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

Abstract

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.

Fig 1. H₂O₂ induces ROS accumulation.

(a) Homogenates of DLA cells from 3 set of each group were pooled separately and used for total ROS measurement (b) Cells were loaded with H₂DCFDA (10μM) for 30min and amount of ROS generated was indicated by shift in fluorescence as detected by flow cytometry. Student’s t-test was used for statistical analysis. Data represent as mean ± S.E.M. of three independent experiments. * denotes significant differences at the level of p < 0.05 between H₂O₂-treated and control group.

Fig 2. H₂O₂ activates PI3K signalling.

(a) Western analysis of p85α, phospho p85α, phospho PDK1, phospho AKT Thr-308, phospho AKT Ser-473 and AKT1 (b) respective densitometric scanning of band after normalization with β-actin. Student’s t-test was used for statistical analysis. Data represent as mean ± S.E.M. of three independent experiments. * denotes significant differences at the level of p < 0.05 between H₂O₂-treated and control group.

Fig 3. H₂O₂ induces the phosphorylation of BAD, ERK1/2 and TNFR1 level and down-regulates PTEN.

(a) Western analysis of phospho BAD, ERK1/2,phospho ERK1/2, TNFR1 and PTEN (b) respective densitometric scanning of band after normalization with β-actin. Student’s t-test was used for statistical analysis. Data represent as mean ± S.E.M. of three independent experiments. * denotes significant differences at the level of p < 0.05 between H₂O₂-treated and control group.

Fig 4. PI-103 and QUE decrease H₂O₂ induced ROS level.

Homogenates of DLA cells from 3 set of each group were pooled separately and used for total ROS measurement. DLA cells were pre-treated with PI-103 and QUE; then post-treated with H₂O₂. Data represent as mean ± S.E.M. * (#) denotes significant differences at level of p < 0.05. # indicates significant difference between control vs H₂O₂-treated group; and * H₂O₂ treated groups vs PI-103/QUE treated group.

Fig 5. PI-103 and QUE attenuate phosphorylation of AKT Thr-308 & AKT Ser-473.

(a) and (b) Western analysis of phospho AKT Thr-308, AKT Ser-473 and respective densitometric scanning of band after normalization with β-actin. (c) Western analysis of AKT1 and respective densitometric scanning of band after normalization with β-actin. DLA cells were pre-treated with PI-103 and QUE; then post-treated with H₂O₂. Data represent as mean ± S.E.M. * (#) denotes significant differences at level of p < 0.05. # indicates significant difference between control vs H₂O₂-treated group; and * H₂O₂ treated groups vs PI-103/QUE treated group.

Fig 6. PI-103 and QUE attenuate H₂O₂ induced phosphorylation of PDK1, BAD, expression of TNFR1 and induce PTEN level.

(a) Western analysis of phospho PDK1, PTEN, phospho BAD and TNFR1 (b) respective densitometric scanning of band after normalization with β-actin. DLA cells were pre-treated with PI-103 and QUE; then post-treated with H₂O₂. Data represent as mean ± S.E.M. * (#) denotes significant differences at level of p < 0.05. # indicates significant difference between control vs H₂O₂-treated groups; and * H₂O₂ treated groups vs PI-103/QUE treated group.

Fig 7. PI-103 & QUE downregulate PKCα level.

(a) and (b) Western analysis of PKCα and respective densitometric scanning of band after normalization with β-actin. DLA cells were pre-treated with PI-103 and QUE; then post-treated with H₂O₂. Data represent as mean ± S.E.M. * (#) denotes significant differences at level of p < 0.05. # indicates significant difference between control vs H₂O₂-treated groups; and * H₂O₂ treated groups vs PI-103/QUE treated group.

Fig 8. Schematic representation showing PI-103 and QUE mediated modulation of PI3K-AKT signaling pathway.

Exposure of H₂O₂ induces ROS accumulation, which in turn activates TNFR1 and PI3K level. Activated PI3K phosphorylates AKT via activated PDK1 and thereby induces downstream survival factor BAD. This is consistent with reduced level of tumor suppressor PTEN—negative regulator of PI3K pathway. Enhanced level of ROS by H₂O₂ is suppressed by both PI-103 & QUE which in turn down-regulates phosphorylation of AKT, PDK1 and BAD. Additionally, QUE and PI-103 increase the level of PTEN and decrease TNFR1 as well as PKCα.