Anindita Dutta

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7.

AIMS The tumor inhibiting property of green tea polyphenol epigallocatechin-3-gallate (EGCG) is well documented. Studies reveal that matrix-metalloproteinases (MMPs) play pivotal roles in tumor invasion through degradation of basement membranes and extracellular matrix (ECM). We studied the effect of EGCG on matrixmetalloproteinases-2 (MMP-2), the factors involved in activation, secretion and signaling molecules that might be involved in the regulation of MMP-2 in human breast cancer cell line, MCF-7. MAIN METHODS MCF-7 was treated with EGCG (20 muM, 24 h), the effect of EGCG on MMP-2 expression, activity and its regulatory molecules were studied by gelatin zymography, Western blot, quantitative and semi-quantitative real time RT-PCR, immunoflourescence and cell adhesion assay. KEY FINDINGS EGCG treatment reduced the activity, protein expression and mRNA expression level of MMP-2. EGCG treatment reduced the expression of focal adhesion kinase (FAK), membrane type-1-matrix metalloproteinase (MT1-MMP), nuclear factor-kappa B (NF-kB), vascular endothelial growth factor (VEGF) and reduced the adhesion of MCF-7 cells to ECM, fibronectin and vitronectin. Real time RT-PCR revealed a reduced expression of integrin receptors alpha5, beta1, alphav and beta3 due to EGCG treatment. SIGNIFICANCE Down regulation of expression of MT1-MMP, NF-kB, VEGF and disruption of functional status of integrin receptors may indicate decreased MMP-2 activation; low levels of FAK expression might indicate disruption in FAK-induced MMP-2 secretion and decrease in activation of phosphatidyl-inositol-3-kinase (PI-3K), extracellular regulated kinase (ERK) indicates probable hindrance in MMP-2 regulation and induction. We propose EGCG as potential inhibitor of expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in MCF-7.

Hypertension with elevated levels of oxidized low-density lipoprotein and anticardiolipin antibody in the circulation of premenopausal Indian women chronically exposed to biomass smoke during cooking.

UNLABELLED This study aims to investigate whether indoor air pollution (IAP) from biomass fuel use was associated with hypertension, platelet hyperactivity, and elevated levels of oxidized low-density lipoprotein (oxLDL) and anticardiolipin antibody (aCL). We enrolled 244 biomass fuel-using (median age 34 year) and 236 age-matched control women who cooked with liquefied petroleum gas (LPG). Enzyme-linked immunosorbent assay was used to measure oxLDL in plasma and aCL in serum, flow cytometry for P-selectin expression on platelet and reactive oxygen species (ROS) generation by leukocytes, aggregometry for platelet aggregation, spectrophotometry for superoxide dismutase (SOD) in erythrocytes, and laser photometer for particulate matter <10 and 2.5 μm in diameter (PM(10) and PM(2.5), respectively) in cooking areas. Biomass users had three times more particulate pollution in kitchen, had higher prevalence of hypertension (29.5 vs. 11.0% in control, P < 0.05), elevated oxLDL (170.6 vs. 45.9 U/l; P < 0.001), platelet P-selectin expression (9.1% vs. 2.4%), platelet aggregation (23.2 vs. 15.9 Ohm), raised aCL IgG (28.7% vs. 2.1%), IgM (8.6% of vs. 0.4%), and ROS (44%) but depleted (13%) SOD. After controlling potential confounders, the changes were positively associated with PM(10) and PM(2.5) in indoor air, suggesting a positive association between IAP and increased cardiovascular risk. PRACTICAL IMPLICATIONS The study showing high risk of developing cardiovascular diseases (CVD) among poor, underprivileged women in their reproductive ages in rural India is important from public health perspectives. It may motivate the government and the regulatory agencies of the country to take a serious note of the indoor air pollution (IAP) from biomass fuel use as it threatens the health of millions of women, children, and the elderly who mostly stay indoor. We hope the findings will strengthen the demand for setting up a standard for indoor air quality in the country in the line of national ambient air quality standard. The findings may also inspire the authorities to take measures for the reduction in IAP by improving housing, kitchen ventilation, and cook stoves. Moreover, the parameters used in this study can be utilized for large, population-based studies to identify women at a higher risk of developing CVD so that medical intervention can be taken at the formative stage of a disease.

Epigallocatechin-3-gallate (EGCG) downregulates gelatinase-B (MMP-9) by involvement of FAK/ERK/NFκB and AP-1 in the human breast cancer cell line MDA-MB-231

Epigallocatechin-3-gallate (EGCG) is effective against the initiation, progression, and invasion of carcinogenesis. Matrix-metalloproteinases (MMPs) are a family of endopeptidases that hydrolyze the majority of extracellular proteins. MMP-9 is one of the most important members of the family and we observed the effect of EGCG on MMP-9 in the human breast cancer cell line, MDA-MB-231. The effect of EGCG on MMP-9 was studied by gelatin zymography, western blot, quantitative and semiquantitative real-time RT-PCR, immunoflourescence, cell adhesion assay, enzyme-linked immunosorbent assay, and electrophoretic mobility shift assay. EGCG treatment reduced the activity, protein, and mRNA expression of MMP-9 and enhanced the expression of the tissue inhibitor of MMP 1 (TIMP-1). EGCG downregulated the activation of focal adhesion kinase (FAK) and extracellular regulated kinase (ERK), reduced the adhesion of MDA-MB-231 cells to fibronectin and vitronectin, and reduced the mRNA expression of the integrin receptors α5β1 and αvβ3. The expression of the nuclear factor kappa B (NFκB), and the DNA binding activity of NFκB and activator protein 1 (AP1) to MMP-9 promoter were noticeably reduced on EGCG treatment. Upregulation of TIMP-1 and disruption of the functional status of integrin receptors may indicate decreased MMP-9 activation; inhibition of FAK and ERK activation might indicate disruption in the FAK/ERK-induced MMP-9 secretion and induction. Decreased DNA binding activity of NFκB and AP1 to MMP-9 promoter might indicate transcriptional deregulation of MMP-9 gene on EGCG treatment. We propose EGCG as a potential inhibitor of the expression and activity of MMP-9 by a process involving FAK/ERK and transcription factors in MDA-MB-231.

Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)

Background. Vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We studied the effect of ATRA on MMP-2 in MCF-7, human breast cancer cells, and the probable signaling pathways which are affected by ATRA on regulating pro-MMP-2 activity and expression. Methods. Gelatin zymography, RT-PCR, ELISA, Western blot, Immunoprecipitation, and Cell adhesion assay are used. Results. Gelatin zymography showed that ATRA caused a dose-dependent inhibition of pro-MMP-2 activity. ATRA treatment downregulates the expression of MT1-MMP, EMMPRIN, FAK, NF-kB, and p-ERK. However, expression of E-cadherin, RAR, and CRABP increased upon ATRA treatment. Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment. Conclusions. The experimental findings clearly showed the inhibition of MMP-2 activity upon ATRA treatment. This inhibitory effect of ATRA on MMP-2 activity in human breast cancer cells (MCF-7) may result due to its inhibitory effect on MT1-MMP, EMMPRIN, and upregulation of TIMP-2. This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

Fibronectin–integrin mediated signaling in human cervical cancer cells (SiHa)

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin–integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin–integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-κB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

Chronic inhalation of biomass smoke is associated with DNA damage in airway cells: involvement of particulate pollutants and benzene.

This study examined whether indoor air pollution from biomass fuel burning induces DNA damage in airway cells. For this, sputum cells were collected from 56 premenopausal rural women who cooked with biomass (wood, dung, crop residues) and 49 age‐matched controls who cooked with cleaner liquefied petroleum gas. The levels of particulate matters with diameters of less than 10 and 2.5 µm (PM10 and PM2.5) in indoor air were measured using a real‐time aerosol monitor. Benzene exposure was monitored by measuring trans,trans‐muconic acid (t,t‐MA) in urine by HPLC‐UV. DNA damage was examined by alkaline comet assay in sputum cells. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in sputum cells were measured by flow cytometry and spectrophotometry, respectively. Compared with controls, biomass users had 4 times higher tail percentage DNA, 37% more comet tail length and 5 times more Olive tail moment (p < 0.001) in inflammatory and epithelial cells in sputum, suggesting extensive DNA damage. In addition, women who cooked with biomass had 6 times higher levels of urinary t,t‐MA and 2‐fold higher levels of ROS generation concomitant with 28% depletion of SOD. Indoor air of biomass‐using households had 2–4 times more PM10 and PM2.5 than that of controls. After controlling potential confounders, positive association was found between DNA damage parameters, particulate pollution, urinary t,t‐MA and ROS. Thus, long‐term exposure to biomass smoke induces DNA damage in airway cells and the effect was probably mediated, at least in part, by oxidative stress generated by inhaled particulate matter and benzene. Copyright

All-trans retinoic acid (ATRA) downregulates MMP-9 by modulating its regulatory molecules.

The vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We aimed to study the effect of ATRA on MMP-9 in MDA-MB-231, human breast cancer cells and the probable molecular mechanisms through which ATRA exerts its effect. Results: Our experimental findings demonstrate that ATRA enters into the nucleus and regulates various signaling pathways viz. Integrin, FAK, ERK, PI-3K, NF-κB and also EGFR and down regulates pro-MMP-9 activity as well as its expression. As a result MDA-MB-231 cell migration on fibronectin medium gets retarded in presence of ATRA. ATRA up regulates TIMP-1 expression. Conclusions: Our study may help to understand the role of ATRA as a regulator of MMP-9 and the possible signaling pathways which are involved in this ATRA mediated down regulation of MMP-9.

Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women in India who cook with biomass fuel.

The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the airways has been investigated in 187 pre-menopausal women (median age 34years) from eastern India who cooked exclusively with biomass and 155 age-matched control women from same locality who cooked with cleaner fuel liquefied petroleum gas. Compared with control, Papanicolau-stained sputum samples showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt (p-Akt(ser473) and p-Akt(thr308)) proteins in AEC of biomass users, especially in metaplastic and dysplastic cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species (ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative stress. There were 2-5 times more particulate pollutants (PM(10) and PM(2.5)), 72% more nitrogen dioxide and 4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-using households, and high performance liquid chromatography estimated 6-fold rise in the concentration of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia, p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-mediated activation of Akt signal transduction pathway.

Extracellular Matrix Protein Laminin Induces Matrix Metalloproteinase-9 in Human Breast Cancer Cell Line MCF-7

Studies on interaction of tumor cells with extracellular matrix (ECM) components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human breast cancer cell line MCF-7-laminin (LM) interaction on MMPs and the underlying signaling pathways. Culturing of MCF-7 cells on LM coated surface upregulated MMP-9 expression as well as reduced tissue inhibitor of metalloproteinases-1 (TIMP-1) expression. LM induced MMP-9 expression is abrogated by the blockade of α2 integrin. Inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI3K), extracellular signal regulated kinase (ERK) and nuclear factor-kappaB (NF-κB) in LM induced signaling. LM treatment also enhanced phosphorylation of FAK (focal adhesion kinase), PI3K, ERK; nuclear translocation of ERK, pERK, NF-κB and cell migration. Our findings indicate that, binding of MCF-7 cells to LM, possibly via α2β1 integrin, induces signaling involving FAK, PI3K, ERK, NF-κB followed by upregulation of MMP-9 and cell migration.

Increased cardiovascular risk in association with chronic airflow obstruction among premenopausal rural women of India who cook exclusively with biomass

We aimed to compare the cardiovascular risk in biomass-using women with or without chronic obstructive pulmonary disease (COPD). A total of 22 biomass-using married women with COPD and 24 matched controls with normal lung function were enrolled for this purpose. Platelet P-selectin (P-sel) expression and platelet–leukocyte aggregation were determined using flow cytometry. Platelet aggregation by collagen was measured by aggregometer. Soluble P-selectin (sP-sel), tumor necrosis factor-alpha (TNF-α), interleukin-8, -6, -10 (IL-8, IL-6, IL-10), neutrophil-activating protein-2 (NAP-2), C-reactive protein (CRP), oxidized low density lipoprotein (oxLDL) in plasma were measured by enzyme-linked immunosorbent assay. Generation of reactive oxygen species (ROS) by leukocytes was measured by flow cytometry, and erythrocyte content of superoxide dismutase (SOD) was measured by spectrophotometry. Particulate matter with a diameter of less than 2.5 μm (PM2.5) in indoor air was measured by real-time aerosol monitor. Compared with control, biomass users with COPD had increased expression of platelet P-selectin, elevated levels of sP-sel, oxLDL, TNF-α, IL-8, IL-6, NAP-2, CRP, lowered IL-10 and more circulating platelet-neutrophil (p < 0.0001) and platelet–monocyte (p < 0.0001) aggregates. ROS generation was increased by 19.5% while SOD was depleted by 32% in women with COPD. Biomass smoke-induced COPD is associated with excess cardiovascular risk via oxidative stress, platelet activation, and inflammation.