Brahma N. Singh

Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications

An expanding body of preclinical evidence suggests EGCG, the major catechin found in green tea (Camellia sinensis), has the potential to impact a variety of human diseases. Apparently, EGCG functions as a powerful antioxidant, preventing oxidative damage in healthy cells, but also as an antiangiogenic and antitumor agent and as a modulator of tumor cell response to chemotherapy. Much of the cancer chemopreventive properties of green tea are mediated by EGCG that induces apoptosis and promotes cell growth arrest by altering the expression of cell cycle regulatory proteins, activating killer caspases, and suppressing oncogenic transcription factors and pluripotency maintain factors. In vitro studies have demonstrated that EGCG blocks carcinogenesis by affecting a wide array of signal transduction pathways including JAK/STAT, MAPK, PI3K/AKT, Wnt and Notch. EGCG stimulates telomere fragmentation through inhibiting telomerase activity. Various clinical studies have revealed that treatment by EGCG inhibits tumor incidence and multiplicity in different organ sites such as liver, stomach, skin, lung, mammary gland and colon. Recent work demonstrated that EGCG reduced DNMTs, proteases, and DHFR activities, which would affect transcription of TSGs and protein synthesis. EGCG has great potential in cancer prevention because of its safety, low cost and bioavailability. In this review, we discuss its cancer preventive properties and its mechanism of action at numerous points regulating cancer cell growth, survival, angiogenesis and metastasis. Therefore, non-toxic natural agent could be useful either alone or in combination with conventional therapeutics for the prevention of tumor progression and/or treatment of human malignancies.

Total phenol, antioxidant and free radical scavenging activities of some medicinal plants

Phenols, a major group of antioxidant phytochemicals, have profound importance due to their biological and free radical scavenging activities. To identify their potential sources, extracts of some plants were studied for their total phenolic content (TPC), antioxidant (AOA) and free radical scavenging activities (FRSA) by different methods at multiple concentrations followed by specific phenolic composition. The amount of TPC varied from 2.8 mg/g (Withania somnifera, roots) to 107.8 mg/g (Cassia fistula, fruits) and the AOA from 24.2% (Curcuma zeoderia, leaves) to 96.9% (Trewia nudiflora, leaves). Bark of Azadirachta indica, fruits of C. fistula, and leaves and fruits of T. nudiflora were found to have high TPC (89.8–107.8 mg/g) and high AOA (84.8–96.9%). Promising plant parts were studied for their FRSA and reducing power (RP), where the bark of A. indica, Casuarina equisetifolia and Cinnamomum zeylanicum, flowers of Indigofera tinctoria, fruits of Lawsonia inermis, and fruits and leaves of T. nudiflora showed a very low inhibitory concentration value ranging from 0.14 to 0.26 mg/ml, efficiency concentration value from 6.1 to 11.6 mg/mg DPPH and reducing power value from 0.6 to 2.8 ascorbic acid equivalents (ASE/ml), and reasonably high values (8.5–16.2) of anti-radical power (ARP), indicating their strong FRSA. They also showed better inhibition of hydroxyl radical induced deoxyribose degradation than that of reference standard. Fruits of C. fistula with high phenols (107.8 mg/g) showed poor reducing power (5.9 ASE/ml) and ARP (4.7); in contrast, the bark of C. equisetifolia and fruits of L. inermis were with comparatively lower phenols (72.1 and 75.8 mg/g) but exhibited good ARP (16.2 and 14.4) and reducing power (0.7 and 0.6 ASE/ml, respectively). Some of the plants were also found effective in protecting plasmid DNA nicking induced by hydroxyl radicals generated by Fentons reaction. They were further assayed for their specific phenolic composition through high-performance liquid chromatography and MS/MS, where the amount of caffeic acid varied from 0.312 to 0.797 mg/g, chlorogenic acid from 0.018 to 2.109 mg/g, ellagic acid from 0.009 to 0.902 mg/g, ferulic acid from 0.036 to 0.078 mg/g, gallic acid from 0.192 to 3.597 mg/g, kaempferol from 0.011 to 0.910 mg/g, quercetin from 0.047 to 1.106 mg/g and rutin from 0.059 to 2.029 mg/g.

Hedgehog Signaling Antagonist GDC-0449 (Vismodegib) Inhibits Pancreatic Cancer Stem Cell Characteristics: Molecular Mechanisms

Background Recent evidence from in vitro and in vivo studies has demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in various human cancer stem cells (CSCs). Therefore, the chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. GDC-0449 (Vismodegib), orally administrable molecule belonging to the 2-arylpyridine class, inhibits SHH signaling pathway by blocking the activities of Smoothened. The objectives of this study were to examine the molecular mechanisms by which GDC-0449 regulates human pancreatic CSC characteristics in vitro. Methodology/Principal Findings GDC-0499 inhibited cell viability and induced apoptosis in three pancreatic cancer cell lines and pancreatic CSCs. This inhibitor also suppressed cell viability, Gli-DNA binding and transcriptional activities, and induced apoptosis through caspase-3 activation and PARP cleavage in pancreatic CSCs. GDC-0449-induced apoptosis in CSCs showed increased Fas expression and decreased expression of PDGFRα. Furthermore, Bcl-2 was down-regulated whereas TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was increased following the treatment of CSCs with GDC-0449. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GDC-0449-treated pancreatic CSCs. Thus, activated Gli genes repress DRs and Fas expressions, up-regulate the expressions of Bcl-2 and PDGFRα and facilitate cell survival. Conclusions/Significance These data suggest that GDC-0499 can be used for the management of pancreatic cancer by targeting pancreatic CSCs.

Antioxidant and anti-quorum sensing activities of green pod of Acacia nilotica L.

The antioxidant and anti-quorum sensing activities of eight extracts were studied in green pods of Acacia nilotica. The specific phenolic compositions and their quantifications were performed by HPLC and MS/MS, which showed that the HEF (pH 4) was higher in gallic acid, ellagic acid, epicatechin, rutin, and GTs. In order to find antioxidant potential of various extracts, their activities were studied for TPC, AOA, FRSA, RP, inhibition of LPO, FIC activity, HO* and O(2)(-) scavenging activities. Among them HEF (pH 4) has shown potent antioxidant activity. HEF (pH 4) was also found effective in protecting plasmid DNA and HAS protein oxidation induced by HO*. Pre-treatment of HEF (pH 4) at 75 and 150 mg/kg body weight for 6 days caused a significant increase in the levels of CAT and SOD and decrease in the level of MDA content in liver, lungs, kidneys and blood when compared to CCl(4)-intoxicated rats. Eventually, the extracts were also screened for anti-QS activity. Of these extracts two showed QS inhibition: HEF (pH 4) and HCE. The results obtained strongly indicate that green pod of A. nilotica are important source of natural antioxidants.

Rottlerin induces autophagy which leads to apoptotic cell death through inhibition of PI3K/Akt/mTOR pathway in human pancreatic cancer stem cells

Multiple lines of evidence support the idea that autophagy plays an essential role in the development of drug resistance, self-renewal, differentiation, and tumorigenic potentials of cancer stem cells (CSCs). Rottlerin (ROT) is widely used as a protein kinase C-delta (PKC-δ) inhibitor. Recent studies revealed that ROT induces apoptosis through engagement of mitochondria. However, it is not known whether ROT-induced apoptosis is associated with other mechanisms such as autophagy. Here we found that ROT induced autophagy followed by induction of apoptosis via inhibition of PI3K/Akt/mTOR pathway and activation of caspase cascade in human pancreatic CSCs. ROT induced a dose- and time-dependent inhibition of cell survival and induction of cytoplasmic vacuolations. The conversion of microtubule-associated protein LC3-I to LC3-II, and increased accumulations of Atg7 and Beclin-1 were also observed in CSCs treated with ROT. Prolonged exposure of CSCs to ROT eventually caused apoptosis which was associated with the suppression of phosphorylated Akt (Ser473) and mTOR (Ser2448), downregulation of XIAP, cIAP-1, Bcl-2 and Bcl-X(L), induction of Bax, activation of caspase-3 and -9, and concomitant degradation of PARP. ROT-induced apoptosis was enhanced by dominant negative AKT, Akt1/2 inhibitor, and rapamycin. Our study also demonstrates that gene silencing of Atg7 and Beclin1, or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited ROT-induced autophagy and accelerated ROT-induced apoptosis. The knockdown of PKC-δ did not block ROT-induced autophagy and cell death, suggesting these effects of ROT were exerted through PKC-δ-independent pathway. In summary, our data demonstrate that ROT can induce autophagy which leads to cell death in pancreatic CSCs.

ROS-dependent anticandidal activity of zinc oxide nanoparticles synthesized by using egg albumen as a biotemplate

Zinc oxide nanoparticles (ZnO NPs) have attracted great attention because of their superior optical properties and wide application in biomedical science. However, little is known about the anticandidal activity of ZnO NPs against Candida albicans (C. albicans). This study was designed to develop the green approach to synthesize ZnO NPs using egg white (denoted as EtZnO NPs) and investigated its possible mechanism of antimicrobial activity against C. albicans 077. It was also notable that anticandidal activity of EtZnO NPs is correlated with reactive oxygen species (ROS) production in a dose dependent manner. Protection of histidine against ROS clearly suggests the implication of ROS in anticandidal activity of EtZnO NPs. This green approach based on egg white-mediated synthesis of ZnO NPs paves the way for developing cost effective, eco-friendly and promising antimicrobial nanomaterial for applications in medicine.

Biosynthesis of Stable Antioxidant ZnO Nanoparticles by Pseudomonas aeruginosa Rhamnolipids

During the last several years, various chemical methods have been used for synthesis of a variety of metal nanoparticles. Most of these methods pose severe environmental problems and biological risks; therefore the present study reports a biological route for synthesis of zinc oxide nanoparticles using Pseudomonas aeruginosa rhamnolipids (RLs) (denoted as RL@ZnO) and their antioxidant property. Formation of stable RL@ZnO nanoparticles gave mostly spherical particles with a particle size ranging from 35 to 80 nm. The RL@ZnO nanoparticles were characterized by UV-visible (UV–vis) spectroscopy, scanning electron microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, X-ray diffraction (XRD), and thermal gravimetric analysis. The UV–vis spectra presented a characteristic absorbance peak at ∼360 nm for synthesized RL@ZnO nanoparticles. The XRD spectrum showed that RL@ZnO nanoparticles are crystalline in nature and have typical wurtzite type polycrystals. Antioxidant potential of RL@ZnO nanoparticles was assessed through 2,2–diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion free radicals with varying concentration and time of the storage up to 15 months, while it was found to decline in bare ZnO nanoparticles. Similarly, the inhibitory effects on β-carotene oxidation and lipid peroxidation were also observed. These results elucidate the significance of P. aeruginosa RL as effective stabilizing agents to develop surface protective ZnO nanoparticles, which can be used as promising antioxidants in biological system.

Mycofabricated biosilver nanoparticles interrupt Pseudomonas aeruginosa quorum sensing systems.

Quorum sensing (QS) is a chemical communication process that Pseudomonas aeruginosa uses to regulate virulence and biofilm formation. Disabling of QS is an emerging approach for combating its pathogenicity. Silver nanoparticles (AgNPs) have been widely applied as antimicrobial agents against human pathogenic bacteria and fungi, but not for the attenuation of bacterial QS. Here we mycofabricated AgNPs (mfAgNPs) using metabolites of soil fungus Rhizopus arrhizus BRS-07 and tested their effect on QS-regulated virulence and biofilm formation of P. aeruginosa. Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR. Treatment of mfAgNPs inhibited biofilm formation, production of several virulence factors (e.g. LasA protease, LasB elastrase, pyocyanin, pyoverdin, pyochelin, rhamnolipid, and alginate) and reduced AHLs production. Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors. The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling.

Quercetin Sensitizes Fluconazole-Resistant Candida albicans To Induce Apoptotic Cell Death by Modulating Quorum Sensing

ABSTRACT Quorum sensing (QS) regulates group behaviors of Candida albicans such as biofilm, hyphal growth, and virulence factors. The sesquiterpene alcohol farnesol, a QS molecule produced by C. albicans, is known to regulate the expression of virulence weapons of this fungus. Fluconazole (FCZ) is a broad-spectrum antifungal drug that is used for the treatment of C. albicans infections. While FCZ can be cytotoxic at high concentrations, our results show that at much lower concentrations, quercetin (QC), a dietary flavonoid isolated from an edible lichen (Usnea longissima), can be implemented as a sensitizing agent for FCZ-resistant C. albicans NBC099, enhancing the efficacy of FCZ. QC enhanced FCZ-mediated cell killing of NBC099 and also induced cell death. These experiments indicated that the combined application of both drugs was FCZ dose dependent rather than QC dose dependent. In addition, we found that QC strongly suppressed the production of virulence weapons—biofilm formation, hyphal development, phospholipase, proteinase, esterase, and hemolytic activity. Treatment with QC also increased FCZ-mediated cell death in NBC099 biofilms. Interestingly, we also found that QC enhances the anticandidal activity of FCZ by inducing apoptotic cell death. We have also established that this sensitization is reliant on the farnesol response generated by QC. Molecular docking studies also support this conclusion and suggest that QC can form hydrogen bonds with Gln969, Thr1105, Ser1108, Arg1109, Asn1110, and Gly1061 in the ATP binding pocket of adenylate cyclase. Thus, this QS-mediated combined sensitizer (QC)-anticandidal agent (FCZ) strategy may be a novel way to enhance the efficacy of FCZ-based therapy of C. albicans infections.

Scaffold of Selenium Nanovectors and Honey Phytochemicals for Inhibition of Pseudomonas aeruginosa Quorum Sensing and Biofilm Formation

Honey is an excellent source of polyphenolic compounds that are effective in attenuating quorum sensing (QS), a chemical process of cell-to-cell communication system used by the opportunistic pathogen Pseudomonas aeruginosa to regulate virulence and biofilm formation. However, lower water solubility and inadequate bioavailability remains major concerns of these therapeutic polyphenols. Its therapeutic index can be improved by using nano-carrier systems to target QS signaling potently. In the present study, we fabricated a unique drug delivery system comprising selenium nanoparticles (SeNPs; non-viral vectors) and polyphenols of honey (HP) for enhancement of anti-QS activity of HP against P. aeruginosa PAO1. The developed selenium nano-scaffold showed superior anti-QS activity, anti-biofilm efficacy, and anti-virulence potential in both in-vitro and in-vivo over its individual components, SeNPs and HP. LasR is inhibited by selenium nano-scaffold in-vitro. Using computational molecular docking studies, we have also demonstrated that the anti-virulence activity of selenium nano-scaffold is reliant on molecular binding that occurs between HP and the QS receptor LasR through hydrogen bonding and hydrophobic interactions. Our preliminary investigations with selenium-based nano-carriers hold significant promise to improve anti-virulence effectiveness of phytochemicals by enhancing effective intracellular delivery.