1. Abstract Matrix metalloproteinases (MMPs) are enzymes in the extracellular matrix that are involved in a variety of functions including differentiation of cells, cell adhesion, and cell-to-cell interactions. A subclass of MMPs known as gelatinases (MMP-2 and -9) are associated with breast cancer metastasis due to their ability to degrade type-IV collagen, a constituent of basement membranes. Therefore, significant interest exists in developing therapeutic agents, which might prevent or decrease cancer metastasis. Breast cancer is the most prevalent invasive cancer in women worldwide. Genistein, a soy isoflavone has anti-breast cancer properties. This project seeks to measure the effect that genistein on the activity of the gelatinases MMP-2 and MMP-9. Here we describe our experiments to (i) establish assays for the gelatinases, (ii) detection of gelatinases in human breast cancer cells and (iii) analyze effects of genistein on gelatinase activity. Mammary carcinoma cells HTB-19 and HTB-20 are grown to log phase, harvested by trypsinization and whole cell extracts were prepared using well-established protocols. Activity of MMP-2 and MMP-9 is detected by gelatin zymography using 10% acrylamide gel electrophoresis followed by reactivation, incubation of the gel in presence of specific metal ions and coomassie blue staining. Presence of MMP-2 and MMP-9 in human breast cancer cells is confirmed by western blot analysis using specific antibodies against these proteins. Results obtained in these studies and their significance for growth of human mammary cancer cells will be discussed. Introduction Proteases in extracellular matrix; contain covalently bound metal ion 24 known members; 5 subgroups based on function: Collagenases, Gelatinases, Stromelysins, Membrane-Type, & Other All have three domains in common: propeptide, catalytic domain, & hemopexin (see Fig. 1a) Gelatinases (MMP-2 & MMP-9) degrade both gelatin and Type-IV collagen, a constituent of basement membranes Degradation of basement membranes leads to cancer metastasis Genistein is an isoflavone found in mainly in soybeans, but also in fava beans and coffee Noted as a tyrosine-kinase inhibitor Member of phytoestrogens, a group of compounds found in plants with structural similarities to estrogen (see Fig. 1b) Can compete with estrogen for estrogen receptors Enables it to mimic some of estrogen’s effects as well Has established properties as an inhibitor of breast cancer cell growth Studies have proven a link between high soy intake and a decrease in risk for breast cancer Mechanism of action for this activity is not precisely known Project Objectives Establish valid assays for use on gelatinases Detect MMP-2 & -9 activity in HTB-19 & HTB-20 cells Analyze effects of genistein treatments in both cell lines and on both gelatinases Methods HTB-19 and HTB-20 breast cancer cells grown in sterile hood SDS-PAGE conducted using standard protocol Western blot run on samples to detect the presence of solely the gelatinases Zymography used to determine level of activity of gelatinases Bradford assay run to determine concentration of proteins in samples (see Fig. 2b) Outline of procedures shown in Figure 2a Scott Kaufmann, Michael Buege, Pramod Mahajan (Mentor), Scott Kaufmann, Michael Buege, Pramod Mahajan (Mentor), Drake University College of Pharmacy and Health Sciences Characterization of matrix metalloproteinases in breast cancer cells Figure 1a. Diagrammatic structure of Gelatinases A and B Results Results & Conclusions Established gelatinase activity of recombinant human MMP-2 protein using zymography Confirmed presence of MMP-2 and MMP-9 in both cell lines through western blot (see Fig. 5) Detected intracellular activity of both gelatinases in HTB-19 and HTB-20 cells using zymography (see Fig. 4) Studies on effects of genistein on intracellular gelatinase activity in HTB-19 and HTB-20 cells are in progress Future Work Involvement of estrogen receptor in mediating effects of genistein in these cell lines remains to be analyzed Detection of intracellular activity of MMPs in HTB-19 and HTB-20 cells raises interesting possibilities for understanding their intracellular functions Future research in the Mahajan Lab will explore the role of MMPs in DNA repair 250 150 100 75 50 37 1 2 About 10 ng of rhMMP-2 in 25 μl of 25 mM Tris-glycine buffer pH 6.8 was mixed with equal volume of 2 x SDS-sample buffer (Life Technologies) without β-mercaptoethanol and incubated at 37 0 C for 10 minutes. Samples were loaded on a 10 % acrylamide -Tris- glycine gel containing 0.1% gelatin (Life Technologies) and electrophoresed using Tris-glycine pH 8.3 containing 0.1% SDS at 90 V for 120 min. Reactivation was performed by incubating the gel with gentle shaking in 40-50 ml of 2.5% Triton X-100 for 15 minutes x 3. For gelatin hydrolysis, the gel was immersed in 40-50 ml of 50 mM Tris- Glycine pH 7.5, 100 mM NaCl, 4 mM CaCl2 and 0.02% Triton X-100 and incubated at 37 0 C for 16 hours. Gelatinolytic activity was detected by staining with 0.05% Commassie Blue for 1 hour, followed by imaging on the ChemiDoc MP Imaging System (BioRad). Lane 1 : rhMMP2 (AbCam) Lane 2: Precision Plus Protein Dual Xtra Standards (BioRad) Molecular mass = kDa Figure 2. (a) (b) About 9μg protein from each sample in 15μL of sample was mixed with 15μL of 2 x SDS-sample buffer and were heated at 95 0 C for 5 minutes. Samples were loaded on a 4-20% acrylamide -Tris-glycine gel and electrophoresed using Tris-glycine pH 8.3 containing 0.1% SDS at 90 V for 120 min. Electrophoresed proteins were transblotted on to a PVDF membrane at 25 V for 180 min. Membrane was blocked with 4% Blotto (w/v) in TBST and incubated overnight at 4-6 o C with rabbit polyclonal antibodies (all procured from Santa Cruz Biotechnologies) specific for MMP-2 (H-76), MMP-9 (H-129) and β-Actin (R-22) at a 1:1000 dilution in Blotto. Excess primary antibody was washed using TBST. Membranes were incubated with goat anti-rabbit IgG – HRP conjugate at a 1:1000 dilution for 12 hours at 4-6oC. Excess secondary antibody was removed by four washes using TBST. Blots were developed using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo) following manufacturer's instructions. Imaging was carried out using the BioRad MPV Imager. Figure 3. Gelatinase activity of rhMMP2 Figure 4. Zymography of breast cancer cell extracts Figure 5. (a)(b) Acknowledgements We would like to thank the College of Pharmacy and Health Sciences for providing the funds and opportunity to work on this project. Work was conducted using the facilities in the Ellis Pharmacogenomics Laboratory. Finally, this work was also supported by the Grow Iowa Values Fund Research Grant to PBM. Bibliography 1. Colotti C, et al. Clin Chem Lab Med. 2007;45(10):1292-8 2. Fernandez-Gomez J, et al. Scand J Urol Nephrol.2011 Apr;45(3):171-6 3. Kolukula S. and Anderson D. J Cancer Sci Ther 2011, S1 4. Shah FD, eta l. Indian J Cancer. 2009 Jul-Sep;46(3):194-202 5. La Rocca G, et al. Br. J Cancer. 2004Apr 5;90(7):1414-21 6. Bahar M, et al. Nutr J. 2010 Nov 30;9:62. doi:10.1186/1475-2891-9-62 7. Dutta A, et al. J Oncol. 2009; 627840. doi: 10.1155/2009/627840. (HTB-19) Experimental schemeProtein analysis using Bradford assay (HTB-20) Breast cancer cell morphology M234M56718 92 72 66 40 Intracellular MMP-9 and MMP-2 in breast cancer cells Figure 1b. Chemical structure of Genistein and Estradiol (c)