Anil M. Limaye

Activation of latent TGF‐β1 by low‐power laser in vitro correlates with increased TGF‐β1 levels in laser‐enhanced oral wound healing

The term Laser “Photobiomodulation” was coined to encompass the pleiotropic effects of low‐power lasers on biological processes. The purpose of this study was to investigate whether transforming growth factor (TGF)‐β had a role in mediating the biological effects of low‐power far‐infrared laser irradiation. We assayed for in vitro activation using various biological forms of cell‐secreted, recombinant, and serum latent TGF‐β using the p3TP reporter and enzyme‐linked immunosorbent assays. We demonstrate here that low‐power lasers are capable of activating latent TGF‐β1 and ‐β3 in vitro and, further, that it is capable of “priming” these complexes, making them more amenable to physiological activation present in the healing milieu. Using an in vivo oral tooth extraction‐healing model, we observed an increased TGF‐β1, but not β3, expression by immunohistochemistry immediately following laser irradiation while TGF‐β3 expression was increased after 14 days, concomitant with an increased inflammatory infiltrate. All comparisons were performed between laser‐irradiated wounds and nonirradiated wounds in each subject essentially using them as their own control (paired T‐test p<0.05). Low‐power laser irradiation is capable of activating the latent TGF‐β1 complex in vitro and its expression pattern in vivo suggests that TGF‐β play a central role in mediating the accelerated healing response.

Epigenetic induction of tissue inhibitor of matrix metalloproteinase-3 by green tea polyphenols in breast cancer cells

Aberrant epigenetic silencing of the tissue inhibitor of matrix metalloproteinase‐3 (TIMP‐3) gene that negatively regulates matrix metalloproteinases (MMPs) activity has been implicated in the pathogenesis and metastasis of breast cancer. In the present study, we demonstrate that green tea polyphenols (GTP) and its major constituent, epigallocatechin‐3‐gallate (EGCG) mediate epigenetic induction of TIMP‐3 levels and play a key role in suppressing invasiveness and gelatinolytic activity of MMP‐2 and MMP‐9 in breast cancer cells. Treatment of MCF‐7 and MDA‐MB‐231 breast cancer cells with 20 µM EGCG and 10 µg/mL GTP for 72 h significantly induces TIMP‐3 mRNA and protein levels. Interestingly, investigations into the molecular mechanism revealed that TIMP‐3 repression in breast cancer cells is mediated by epigenetic silencing mechanism(s) involving increased activity of the enhancer of zeste homolog 2 (EZH2) and class I histone deacetylases (HDACs), independent of promoter DNA hypermethylation. Treatment of breast cancer cells with GTP and EGCG significantly reduced EZH2 and class I HDAC protein levels. Furthermore, transcriptional activation of TIMP‐3 was found to be associated with decreased EZH2 localization and H3K27 trimethylation enrichment at the TIMP‐3 promoter with a concomitant increase in histone H3K9/18 acetylation. Our findings highlight TIMP‐3 induction as a key epigenetic event modulated by GTPs in restoring the MMP:TIMP balance to delay breast cancer progression and invasion.

Enhanced production of optically pure d (–) lactic acid from nutritionally rich Borassus flabellifer sugar and whey protein hydrolysate based–fermentation medium

The aim of this study is to optimize the production of optically pure d (–) lactic acid (DLA) employing a cost‐effective production medium. Based on the designed biomass approach, Sporolactobacillus inulinus NBRC 13595 was found to exhibit high DLA titer (19.0 g L−1) and optical purity (99.6%). A cost‐effective medium was constituted using Palmyra palm jaggery (PJ) from Borassus flabellifer and whey protein hydrolysate (WPH) as carbon and nitrogen sources, respectively. Plackett–Burman design indicated that PJ, WPH, and MnSO4 as significant variables influence DLA production. A rotatable central composite design and response surface methodology were used to optimize the PJ and WPH concentrations. A maximum DLA titer (170.14 g L−1) was predicted for 222.24 g L−1 of PJ and 11.99 g L−1 of WPH, respectively. Fermentation experimental results exhibited a maximum DLA titer (189.0 ± 8.53 g L−1) and productivity (5.25 ± 0.24 g L−1 H−1), which is the highest ever reported for DLA production from a renewable feedstock in the batch process. The present investigation substantiates that the potential application of economically viable raw feedstocks (PJ and WPH) for enhanced DLA production, which is attributed to 2.5‐fold reduction in DLA production cost compared with conventional medium.

Expression profiling of genes modulated by estrogen, EGCG or both in MCF-7 breast cancer cells.

(−)-Epigallocatechin-3-gallate (EGCG) is one of the most potent and the most studied green tea catechin. Reports on mechanisms of EGCG action and its cellular targets are plenty. Compelling evidences in the literature in favor of ER being one of its targets suggest that EGCG may have a significant impact on estrogen regulated gene expression. Despite the possible implications on breast cancer chemoprevention or therapy, this aspect of EGCG action has not been adequately investigated. In order to address this issue, we have obtained gene expression profiles of MCF-7 breast cancer cells treated with ethanol (vehicle control) and those treated with estrogen, EGCG or both, using microarrays. Here, we have presented in detail the design and execution of the microarray experiment, quality control checks and analysis of microarray data. The utility and importance of the data generated in this work have been discussed in the context of the background literature. Our data is available in the Gene Expression Omnibus (GEO) database with the identifier GSE56245.

Direct inhibition of matrix metalloproteinase-2 (MMP-2) by (−)-epigallocatechin-3-gallate: A possible role for the fibronectin type II repeats

Matrix metalloproteinases (MMPs) -2 and -9, also called gelatinases, constitute a distinct subgroup within the MMP family of extracellular matrix remodeling proteases. Gelatinases are implicated in tumor cell invasion and metastasis, and are attractive therapeutic targets. Several synthetic small molecule inhibitors of MMPs developed till date have failed in clinical trials. This has prompted explorations into the gamut of dietary compounds and nutraceuticals for specific inhibitors of MMPs with desirable properties. (-)-epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, is popular as a potential chemotherapeutic agent with demonstrable anti-metastatic and MMP inhibitory activities. Here, we have addressed the mechanism of EGCG-mediated inhibition of MMP-2 using in silico molecular docking approach. We show for the first time that EGCG targets the fibronectin type II repeat regions 1 and 3 of MMP-2, binds amino acids that constitute the exosite of this enzyme and hinders proper positioning of the substrate. This study offers a novel insight into the inhibition of MMP-2 by EGCG and presents a starting point for development of novel therapeutic molecules that can specifically target the gelatinases.

Changes in gene expression following androgen receptor blockade is not equivalent to androgen ablation by castration in the rat ventral prostate

Involution of the rat ventral prostate and concomitant modulation of gene expression post-castration is a well-documented phenomenon. While the rat castration model has been extensively used to study androgen regulation of gene expression in the ventral prostate, it is not clear whether all the gene expression changes post-castration are due to androgen depletion alone. To obtain insights into this, we performed differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) which resulted in the identification of castration and/or flutamide-regulated genes in the rat ventral prostate. These include clusterin, methionine adenosyl transferase IIα, and prostate-specific transcripts such as PBPC1BS, S100RVP and A7. While clusterin, PBPC1BS and methionine adenosyl transferase IIα are regulated by both castration and flutamide, S100 RVP and A7 are regulated by castration alone. Interestingly, we show that flutamide, unlike castration, does not induce apoptosis in the rat ventral prostate epithelium, which could be an underlying cause for the differential effects of castration and flutamide treatment. We propose that castration leads to enrichment and depletion of stromal and epithelial cell types, respectively, resulting in erroneous conclusions on some of the cell type-specific transcripts as being androgen regulated.

Validation data of a rabbit antiserum and affinity purified polyclonal antibody against the N-terminus of human GPR30

Rabbit antiserum was generated against the N-terminus of human GPR30 followed by peptide affinity purification. In this article, the methodology used and validation data are presented. The peptide affinity purified polyclonal antibody specifically detects human GPR30 in ELISA and on western blots of total protein prepared from human breast cancer cell lines.

Abstract 253: Green tea polyphenol-mediated epigenetic reactivation of TIMP-3 reduces invasiveness and gelatinolytic activity in human breast cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The molecular pathway from tumor development to clinically evident metastasis is complex and consists of multiple sequential steps. Substantial data indicate the involvement of gelatinases/ type IV collagenases (MMP-2/9) secreted by tumor cells during cancer cell invasion and metastasis. MMP-2/9 are produced in a latent form as pro-matrix metalloproteinases that require activation and are inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). TIMPs are natural inhibitors of MMPs and are involved in diverse biological processes including cell proliferation, apoptosis, invasion, metastasis and angiogenesis. Four TIMP genes (TIMP-1, -2, -3, -4) and proteins are known in human which inhibit active form of most MMPs but with different affinities. TIMPs inhibit MMPs by binding in a 1:1 stoichiometry through strong non-covalent interaction. The balance between active MMPs and TIMPs determine tumor progression by regulating the net activity of MMPs. Aberrant epigenetic silencing of TIMP-3 has been implicated in the pathogenesis and metastasis of breast cancer. In the present study, we demonstrate that green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) mediate epigenetic induction of TIMP-3 levels and play a key role in suppressing invasiveness and gelatinolytic activity of MMP-2/9 in breast cancer cells. Treatment of MCF-7 and MDA-MB-231 breast cancer cells with 20μM EGCG and 10μg/ml GTP for 72 h significantly induces TIMP-3 mRNA and protein levels. Interestingly, investigations into the molecular mechanism revealed that TIMP-3 repression in breast cancer cells is mediated by epigenetic silencing mechanism(s) involving increased activity of the enhancer of zeste homolog 2 (EZH2) and class I histone deacetylases, independent of promoter DNA hypermethylation. Treatment of breast cancer cells with GTP/EGCG significantly reduced EZH2 and class I HDAC protein levels. Furthermore, transcriptional activation of TIMP-3 was found to be associated with decreased EZH2 localization and H3K27 trimethylation enrichment at the TIMP-3 promoter with a concomitant increase in histone H3K9/18 acetylation. Our findings highlight TIMP-3 induction as a key epigenetic event modulated by GTP/EGCG in restoring the MMP:TIMP balance to delay breast cancer progression and invasion. Although case-control and epidemiological studies provide some clues that regular consumption of GTP and EGCG could decrease the risk of breast cancer invasion and/or progression however these studies do not provide further insight into the molecular mechanism, related to tumor inhibition. Based on our present findings, TIMP-3 can be used as a surrogate marker of GTP/EGCG response and warrants further investigation through well designed clinical trial. Citation Format: Gauri Deb, Vijay S. Thakur, Anil M. Limaye, Sanjay Gupta. Green tea polyphenol-mediated epigenetic reactivation of TIMP-3 reduces invasiveness and gelatinolytic activity in human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 253. doi:10.1158/1538-7445.AM2014-253

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.