoxicity • Gene expression study • Microarray

4. Definition of the problem To study the genes expression induced by concanavalin A in human lymphocytes and cancer cells using microarray.

5. Objectives • To optimize the concentration of Con A for mitogenicity and anti-proliferative effect in human lymphocytes and cancer cells. • To study gene expression in untreated lymphoctes and Con A treated lymphocytes using microarray. • To study gene expression in untreated cancer cells and Con A treated cancer cells using microarray.

6. Review of status of Research and Development in the subject International status • Con A enhanced osteogenesis in human bone marrow MSC- osteocalcin, RUNX2, BMP-2, BMP-4, and BMP-6 mRNA expression (Sekiya K et al., 2008) • Increased in telomere length and replicative capacity of peripheral blood mononuclear cells (Murillo- Ortiz B et al., 2013) • Induced apoptosis by p73 regulated Akt-Foxo1a-Bim pathway in p53 deficient cells (Amin AR et al., 2007) • ConAand Sophoraflavescenslectin (SFL) induces apoptosis in Human breast carcinoma MCF-7 cells through p53 dependent pathway by reducing NF-κB, ERK, and JNK levels, and increasing p53 and p21 levels (Shi Z et al., 2014) • Con A induced BNIP3-mediated mitochondrial autophagy in hepatomacells(Chang CP et al., 2007) National status Con A induced autophagy through MEK/Extracellular signal-regulated kinases (ERK) pathway in Human cervical cancer (HeLa) cells was studied by IIT Kharagpur (Roy B et al., 2014)

7. Novelty Importance of the proposed project • Con A induced gene expression study using microarray technique • Since this genomic study compares the normal with the cancerous cells, it will give the differential pattern in expression gene which is basis for tumorigenesis. Such genes can be used as a therapeutic marker in diagnosis of cancer.

8. Target beneficiaries of the proposed work • This in vitro study can be translated to clinical practice in patient tumor samples to analyse the biological markers for an early detection, monitoring, prognosis of the disease and response to therapy.

9. Methodology: • Materials: • Method: • To optimize the concentration of Con A for mitogenicity and anti-proliferative effect in human lymphocytes and cancer cells • Mitogenic potential of Con A is determined against human lymphocytes as described by Singh RS et al. • Antiproliferative effect Con A on cancer cells(HL-60 Cell line) is studied using MTT assay (Yan Q et al., 2009) • To study gene expression in lymphocytes and cancer cells using microarray (Marimuthu A et al., 2011) • Sample goups: untreated lymphoctes and Con A treated lymphocytes • untreated cancer cells and Con A treated cancer cells( HL-60)

11. The development "Outcomes" and "Outputs' of the project • Research publications in high impact journals. • This study opens multidisciplinary research areas like genomics, proteomics, Cancer research for future studies • NCBI database can be updated by adding the microarray data obtained from the study. • The microarray data can be used to detect a biomarker for an early detection, monitoring, prognosis of the disease and response to therapy.

12. BUDGET ESTIMATES: SUMMARY

13. BUDGET FOR SALARIES/WAGES

14. BUDGET FOR CONSUMABLE MATERIALS

15. Justification for costly consumable

16. BUDGET FOR OTHER COSTS/CONTINGENCIES

17. BUDGET FOR EQUIPMENT

18. Time Schedule of Activities through BAR Diagram

19. Infrastructural Facilities

20. File No**_______________ 1. Title: A study on effect of concanavalin on differential gene expression in human cell lines.                    2. Proposed Budget for 3 years: 2.1 Total: 22, 50,000 2.2 Equipment: Agilent microarray scanner (G2505B) 2.3 Staff (proposed research staff) : 2 JRFs 2.4 Other Recurring costs: Consumable: 3, 83,000 Travel: NA Contingency : 27,000 Any other expenses : NA 3. Date of receipt**: 4. PI Name, Designation & Address : Dr. Bhagya B. Sharma, Assistant Professor, Genetics unit, Dept of Anatomy, Yenepoya Medical College, Yenepoya University, University Road, Nithyanandanagara, Deralakatte, Mangalore-575018. 5. Co-Investigator(s) details : Dr. ArunA.B.,Dy director, Yenepoya Research center, Yenepoya University, University Road, Nithyanandanagara, Deralakatte, Mangalore-575018

21. References: • Hamid R, Masood A, Wani IH and Rafiq S. Lectins: proteins with diverse applications. J. App. Pharm Sc. 2013; 3: 93-103. • Ashraf MT, Khan RH. Mitogenic lectins. Med SciMonit. 2003; 9(11):RA265-9. • Sherwani AF, Mohmood S, Khan F, Khan RH, Azfer MA. Characterization of lectins and their specificity in carcinomas-An appraisal. Indian J ClinBiochem. 2003; 18(2):169-80. • Lei HY, Chang CP. Lectin of Concanavalin A as an anti-hepatoma therapeutic agent. J Biomed Sci. 2009;16:10. • Sekiya K, Nishimura M, Suehiro F, Nishimura H, Hamada T, Kato Y. Enhancement of osteogenesis by concanavalin A in human bone marrow mesenchymal stem cell cultures. Int J Artif Organs. 2008; 31(8):708-15. • Murillo- Ortiz B, Tamayo FA, Briones SL, Garza SM,Bribiesca LB and Aranda DA. Increased telomere length and proliferative potential in peripheral blood mononuclear cells of adults of different ages stimulated with concanavalin A. BMC Geriatrics. 2013; 13:99. • Amin AR, Paul RK, Thakur VS, Agarwal ML. A novel role for p73 in the regulation of Akt-Foxo1a-Bim signaling and apoptosis induced by the plant lectin, Concanavalin A. Cancer Res. 2007;67(12):5617-21. • Shi Z, Chen J, Li CY, An N, Wang ZJ, Yang SL, et al. Antitumor effects of concanavalin A and Sophoraflavescenslectin in vitro and in vivo. ActaPharmacologicaSinica. 2014; 35 (2):248. • Chang CP, Yang MC, Liu HS, Lin YS, Lei HY. Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model. Hepatology. 2007;45(2):286-96. • Liu B, Cheng Y, Zhang B, Bian HJ, Bao JK. Polygonatumcyrtonemalectin induces apoptosis and autophagy in human melanoma A375 cells through a mitochondria-mediated ROS-p38-p53 pathway. Cancer Lett. 2009;275(1):54-60.

22. 11. Liu B, Wu JM, Li J, Liu JJ, Li WW, Li CY, et al. Polygonatumcyrtonemalectin induces murinefibrosarcoma L929 cell apoptosis and autophagy via blocking Ras-Raf and PI3K-Akt signaling pathways. Biochimie. 2010 ;92(12):1934-8. • 12. Fang EF, Zhang CZ, Ng TB, Wong JH, Pan WL, Ye XJ, et al. MomordicaCharantialectin, a type II ribosome inactivating protein, exhibits antitumor activity toward human nasopharyngeal carcinoma cells in vitro and in vivo. Cancer Prev Res (Phila). 2012;5(1):109-21. • 13. Roy B, Pattanaik AK, Das J, Bhutia SK, Behera B, Singh P, et al. Role of PI3K/Akt/mTOR and MEK/ERK pathway in Concanavalin A induced autophagy in HeLa cells. ChemBiol Interact. 2014;210:96-102. • 14. Mukhopadhyay S, Panda PK, Behera B, Das CK, Hassan MK, Das DN, et al. In vitro and in vivo antitumor effects of Peanut agglutinin through induction of apoptotic and autophagic cell death. Food ChemToxicol. 2014;64:369-77. • 15. Panda PK, Mukhopadhyay S, Behera B, Bhol CS, Dey S, Das DN, et al. Antitumor effect of soybean lectin mediated through reactive oxygen species-dependent pathway. Life Sci. 2014;111(1-2):27-35. • 16. Mukhopadhyay S, Panda PK, Das DN, Sinha N, Behera B, Maiti TK, et al. Abrus agglutinin suppresses human hepatocellular carcinoma in vitro and in vivo by inducing caspase-mediated cell death. ActaPharmacol Sin. 2014;35(6):814-24. • 17. Behera B, Mishra D, Roy B, Devi KS, Narayan R, Das J, et al. Abrusprecatorius agglutinin-derived peptides induce ROS-dependent mitochondrial apoptosis through JNK and Akt/P38/P53 pathways in HeLa cells. ChemBiol Interact. 2014;222C:97-105. • 18. Singh RS, Kaur HP, Singh J. Purification and characterization of a mucin specific myceliallectin from Aspergillusgorakhpurensis: application for mitogenic and antimicrobial activity. PLoS One. 2014;9(10):e109265. • 19. Yan Q, Li Y, Jiang Z, Sun Y, Zhu L, Ding Z. Antiproliferation and apoptosis of human tumor cell lines by a lectin (AMML) of Astragalusmongholicus. Phytomedicine. 2009; 16: 586–593. • 20. Marimuthu A, Jacob HKC, Jakharia A, Subbannayya Y, Keerthikumar Y, Kashyap MK, et al. Gene expression profiling of gastric cancer. J Proteomics Bioinform. 2011; 4(4): 074-082.

23. THANK YOU