Akhilendra Kumar Maurya

Anticarcinogenic action of quercetin by downregulation of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) via induction of p53 in hepatocellular carcinoma (HepG2) cell line

Protein kinase C (PKC) is a key regulator of cell growth and differentiation in mammalian cells and hyperactivation of PKC is believed to play an important role in tumor progression. PKC is downstream to signaling protein of phosphatidylinositol 3-Kinase (PI3K), a known up-regulator of cell proliferation and survival. Accumulation of reactive oxygen species (ROS) triggers oxidative stress in the tumor microenvironment, leading to the hyperactivation of various oxidative stress-stimulated signaling molecules. Quercetin (QUE) is a naturally occurring dietary flavonoid having antioxidant properties. QUE is reported to show antitumor activity both in vitro and in vivo; however, the molecular mechanism is yet to be thoroughly explored. HepG2 cells display cellular functions similar to the normal hepatocytes with high degree of morphological and functional differentiation, therefore HepG2 cell line is chosen as the suitable model for drug targeting. Present study is aimed to establish the signaling pathway involved in the anticarcinogenic action of QUE in HepG2 cell line. HepG2 cells were treated with different doses of QUE. Protein level and gene expression were analysed by Western blotting and RT-PCR, respectively. PKC activity was measured by non-radioactive-tagged phosphorylation. Results showed downregulation of expression of PI3K, PKC, COX-2 and ROS caused by QUE. Additionally, QUE enhanced the expression of p53 and BAX in HepG2 cells. Overall, results of the current study suggested that QUE elicited anticarcinogenic action by upregulation of p53 and BAX in HepG2 cells via downregulation of ROS, PKC, PI3K and COX-2, confirming our earlier report on the animal model.

Quercetin Regresses Dalton's Lymphoma Growth via Suppression of PI3K/AKT Signaling Leading to Upregulation of p53 and Decrease in Energy Metabolism

Various oncogenes are associated with deregulation in cell proliferation, apoptosis, and cell survival, which ultimately cause cancerous growth. Phosphatidylinositol 3-kinase (PI3K) mediated signaling plays a key role in malignant transformation. Cell proliferation and cell survival of tumor cell are induced by hyper activation of PI3K, AKT1, glycolytic enzyme LDH-A, and inactivation of tumor suppressor gene p53. Dietary flavonoids such as quercetin are considered a powerful modulator of different cellular signaling pathways. The present study is focused on the role of quercetin on regulation of PI3K/AKT pathways in Daltons lymphoma mice. Effect of quercetin was analyzed in ascite cells in terms of cell viability, glycolytic metabolism as well as expression, and level of PI3K (regulatory and catalytic subunit), AKT1, and p53 using standard methods. Results reflect hyperactivation of PI3K signaling in ascite cells of Daltons lymphoma mice, leading to activation of AKT1 and inactivation of p53. Quercetin modulates the pathway toward suppression of lymphoma. Glycolytic metabolism was also downregulated by quercetin. Its tumor suppressor activity was confirmed by morphological parameters and longevity of mice. The findings suggest that quercetin may contribute to lymphoma prevention by downregulating PI3K–AKT1–p53 pathway as well as by glycolytic metabolism.

Modulation of PKC signaling and induction of apoptosis through suppression of reactive oxygen species and tumor necrosis factor receptor 1 (TNFR1): key role of quercetin in cancer prevention

Cancer cells are characterized by increased production of reactive oxygen species (ROS) and an altered redox environment as compared to normal cells. Continuous accumulation of ROS triggers oxidative stress leading to hyper-activation of signaling pathways that promote cell proliferation, survival, and metabolic adaptation to the tumor microenvironment. Therefore, antioxidants are proposed to contribute to cancer prevention. Protein kinase C (PKC) is a crucial regulator of diverse cellular processes and contributes to cancer progression. The activation of PKC is partially dependent on ROS signaling. In the present study, cancer preventive activity of natural flavonoid quercetin is analyzed in ascite cells of Dalton’s lymphoma-bearing mice. The total ROS level and activity of PKC were downregulated after quercetin treatment in lymphoma-bearing mice. Quercetin modulates the expression of almost all isozymes of classical, novel, and atypical PKC as well as downregulates the level and expression of PKCα. Further, quercetin improves apoptotic potential, as observed by the levels of caspase 3, caspase 9, PARP, PKCδ, and nuclear condensation. Additionally, quercetin reduces cell survival and promotes death receptor-mediated apoptosis via differential localization of the TNFR1 level in ascite cells. The overall result suggests the cancer preventive activity of quercetin via the induction of apoptosis and modulates PKC signaling with the reduction of oxidative stress in ascite cells of lymphoma-bearing mice.

Controlled release of drug and better bioavailability using poly(lactic acid-co-glycolic acid) nanoparticles

Tamoxifen (Tmx) embedded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-Tmx) is prepared to evaluate its better DNA cleavage potential, cytotoxicity using Daltons lymphoma ascite (DLA) cells and MDA-MB231 breast cancer cells. PLGA-Tmx nanoparticles are prepared through emulsified nanoprecipitation technique with varying dimension of 17-30nm by changing the concentrations of polymer, emulsifier and drug. Nanoparticles dimension are measured through electron and atomic force microscopy. Interactions between tamoxifen and PLGA are verified through spectroscopic and calorimetric methods. PLGA-Tmx shows excellent DNA cleavage potential as compared to pure Tmx raising better bioavailability. In vitro cytotoxicity studies indicate that PLGA-Tmx reduces DLA cells viability up to ∼38% against ∼15% in pure Tmx. Hoechst stain is used to detect apoptotic DLA cells through fluorescence imaging of nuclear fragmentation and condensation exhibiting significant increase of apoptosis (70%) in PLGA-Tmx vis-à-vis pure drug (58%). Enhanced DNA cleavage potential, nuclear fragmentation and condensation in apoptotic cells confirm greater bioavailability of PLGA-Tmx as compared to pure Tmx in terms of receptor mediated endocytosis. Hence, the sustained release kinetics of PLGA-Tmx nanoparticles shows much better anticancer efficacy through enhanced DNA cleavage potential and nuclear fragmentation and, thereby, reveal a novel vehicle for the treatment of cancer.

PI-103 and Quercetin Attenuate PI3K-AKT Signaling Pathway in T- Cell Lymphoma Exposed to Hydrogen Peroxide.

Phosphatidylinositol 3 kinase—protein kinase B (PI3K-AKT) pathway has been considered as major drug target site due to its frequent activation in cancer. AKT regulates the activity of various targets to promote tumorigenesis and metastasis. Accumulation of reactive oxygen species (ROS) has been linked to oxidative stress and regulation of signaling pathways for metabolic adaptation of tumor microenvironment. Hydrogen peroxide (H2O2) in this context is used as ROS source for oxidative stress preconditioning. Antioxidants are commonly considered to be beneficial to reduce detrimental effects of ROS and are recommended as dietary supplements. Quercetin, a ubiquitous bioactive flavonoid is a dietary component which has attracted much of interest due to its potential health-promoting effects. Present study is aimed to analyze PI3K-AKT signaling pathway in H2O2 exposed Dalton’s lymphoma ascite (DLA) cells. Further, regulation of PI3K-AKT pathway by quercetin as well as PI-103, an inhibitor of PI3K was analyzed. Exposure of H2O2 (1mM H2O2 for 30min) to DLA cells caused ROS accumulation and resulted in increased phosphorylation of PI3K and downstream proteins PDK1 and AKT (Ser-473 and Thr-308), cell survival factors BAD and ERK1/2, as well as TNFR1. However, level of tumor suppressor PTEN was declined. Both PI-103 & quercetin suppressed the enhanced level of ROS and significantly down-regulated phosphorylation of AKT, PDK1, BAD and level of TNFR1 as well as increased the level of PTEN in H2O2 induced lymphoma cells. The overall result suggests that quercetin and PI3K inhibitor PI-103 attenuate PI3K-AKT pathway in a similar mechanism.

Quercetin Attenuates Cell Survival, Inflammation, and Angiogenesis via Modulation of AKT Signaling in Murine T-Cell Lymphoma

ABSTRACT AKT signaling is important to maintaining normal physiology. Hyperactivation of AKT signaling is frequent in cancer, which maintains a high oxidative state in a tumor microenvironment that is needed for tumor adaptation. Therefore, antioxidants are proposed to exhibit anticancer properties by interfering with the tumor microenvironment. Quercetin is an ubiquitous bioactive antioxidant rich in vegetables and beverages. The present study aimed to analyze cancer preventive property of quercetin in ascite cells of Daltons lymphoma-bearing mice. Protein level was determined by Western blotting. Nitric oxide (NO) level was estimated spectrophotometrically using Griess reagent. Results show downregulation in phosphorylation of AKT and PDK1 by quercetin, which was consistent with decreased phosphorylation of downstream survival factors such as BAD, GSK-3β, mTOR, and IkBα. Further, quercetin attenuated the levels of angiogenic factor VEGF-A and inflammatory enzymes COX-2 and iNOS as well as NO levels, whereas it increased the levels of phosphatase PTEN. Overall results suggest that quercetin modulates AKT signaling leading to attenuation of cell survival, inflammation, and angiogenesis in lymphoma-bearing mice.

New planar trans-copper(II) β-dithioester chelate complexes: synthesis, characterization, anticancer activity and DNA-binding/cleavage studies

Abstract New planar trans-copper(II) β-dithioester complexes, [Cu(L)2] (L = methyl-3-hydroxy-(3-pyridyl)-2-propenedithioate (L1 in 1), methyl-3-hydroxy-(2-naphthyl)-2-propenedithioate (L2 in 2), methyl-3-hydroxy-3-(p-methoxyphenyl)-2-propenedithioate (L3 in 3), methyl-3-hydroxy-3-(p-fluorophenyl)-2-propenedithioate (L4 in 4), and methyl-3-hydroxy-3-(p-bromophenyl)-2-propenedithioate (L5 in 5)), have been synthesized and characterized by elemental (C, H, N and S) analysis, ESI-MS, IR, and UV-vis spectra. The structures of HL3 and its corresponding complex 3 have been determined by X-ray crystallography. Electrochemical behavior of all complexes has been studied by cyclic voltammetry. All five planar complexes show efficient DNA-binding and DNA (PBR322)-cleavage in a concentration-dependent manner (1 > 3 > 5 > 2 > 4). Cleavage efficiency is enhanced in the presence of H2O2 as well as ascorbic acid. However, the order of increased efficiency of Cu(II) chelates differs in the presence of H2O2 as 4 > 1 > 2 > 3 > 5. Among these complexes, the pyridyl- and methoxy-functionalized 1 and 3 have shown higher self-activating capability in DNA-cleavage. All complexes show significant variation in IC50 on MCF-7 cell line. Additionally, treatment with the complexes gradually increases apoptotic cell death in dose-dependent manner in RAW 264.7 cell line. These findings highlight potential cancer protective nature of these complexes.

PI-103 attenuates PI3K-AKT signaling and induces apoptosis in murineT-cell lymphoma

Abstract Aberrant activation of PI3K-AKT signaling in many pathological conditions including cancer has attracted much of interest for drug targeting. Various isoforms are known from three classes of PI3K. Targeting selective isoform is advantageous to overcome the global deleterious effects of drug. PI-103 is a specific inhibitor of p110α of class I PI3K. The present study is aimed to analyze anti-carcinogenic activity of PI-103 in Dalton’s lymphoma ascite (DLA) cells. Result shows regression in cell proliferation and increased apoptosis in terms of increased Annexin V binding, nuclear fragmentation and active caspase 3 level. It is correlated with attenuation of PI3K-AKT signaling by PI-103 via downregulation of the level of p110α, phospho-p85α, phospho- AKT, and PKCα in DLA cells as well as in H2O2 induced DLA cells. Additionally, ROS accumulation is declined in H2O2 induced DLA cells. Overall result suggests that PI-103 attenuates PI3K-AKT signaling via induction of apoptosis in murine T-cell lymphoma.

Abstract A07: Decline in the growth of murine T-cell lymphoma via modulation of PI3K signaling pathway: Key role of quercetin and PI-103

Deregulation in cell proliferation, oxidative status, antiapoptosis and survival are involved in development of cancer. The cellular signaling cascades mediated by Phosphatidylinositol 3-kinase (PI3K) with their ability to stimulate cell proliferation and suppress apoptosis, has been considered to play key role in tumor initiation, promotion, and progression. Therefore, use of inhibitors or antioxidants like natural flavonoids could be promising strategy to target PI3K pathway for suppression of carcinogenic process. The present study was aimed to investigate the anti-cancer activity of quercetin, a natural flavonoid and PI-103, a synthetic inhibitor in transplantable murine T-cell lymphoma. Cytotoxicity, apoptotic potential, cell survival, cell proliferation as well as antioxidant defense are evaluated in ascite cells of murine T-cell lymphoma in vivo and in primary culture. Significant decline in level of AKT1, PKCα, COX 2 as well as regulatory domain of PI3K (p85α), AKT1 and Bad, suggested implication of quercetin in regression of lymphoma by down regulation of cell proliferation and survival. Improvement of active PKCδ, p53, active caspase 3, 9 and cleaved PARP supported its cell apoptotic mechanism in cancer prevention. Additionally, decreased ROS level and LDH A activity contributes to reduced survival of cancer cells. Inhibition of p110α by PI-103 resulted in suppression of phospho-AKT and p85α level in primary culture. Modulation of various molecular targets including PI3K/AKT signaling by quercetin and PI-103 in vitro and in vivo tumor models suggests the importance and contribution of antioxidant quercetin in the prevention of cancer. Citation Format: Akhilendra Kumar Maurya, Manjula Vinayak. Decline in the growth of murine T-cell lymphoma via modulation of PI3K signaling pathway: Key role of quercetin and PI-103. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A07.

Synthesis, characterization, DNA binding and cleavage activity of homoleptic zinc(II) β-oxodithioester chelate complexes

Abstract New homoleptic zinc(II) complexes, [Zn(L)2], where L = methyl-3-hydroxy-(3-pyridyl)-2-propenedithioate L1 1, and methyl-3-hydroxy-(4-pyridyl)-2-propenedithioate L2 2, have been synthesized and characterized by elemental (C, H, and N) analysis, ESI-MS, and (IR, UV–vis, NMR) spectroscopy; the structure of 1 has been deduced by X-ray crystallography. The DNA binding and cleavage activity of the complexes have been studied. The cleavage potential of pBR322 DNA by 1 and 2 has been checked. Complex 1, which contains nitrogen of the pyridine group in the 3-position enhances DNA cleavage potential in the presence of ascorbic acid; however, the complex is protective against DNA cleavage in the presence of DMSO or H2O2. Also, 1 causes cytotoxicity against the MCF-7 breast cancer cell line. The efficient cytotoxic activity and DNA cleavage ability of 1 in the presence of ascorbic acid shows its potential anticancer properties and the need for further investigations of its potential as an anticancer drug.