Data in support of the negative influence of divalent cations on (−)-epigallocatechin-3-gallate (EGCG)-mediated inhibition of matrix metalloproteinase-2 (MMP-2)

In this data article we have provided evidence for the negative influence of divalent cations on (−)‐epigallocatechin-3-gallate (EGCG)-mediated inhibition of matrix metalloproteinase-2 (MMP-2) activity in cell-free experiments. Chelating agents, such as EDTA and sodium citrate alone, did not affect MMP-2 activity. While EDTA enhanced, excess of divalent cations interfered with EGCG-mediated inhibition of MMP-2.


a b s t r a c t
In this data article we have provided evidence for the negative influence of divalent cations on ( À )-epigallocatechin-3-gallate (EGCG)-mediated inhibition of matrix metalloproteinase-2 (MMP-2) activity in cell-free experiments. Chelating agents, such as EDTA and sodium citrate alone, did not affect MMP-2 activity. While EDTA enhanced, excess of divalent cations interfered with EGCGmediated inhibition of MMP-2.
& 2016 Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Relevant for researchers investigating the bioavailability and therapeutic efficacy of EGCG.

Cell culture
HT1080 human fibrosarcoma cells were obtained from the National Centre for Cell Science, Pune, India. They were routinely cultured in 25 cm 2 flasks (Greiner Bio-one, GmbH, Germany) in a humidified incubator maintained at 37°C with 5% CO 2 . Cells were fed with phenol red-containing DMEM, which was supplemented with 10% FBS (PAA Laboratories, GmbH, Austria), 100 U/mL penicillin and 100 mg/mL streptomycin (HiMedia, Mumbai, India). When confluent, the HT1080 conditioned medium (HT1080-cm), which is a source of gelatinases namely MMP-2 and MMP-9, was aspirated out and centrifuged at 12,000 rpm for 5 min to remove cell debris. The supernatant (HT1080-cm) was stored in aliquots at À 80°C until use.

Experiments to study the effect of EGCG on MMP-2 activity in vitro
Aliquots of HT1080-cm were mixed with appropriate volumes of EGCG stock solution to maintain the desired concentrations. Aliquots mixed with equal volume of ethanol alone served as controls. The mixtures were incubated at 37°C for 45 min and analyzed by gelatin zymography. Alternatively, equal quantities (10 ng) of active human MMP-2 were added to a reaction buffer containing indicated concentrations of EGCG. After incubation at 37°C for 45 min, the mixtures were analyzed by gelatin zymography. The effect of EGCG on active MMP-2 was also studied using FRET-peptide based fluorogenic substrate assay. This assay is based on cleavage of an MMP-2 specific fluorogenic peptide substrate. In each well of a 96-well plate, 10 ng of active MMP-2 was mixed with indicated concentrations of EGCG in assay buffer (10 mM Tris-HCl at pH 7, 5 mM CaCl 2 , 1 mM ZnCl 2 ) in a total volume of 150 mL and incubated at 37°C for 30 min. This was followed by addition of peptide substrate to a final concentration of 20 mM and further incubation of the reaction mixture at 37°C for 1 h.
Fluorescence was measured using Synergy HT Multimode Microplate reader (BioTeK) using λ max excitation of 324 nm and λ max emission of 393 nm.

Experiments to study the effect of EDTA and sodium citrate on EGCG-mediated suppression of MMP-2 activity
These experiments were performed exactly as described in the previous section except that HT1080-cm or pure active MMP-2 protein was pre-incubated with indicated concentrations of EDTA or sodium citrate at 37°C for 30 min prior to addition of EGCG.

Gelatin zymography
Gelatin zymography was performed as described earlier with minor modifications [1]. Samples were resolved in 7.5% SDS-polyacrylamide gels containing 0.1% gelatin under nondenaturing conditions. The gels were washed in 2.5% Triton X-100 to remove SDS and incubated overnight in the activation buffer (10 mM Tris-HCl at pH 7, 5 mM CaCl 2 , 1 mM ZnCl 2 , and 1.5% Triton X-100). The gels were stained with 0.5% coomassie brilliant blue followed by destaining with methanol:glacial acetic acid:water (30:10:60) until clear bands were visible against blue background. The gels were either scanned or photographed using Kodak gel imaging system. The digital images were analyzed by ImageJ software [2]. It should be noted that HT1080-cm samples used in gelatin zymography experiments were not treated with 4-aminophenylmercuric acetate (APMA). Hence the bands detected are of pro-MMP-2 and pro-MMP-9.

Data presentation and analysis
Bar graphs were plotted in MS Excel. Data were analyzed by R statistical package [3]. ANOVA and comparison of group means was done using Tukey's test at 5% level of significance.