Biomedical subjects

Sivaprakasam Balasubramanian

Publications and source records attributed to Sivaprakasam Balasubramanian.

13 recordsLinked to original sources

PubMed2006

Curcumin suppresses AP1 transcription factor-dependent differentiation and activates apoptosis in human epidermal keratinocytes.

The diet-derived cancer preventive agent, curcumin, inhibits skin cancer cell proliferation and tumor formation. However, its effect on normal human keratinocyte differentiation, proliferation, and apoptosis has not been adequately studied. Involucrin (hINV) is a marker of keratinocyte differentiation and a useful model for the study of chemopreventive agent action. We show that curcumin suppresses the differentiation agent-dependent activation of hINV gene expression and that an AP1 transcription factor DNA binding site in the hINV gene is required for this regulation. A protein kinase C, Ras, MEKK1, MEK3 signaling cascade controls hINV expression by regulating AP1 factor level. Curcumin treatment inhibits the novel protein kinase C-, Ras-, and MEKK1-dependent activation of hINV promoter activity and reduces the differentiation agent-dependent increase in AP1 factor level and DNA binding. This reduction requires proteasome function. In addition, curcumin treatment reduces cell number, which is associated with a reduced cyclin and cdk1 levels. Curcumin treatment also suppresses the Bcl-xL level, leading to reduced mitochondrial membrane potential and increased cleavage of procaspases and poly(ADP-ribose) polymerase. These studies provide important insights regarding the mechanism whereby curcumin acts as a chemopreventive agent in normal human epidermis.

Apoptosis↗

PubMed2006

Apigenin inhibition of involucrin gene expression is associated with a specific reduction in phosphorylation of protein kinase Cdelta Tyr311.

Apigenin is a plant-derived flavanoid that has significant promise as a skin cancer chemopreventive agent. In the present study, we examine the mechanism whereby apigenin regulates normal human keratinocyte differentiation. Expression of involucrin (hINV), a marker of keratinocyte differentiation, is increased by differentiating agents via a protein kinase Cdelta (PKCdelta), Ras, MEKK1, MEK3 cascade that increases AP1 factor level and AP1 factor binding to DNA elements in the hINV promoter. We show that apigenin inhibits this response. Apigenin suppresses the 12-O-tetradeconylphorbol-13-acetate-dependent increase in AP1 factor expression and binding to the hINV promoter and the increase in hINV promoter activity. Apigenin also inhibits the increase in promoter activity observed following overexpression of PKCdelta, constitutively active Ras, or MEKK1. The suppression of PKCdelta activity is associated with reduced phosphorylation of PKCdelta-Y311. The physiological importance of this phosphorylation event was confirmed by showing that the PKCdelta phosphorylation-defective mutant, PKCdelta-Y311F, is less able to increase hINV promoter activity. Activation of hINV promoter activity by the green tea polyphenol, (-)-epigellocathecin-3-gallate, is also inhibited by apigenin, suggesting that the two chemopreventive agents can produce opposing actions in keratinocytes. Additional studies show that the apigenin-dependent suppression of differentiation is associated with reduced cell proliferation but that there is no evidence of apoptosis.

Apigenin↗

PubMed2006

Opposing action of curcumin and green tea polyphenol in human keratinocytes.

Persistent environmental insult can convert a normal cell into a cancer cell. However, various natural chemopreventive agents called antioxidants can retard this progression. We have recently explored the effects of several chemopreventive agents, including green tea polyphenol and curcumin, on normal human keratinocyte function. Our findings suggest that a bioactive polyphenol from green tea, (-)-epigallocatechin-3-gallate (EGCG), acts to increase involucrin gene expression, suggesting that EGCG treatment enhances normal human keratinocyte differentiation. Mechanistic studies indicate that EGCG alters mitogen-activated protein kinase cascade function to activate involucrin gene transcription via a Ras, MEKK1, MEK3, ERK1/2-p38delta cascade that targets AP1 and CAATT enhancer binding protein transcription factors. These findings suggest that EGCG may inhibit disease progression by promoting keratinocyte differentiation. Parallel studies indicate that not all antioxidants produce a similar response. Curcumin, an antioxidant derived from the turmeric, antagonizes the EGCG-dependent response by interfering in this signaling pathway. These studies suggest that different antioxidant may produce antagonistic effects in tissues.

Antioxidants↗

PubMed2005

A novel retinoid-related molecule inhibits pancreatic cancer cell proliferation by a retinoid receptor independent mechanism via suppression of cell cycle regulatory protein function and induction of caspase-associated apoptosis.

Retinoid-related molecules are important potential agents for the treatment of cancer. In the present study, we test the effect of a novel retinoid-related ligand, AGN193198 (4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-prophenyl] benzoic acid), on pancreatic cancer cell proliferation and survival. AGN193198 treatment reduces BxPC-3 cell proliferation more efficiently than high-affinity retinoid acid receptor (RAR)- or retinoid X receptor (RXR)-selective retinoids. Moreover, AGN193198 does not activate transcription from RAR or RXR response elements and its effects on cell survival are not reversed by treatment with RAR- or RXR receptor-selective antagonists. These results suggest that the AGN193198-dependent inhibition of BxPC-3 cell function is not mediated via activation of the classical retinoid receptors. Cell cycle analysis of AGN193198-treated BxPC-3 cells indicates that AGN193198 causes accumulation of cells in G2/M. This change is associated with a marked reduction in regulators of S (cyclin A, cyclin-dependent kinase (cdk)2), G2/M (cyclin B1, cdk1, cdc25c) and G1 (cyclin D1, cyclin E, cdk2, cdk4) phase, and an increase in p21 and p27 level. Kinases assays reveal that cdk1, cdk2 and cdk4 activity are suppressed in AGN193198-treated cells. In addition, reduced cell proliferation is associated with enhanced procaspase (3, 8 and 9) and PARP cleavage. Z-VAD-FMK, a pancaspase inhibitor, inhibits AGN193198-dependent caspase activation and attenuates cell death. Z-VAD-FMK inhibits PARP cleavage, but does not alter the AGN193198-dependent reduction in cell cycle regulatory protein expression and activity, suggesting that caspase activation and suppression of cell cycle regulatory protein levels are independent processes. AGN193198 produces similar responses in other pancreatic cancer cell lines including AsPC-1 and MIA PaCa-2. These studies suggest that AGN193198 may be useful for the treatment of pancreatic cancer.

Apoptosis↗

PubMed2005

Human epidermal keratinocytes undergo (-)-epigallocatechin-3-gallate-dependent differentiation but not apoptosis.

Epigallocatechin-3-gallate (EGCG) is an important chemopreventive agent derived from green tea. We recently reported that EGCG treatment enhances keratinocyte differentiation as evidenced by increased human involucrin promoter activity [Balasubramanian,S., Efimova,T. and Eckert,R.L. (2002) J. Biol. Chem., 277, 1828-1836]. In the present paper, we extend these findings and show that EGCG also increases the expression of other differentiation markers-procaspase 14 and type I transglutaminase (TG1). Both TG1 mRNA and protein level, and activity are increased by treatment with EGCG. Increased TG1 activity is evidenced by a direct transglutaminase assay, and by the ability of EGCG to stimulate the covalent incorporation of fluorescein cadaverine substrate into crosslinked intracellular structures. In contrast, type II transglutaminase levels are not altered by EGCG treatment. We also assessed whether EGCG promotes keratinocyte apoptosis. We show that EGCG treatment does not promote the cleavage of procaspase-3, -8, -9 or poly(ADP-ribose) polymerase. Moreover, treatment with the pan-caspase inhibitor, Z-VAD-FMK, does not reverse the EGCG-associated reduction in cell viability. In addition, there is no increase in cells having sub-G(1)/S DNA content, and no evidence for the release of cytochrome c from the mitochondria. These findings confirm, using several endpoints, that EGCG treatment enhances normal keratinocyte differentiation but does not promote apoptosis.

Apoptosis↗

PubMed2004

Antioxidants regulate normal human keratinocyte differentiation.

Cancer begins with a normal cell that, due to persistent environmental insult, is transformed, via a series of progressively more insidious steps, into a cancer cell. A major goal of chemopreventive therapy is to alter the normal cell response to the environmental agent with the goal of inhibiting disease progression. (-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive green tea antioxidant that possesses remarkable cancer chemopreventive properties. We have recently explored the hypothesis that EGCG prevents cancer by promoting keratinocyte differentiation. Based on our findings, we argue that EGCG acts to enhance the differentiation of normal keratinocytes. This is a potentially important finding, as it represents a novel mechanism of disease inhibition by EGCG--cancer preventive "differentiation therapy". However, not all antioxidant chemopreventive agents work by this mechanism. Curcumin, for example, inhibits the differentiation-promoting activity of EGCG. This report discusses the mechanism of EGCG and curcumin action in regulating expression of involucrin, a marker of keratinocyte differentiation.

Anticarcinogenic Agents↗

PubMed2004

Green tea polyphenol and curcumin inversely regulate human involucrin promoter activity via opposing effects on CCAAT/enhancer-binding protein function.

Antioxidants are important candidate agents for the prevention of disease. However, the possibility that different antioxidants may produce opposing effects in tissues has not been adequately explored. We have reported previously that (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol antioxidant, stimulates expression of the keratinocyte differentiation marker, involucrin (hINV), via a Ras, MEKK1, MEK3, p38delta signaling cascade (Balasubramanian, S., Efimova, T., and Eckert, R. L. (2002) J. Biol. Chem. 277, 1828-1836). We now show that EGCG activation of this pathway results in increased CCAAT/enhancer-binding protein (C/EBPalpha and C/EBPbeta) factor level and increased complex formation at the hINV promoter C/EBP DNA binding site. This binding is associated with increased promoter activity. Mutation of the hINV promoter C/EBP binding site eliminates the regulation as does expression of GADD153, a dominant-negative C/EBP factor. In contrast, a second antioxidant, curcumin, inhibits the EGCG-dependent promoter activation. This is associated with inhibition of the EGCG-dependent increase in C/EBP factor level and C/EBP factor binding to the hINV promoter. Curcumin also inhibits the EGCG-dependent increase in endogenous hINV levels. The curcumin-dependent suppression of C/EBP factor level is inhibited by treatment with the proteasome inhibitor MG132, suggesting that the proteasome function is required for curcumin action. We conclude that curcumin and EGCG produce opposing effects on involucrin gene expression via regulation of C/EBP factor function. The observation that two antioxidants can produce opposite effects is an important consideration in the context of therapeutic antioxidant use.

Antioxidants↗

PubMed2004

Suppression of human pancreatic cancer cell proliferation by AGN194204, an RXR-selective retinoid.

Retinoids may be useful agents for the treatment of pancreatic cancer. However, retinoic acid receptor (RAR)-selective retinoids produce unwanted side effects. In contrast, retinoid X receptor (RXR)-selective retinoids produce fewer side effects; however, it was not known whether RXR-selective retinoids could reduce pancreatic tumor cell proliferation. In the present study, the novel RXR-selective retinoid, AGN194204, was compared with that of other retinoids for the ability to suppress pancreatic cancer cell proliferation. We treated various pancreatic cancer cell lines with receptor-selective ligands and cytotoxic agents and monitored the effects on cell proliferation, markers of apoptosis and cell cycle. Our results indicate that AGN194204, at concentrations >10 nM, inhibits proliferation of MIA PaCa-2 and BxPC-3 cells but not the proliferation of AsPC-1 cells. Moreover, in BxPC-3 and MIA PaCa-2 cells, AGN194204 was 10-100 times more effective than RAR-selective retinoids. AGN194204-dependent suppression of MIA PaCa-2 cell proliferation is associated with reduced cyclin E and cyclin-dependent kinase 6 (cdk6) level, but cyclin D1, cdk2 and cdk4 content is not altered. In addition, p27 level increases 2-fold. The RXR-selective antagonist, AGN195393, reverses the AGN194204-dependent growth inhibition and the decline in cyclin E and cdk6 levels. In contrast, these changes are not reversed by treatment with the RAR antagonist, AGN193109. AGN194204 did not appear to alter cell apoptosis as measured by change in cleavage of procaspase-3, -8 or -9. We also examined the effects AGN194204 co-treatment with cytotoxic agents. Treatment of MIA PaCa-2 cells with AGN194204 + cisplatin, gemcitabine, 5-fluorouracil, interferon (IFN)alpha or IFNgamma resulted in an additive but not synergistic reduction in MIA PaCa-2 cell number. These results indicate that AGN194204, an RXR-selective retinoid, is a more effective inhibitor of pancreatic cell proliferation than the RAR-selective retinoids, and further indicate that AGN194204 produces an additive reduction in cell number when given with other agents. Our results suggest that RXR-selective ligands, which are less toxic than RAR-selective ligands, may be suitable agents for the treatment of pancreatic cancer.

Antimetabolites, Antineoplastic↗

PubMed2004

Regulation of involucrin gene expression.

The epidermis is a dynamic renewing structure that provides life-sustaining protection from the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a carefully choreographed program of differentiation. Alteration of these events results in a variety of debilitating and life-threatening diseases. Understanding how this process is regulated is an important current goal in biology. In this review, we summarize the literature regarding regulation of involucrin, an important marker gene that serves as a model for understanding the mechanisms that regulate the differentiation process. Current knowledge describing the role of transcription factors and signaling cascades in regulating involucrin gene expression are presented. These studies describe a signaling cascade that includes the novel protein kinase C isoforms, Ras, MEKK1, MEK3, and a p38delta-extracellular signal regulated kinase 1/2 complex. This cascade regulates activator protein one, Sp1, and CCATT/enhancer-binding protein transcription factor DNA binding to two discrete involucrin promoter regions, the distal- and proximal-regulatory regions, to regulate involucrin gene expression.

Gene Expression Regulation↗

PubMed2003

Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases.

Epidermal growth factor receptor (EGFR) activation is absolutely required for cervical cell proliferation. This suggests that EGFR-inhibitory agents may be of therapeutic value. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a bioactive green tea polyphenol, on EGFR signaling in cervical cells. EGCG inhibits epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibits EGFR-dependent AKT activity. The EGCG-dependent reduction in ERK and AKT activity is associated with reduced phosphorylation of downstream substrates, including p90RSK, FKHR, and BAD. These changes are associated with increased p53, p21(WAF-1), and p27(KIP-1) levels, reduced cyclin E level, and reduced CDK2 kinase activity. Consistent with these findings, flow cytometry and TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling) staining revealed EGCG-dependent G(1) arrest. Moreover, sustained EGCG treatment caused apoptotic cell death. In addition to inhibiting EGFR, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and AKT, suggesting that EGCG acts simultaneously at multiple levels to inhibit EGF-dependent signaling. Importantly, the EGCG inhibition is selective, as EGCG does not effect the EGFR-dependent activation of JNK. These results suggest that EGCG acts to selectively inhibit multiple EGF-dependent kinases to inhibit cell proliferation.

Antineoplastic Agents, Phytogenic↗

PubMed2003

p38 Mitogen-activated protein kinases on the body surface--a function for p38 delta.

The p38 family of mitogen-activated protein kinases includes p38 alpha (SAPK2a, CSBP), p38 beta (SAPK2b), p38 delta (SAPK4), and p38 gamma (SAPK3/ERK6). p38 alpha and p38 beta are widely expressed p38 isoforms that are involved in regulation of cell proliferation, differentiation, development, and response to stress. Relatively less is known regarding the function of the p38 delta isoform. In this review, we discuss the role of the p38 alpha, p38 beta, and p38 gamma isoforms and then present recent findings that define a role for p38 delta as a regulator of differentiation-dependent gene expression in keratinocytes.

Animals↗

PubMed2002

Keratinocyte survival, differentiation, and death: many roads lead to mitogen-activated protein kinase.

The epidermis is a dynamic and continually renewing surface that provides and maintains a life-sustaining interface with the environment. The epidermal keratinocyte, the major cell type of the epidermis, undergoes a complex and carefully choreographed program of differentiation. This process requires a balance between keratinocyte proliferation, differentiation, and apoptosis. This overview will concentrate on cascades that regulate the balance between keratinocyte cell proliferation and survival, and apoptosis and cell differentiation, with a particular emphasis on the role of the mitogen-activated protein kinase cascades. A summary of the literature suggests that extracellular regulated kinases function to promote keratinocyte proliferation and survival, whereas p38 mitogen-activated protein kinase functions to promote differentiation and apoptosis.

Animals↗

PubMed2001

Green tea polyphenol stimulates a Ras, MEKK1, MEK3, and p38 cascade to increase activator protein 1 factor-dependent involucrin gene expression in normal human keratinocytes.

(-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea that efficiently reduces epidermal cancer cell proliferation. This inhibition is associated with a reduction in activator protein 1 (AP1) transcription factor level and activity. However, its effects on AP1 function in normal epidermal cells have not been extensively explored. Our present studies show that EGCG regulates normal keratinocyte function. To understand the mechanism of action, we examined the effects of EGCG on AP1 factor activity, MAPK signal transduction, and expression of the AP1 factor-regulated human involucrin (hINV) gene. EGCG increases hINV promoter activity in a concentration-dependent manner that requires the presence of an intact hINV promoter AP1 factor binding site. This response appears to be physiologic, as endogenous hINV gene expression is also increased. Fra-1, Fra-2, FosB, JunB, JunD, c-Jun, and c-Fos levels are increased by EGCG treatment, as is AP1 factor binding to hINV promoter AP1 site. Gel mobility shift studies show that this complex contains Fra-1 and JunD. Signal transduction analysis indicates that the EGCG response requires Ras, MEKK1, MEK3, and p38 kinases. Kinase assays and inhibitor studies suggest that p38delta is the p38 isoform responsible for the regulation. These changes are also associated with a cessation of cell proliferation and enhanced cornified envelope formation. These studies show that in normal human keratinocytes EGCG markedly increases, via a MAPK signaling mechanism, AP1 factor-associated responses.

Catechin↗