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Hepatocyte growth factor, a potent cytokine of mesenchymal origin exhibiting slews of physiological responses in the wide variety of cells like proliferation, migration and invasion upon binding to its receptor c-Met, but the mechanisms were not clearly understood where upon improper secretions of hepatocyte growth factor and activation of the c-Met results to the development of cancers. The human HGF variants are the natural occurring splice variants composed of N-terminal domain with their respective Kringle domains designated as K1, K2, K3 and K4. The biological properties of all the variants (NK1-NK4) of HGF have been controversial, and in-vitro studies suggest that these variants might have the therapeutic role as an HGF/SF antagonist. In order to clarify the NK-mediated inhibitory mechanism, we isolated and purified the NK variants of HGF (NK1-NK4) as the soluble form, from E.coli with GST as their tags. The topical application of these recombinant GST tagged HGF variant proteins (fusion proteins) on Human lung cancer cells (A549) Proliferation, migration, invasion and expression of different MMPs were assayed at 4nM concentration and compared to the medium control and second control GST (4nM). Our result demonstrated that these recombinant fusion NKs (GST with NKs) have significantly inhibited mitogen, motogen, morphogenesis of A549 at 4nM with downhill regulation of MMP-2 and MMP-9 and over expression of MMP-8. Therefore, our results suggest that in-vitro over expression of MMP-8 with downward regulation of MMP-9 can be considered as vital signals against metastasis of lung cancer cells, underlying the possible development of new therapeutic drug.

Contents Page no
Chapter 1: Introduction to Cancers
1.1) Basic epidemiology of lung cancers 1
1.2) Basic overview of metastasis 2
1.2.1) Genome protection 3
1.2.2) Immortalization of cancer cells 4
1.2.3) Invasion and metastasis. 5
1.3) General overview of Matrix metalloproteinases 6
1.3.1) Structure of MMP`s 6
1.3.2) Detection of MMP`s by substrate Zymography 7
1.3.3) Gelatinases A and B 10
1.3.4) Collagenases-2 12
1.3.5) TIMP`s 13
Chapter 2:Aims of the thesis. 15
Chapter 3:Hepatocyte growth factor 16
3.1) Basic structure of HGF 16
3.2) Basic structure of c-Met 17
3.3) Interaction of HGF/c-Met 18
3.4) Biological functions elicited by HGF/c-Met 18
3.5) Inhibitors of HGF/c-Met 22
Chapter 4:Materials and methods 36
4.1) Chemicals and Methods 27
4.2) Molecular Biological Methods 28
4.2.1) clones from Shiping He et al 28
4.2.2) Transformation 28
4.2.3) Plasmid isolations and restriction digestion 29
4.2.4) Expression of recombinant HGF variants 30
4.2.5) Protein purification 31
4.2.6) SDS-Polyacrylamide gel electrophoresis 32
4.3) Biochemical methods 33
4.3.1) Western blotting 33
4.3.1a) Protein transfer 33
4.3.1b) Immunoassay 34
4.4) Cellular -Biological Methods 35
4.4.1) Cell lines and Culturing 35
4.4.2) Invitro Proliferation Assay 36
4.4.3) Invitro Migration assay 36
4.4.4) Invitro Invasion assay 37
4.4.5) Invitro GST binding assay 38
4.4.6) Gelatin Zymography 38
4.4.7) Quantification of Gelatinases 39
4.4.8) Casein Zymography 40
4.4.9) Quantification of collagenases 40
4.4.10) Statistical Analysis 40



Chapter 5: Results 41
5.1) Restriction digestion of clones,transformation,
protein expression and Western blotting 41
5.2) Recombinant HGF variants inhibit proliferation of A549 proliferation assay 42
5.3) Recombinant HGF variants inhibit migration assay 42
5.4) Recombinant HGF variants inhibit Invasion assay 43
5.5) sjGST doesn`t bind to A549 43
5.6) Up and down regulation of MMP-8, and MMP-9 of A549 in
presence of HGF variants 44
Chapter 6: Discussion 46
Chapter 7: Conclusion 51
Chapter 8; References 52
Chapter 9: Appendix 64
Chapter 10: Figures and Photographs 82
Figure: 6 Clones PGEX-2T with respectiveHGF truncated fragments 82
Figure: 7 SDS page gel for GST 83
Figure: 8 SDS-PAGE gels for NK1 84
Figure: 9 SDS-PAGE gels for NK2 85
Figure: 10 SDS-PAGE gels for NK3 86
Figure: 11 SDS-PAGE gels for NK4 87
Figure: 12 Quantification of NKs fusion proteins 88
Figure: 13Western blotting of all NKs proteins 89
Figure: 14 western blotting of GST 90
Figure: 15.Invitro-proliferation Assay 91
15. 1. 0 day photos of the proliferation assay 92
15. 2. 2 day photos of the proliferation assay 93
15. 3. 4 days photos of the proliferation assay 94
15. 4. 6 days photos of the proliferation assay 95
15. 5. 8 days photos of the proliferation assay 96
Figure: 16.Invitro- migration Assay 97
16.1 Photos of migration assay for 0 hr 98
16.2. Photos of migration assay for 6 hr 99
16.3. Photos of migration assay for 12 hr 100
16.4. Photos of migration assay for 24 hr 101
16.5 Photos of migration assay for 36 hr 102
16.6. Photos of migration assay for 48 hr 103
Figure: 17. . Invitro-Invasion Assay 104
17. 1. Photos of the invasion assay for 36 hr 105
Figure: 18 GST Binding Assay 106
Figure: 19. Gelatin Zymography 107
19. 1 Quantification of MMP-2 by image J 108
19. 2 Quantification of MMP-9 by image J 109
Figure: 20. Casein Zymography 110
20. 1 Quantification of MMP-8 in casein gel 111
20. 2 Quantification of MMP-9 in casein zymography 112
Paper Accepted in the Journal 113

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