Karunagaran Devarajan

Human papilloma virus is associated with cervical and other tumors, and several cellular conditions also play an important role in carcinogenesis. Human papilloma virus (HPV)-infected cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but little is known about their regulation by HPV. This study attempts to understand the role of HPV16 E2, an important early protein of HPV16, in the regulation of NF-κB and STAT3 by reporter assays, quantitative reverse transcriptase-polymerase chain reaction, and immunoblotting. We demonstrate that E2 enhances NF-κB activation induced by TNF-α, a proinflammatory cytokine, in both non-tumor- and tumor-derived epithelial cell lines besides potentiating STAT3 transcriptional activity induced by TNF-α in HEK293 cells. E2 increases the expression of RelA and its transcriptional activation, and retention of E2 was observed in the nucleus with significant interaction with RelA (immunoprecipitation) upon TNF-α treatment. Transfection with shRNA-RelA or pretreatment with a STAT3 inhibitor had a negative effect on the ability of E2 to enhance TNF-α-induced NF-κB activation. Experiments with co-expression of a mutant of STAT3 with E2 also suggested that the activation of STAT3 is indispensible for TNF-α-induced NF-κB activation. Inhibition of STAT3 activation enhanced E2-induced apoptosis, whereas parallel activation of NF-κB and STAT3 by the combined action of E2 and TNF-α increased the expression of their common targets, cyclin-D1, c-Myc, survivin, and Bcl-2, leading to a decrease in E2-induced apoptosis (viability and cell cycle). Our results reveal novel mechanisms by which E2 may regulate NF-κB and STAT3 activation in the presence of TNF-α with implications on the survival of HPV-infected cells.

With increasing use of plant-derived cancer chemotherapeutic agents, exploring the antiproliferative effects of phytochemicals has gained increasing momentum for anticancer drug design. The dietary phytochemical quercetin, modulates several signal transduction pathways associated with cell proliferation and apoptosis. The present study was undertaken to examine the effect of quercetin on cell viability, and to determine the molecular mechanism of quercetin-induced cell death by investigating the expression of Bcl-2 family proteins (Bcl-2, Bcl-xL, Mcl1, Bax, Bad, p-Bad), cytochrome C, Apaf-1, caspases, and survivin as well as the cell cycle regulatory proteins (p53, p21, cyclin D1), and NF-κB family members (p50, p65, IκB, p-IκB-α, IKKβ and ubiquitin ligase) in human cervical cancer (HeLa) cells. The results demonstrate that quercetin suppressed the viability of HeLa cells in a dose-dependent manner by inducing G2/M phase cell cycle arrest and mitochondrial apoptosis through a p53-dependent mechanism. This involved characteristic changes in nuclear morphology, phosphatidylserine externalization, mitochondrial membrane depolarization, modulation of cell cycle regulatory proteins and NF-κB family members, upregulation of proapoptotic Bcl-2 family proteins, cytochrome C, Apaf-1 and caspases, and downregulation of antiapoptotic Bcl-2 proteins and survivin. Quercetin that exerts opposing effects on different signaling networks to inhibit cancer progression is a classic candidate for anticancer drug design. © 2010 Elsevier B.V.

Antiproliferative and apoptosis inducing effects of black tea polyphenols (Polyphenon-B) on HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo were investigated. Viability of HepG2 cells was evaluated by the MTT assay, and apoptosis by AO-EB and DAPI staining, cell cycle analysis, and annexin V-PI assay. For the in vivo study, male Sprague-Dawley rats treated with dimethylaminoazobenzene (DAB) (0.06%) were used. The expression of Bcl-2 and NF-κB family members were analyzed by immunoblotting. Administration of Polyphenon-B induced dose-dependent inhibition of growth of HepG2 cells and reduced tumor incidence in DAB administered animals. HepG2 cells also exhibited morphological features characteristic of apoptotic cell death. In addition, administration of Polyphenon-B increased the expression of Bax, tBid, Smac/Diablo, cytochrome C, Apaf-1, caspases, and IκB with PARP cleavage, and decreased the expression of Bcl-2, Bcl-xL, pBad, NF-κB, p-IκB-α, IKKβ and Ub in both HepG2 cells and in DAB-treated animals. These results provide evidence that Polyphenon-B effectively inhibits proliferation and induces apoptosis both in vitro and in vivo by inhibiting NF-κB, and inducing intrinsic apoptosis by modulating the expression of a network of interrelated molecules eventually culminating in caspase-mediated cell death. © 2010 Elsevier Ltd.

Aims: High-mobility group (HMG) proteins are oncogenic in different cancers, including cervical cancer; silencing their individual expression using sh-RNAs, siRNAs, and miRNAs has had anti-tumorigenic effects, but the consequences of their collective downregulation are not known. Since multiple gene targeting is generally very effective in cancer therapy, the present study highlighted the consequences of silencing the expression of HMGA1, A2, B1, and B3 using sh-RNAs or miR-142-3p (that can potentially target HMGA1, A2, B1, and B3) in cervical cancer cell lines. Main methods: 3′ UTR luciferase reporter assays were performed to validate HMGA1, A2, B1, and B3 as targets of miR-142-3p in human cervical cancer cells. Annexin V/PI dual staining and flow cytometry analyses were used to detect apoptotic cells. miR-142-3p-mediated regulation of cell death, colony formation, migration, and invasion was investigated in human cervical cancer cells together with in vivo metastasis in zebrafish. Key findings: Concurrent knockdown of HMGA1, A2, B1, and B3 through their corresponding sh-RNAs inhibited cell viability and colony formation but induced apoptosis, and these effects were relatively reduced upon their individual knockdown. miR-142-3p targeted HMGA1, A2, B1, and B3 by binding to their 3′UTRs and induced apoptosis but inhibited proliferation, migration, and invasion of human cervical cancer cells. In addition, miR-142-3p expression decreased phospho-p65 and EMT-related proteins in cervical cancer cells and their in vivo metastatic potential upon implantation in zebrafish. Significance: These findings suggest that miR-142-3p acts as a tumor-suppressive miRNA by targeting HMGA1, A2, B1, and B3 and may serve as a potential therapeutic agent in human cervical cancer.

Nuclear factor kappa B (NF-κB), an oncogenic signaling factor plays a critical role in the development and progression of various cancers. The objective of this study was to investigate the effect of nimbolide, a neem derived tetranortriterpenoid on NF-κB signaling and its downstream events - Wnt/β-catenin activation and apoptosis evasion in human hepatocarcinoma (HepG2) cells by evaluating NF-κB family members (NF-κB-p50, p65, IκB-α, p-IκB-α, and IKKβ), members of Wnt signaling (GSK-3β and β-catenin), and intrinsic apoptosis (Bcl-2, Bax, cytochrome c, Smac/DIABLO, caspase-3, and caspase-9). Our results demonstrate that nimbolide concurrently abrogates canonical NF-κB and Wnt signaling and induces intrinsic apoptosis in HepG2 cells. These data suggest that phytochemicals such as nimbolide that can target multiple steps along the NF-κB signaling circuit are promising candidates for future phytochemical-based mechanistic pathway targeted anticancer regimens. © 2012 Elsevier B.V. All rights reserved.

HPV-transformed cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but very little is known about their regulation under inflammatory conditions before HPV integration. This study reports that cervical tissues with stromal inflammation and intact HPV16 E2 gene show increased expression of target genes of NF-κB and/or STAT3 which can regulate cell survival (cyclin D1, c-Myc, survivin and Bcl2) and inflammatory responses (TNF-α, IL-1β, IL-6, IL-8 and CCR2). Increased expression of RelA, p-IκBα, STAT3, p-STAT3 (Ser727), Pin1 (peptidyl-prolyl cis/trans isomerase) and MCM2 in the squamous epithelia of cervices with stromal inflammation supports early activation of NF-κB-STAT3. Furthermore, HPV16 E2 potentiated NF-κB activation induced by inflammatory mediators, IL-1β and SDF-1α, in HEK293 cells. These results reveal a novel role for E2 in regulating the activities of NF-κB and STAT3 that may have implications in carcinogenic progression of HPV16-infected cells under conditions of stromal inflammation.

Monocyte Chemoattractant Protein-1 (MCP-1) is involved in the diapedesis of blood monocytes into the arterial intima, an early critical event in atherogenesis. Modulating MCP-1 expression can be a key strategy to decrease the risk for atherosclerosis in diabetes. We hypothesized that quercetin, an anti-inflammatory molecule could modulate high glucose concentration (HG) induced MCP-1 expression in aortic endothelial cells in vitro because of its regulatory effects on Activator Protein-1 (AP-1) and Nuclear Factor-κB (NF-κB). Rat aortic endothelial cells (RAECs) were exposed to HG in the presence or absence of quercetin. Quercetin attenuated HG induced MCP-1 mRNA (42%) and protein synthesis (45%) when estimated using real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Western blot analysis found quercetin to maintain cytosolic p65 protein levels to that seen in control. Quercetin was found to attenuate HG induced increased NF-κB and AP-1 DNA binding activity in electrophoretic mobility shift assay. Immunofluorescence studies revealed quercetin to prevent HG induced nuclear localization of p65 and c-jun. Quercetin was also found to decrease HG induced activation of NF-κB (71% ± 14%), AP-1 (69% ± 24%) and MCP-1 promoter (79% ± 25%) in EA.hy926 cells when analyzed using luciferase reporter assay.We conclude that quercetin attenuates MCP-1 expression in HG treated RAECs, probably by regulating both NF-κB and AP-1 pathways. The findings provide new insights into HG induced MCP-1 gene regulation in aortic endothelial cells and the potential of quercetin in abating the risk for atherosclerosis in diabetes. © 2010 Elsevier Ltd.

Marine organisms provide several biologically active compounds that include alkaloids with high cytotoxic activity but only a few of them have so far reached clinical stage, due partly to their limited supply and complex structural features. In an attempt to develop novel anticancer compounds, we have now synthesized diaminoindoloylthiazoles (4a-c; DIT1-3) and diaminocinnamoylthiazoles (5a,b; DCT1-2) as analogs based on a topsentin scaffold and investigated the cytotoxic and apoptotic activities of these compounds in HeLa cells. The results suggest that diaminoindoloylthiazoles (DIT1-3) inhibit cell growth and among these, DIT3 is the most cytotoxic against HeLa cells (IC50 1 μM). The diaminocinnamoylthiazoles DCT1 and DCT2, which can be viewed as curcumin-diaminothiazole hybrids, also inhibited cell growth but at relatively higher concentrations with IC50 values of 60 and 30 μM, respectively. These compounds induced apoptosis through the intrinsic pathway by reducing the mitochondrial membrane potential and activating caspases, 9 and 3, but not caspase 8. Among the marine alkaloid analogs tested in this study, DIT1-3 are very effective in inducing apoptosis of HeLa cells followed by DCT2 and DCT1. The treated cells were arrested in G 2/M phase followed by accumulation of the cells in the Sub G 0 phase. The curcumin-diaminothiazole hybrid DCT1 had the maximum effect in downregulating TNF-induced NF-κB activation among the compounds tested in this study. Thus, we demonstrate that diaminoindoloylthiazoles and diaminocinnamoylthiazoles induce apoptosis, regulate cell cycle and NF-κB signaling and thus show promising anticancer effects that warrant further investigation. © 2013 Elsevier Masson SAS. All rights reserved.