Abstract
Background/Aim: (–)-Epigallocatechin-3-gallate (EGCG) has been indicated to regulate the function of P-glycoprotein (P-gp), which is a drug transporter encoded by the MDR1 (ABCB1) gene. P-gp expression is induced by doxorubicin (DOX). We aimed to clarify the mechanisms and inhibitory effects of EGCG on DOX-induced P-gp expression in HepG2 cells. Materials and Methods: Rhodamine 123 (Rho123) was used for P-gp substrate. Western blotting and polymerase chain reactions (PCRs) were conducted using specific antibodies and primer sets. Results: The DOX-pretreated cells accumulated a significantly decreased amount of Rho123), than control cells; however, the cells pretreated with EGCG and DOX, in combination, accumulated Rho123 more than DOX-pretreated cells. DOX induced the overexpression of MDR1 mRNA and increased the phosphorylation of Akt, ERK1/2, p38 MAPK and JNK. EGCG significantly inhibited the phosphorylation of Akt and ERK. The DOX-induced P-gp overexpression was partially suppressed by an inhibitor of MEK1/2 (U0126), but not by a PI3K inhibitor (LY294002). Interestingly, the expression of P-gp was synergistically inhibited by combined treatment of U0126 with LY294002 and also inhibited by an mTORC1 inhibitor, rapamycin. Conclusion: EGCG inhibited DOX-induced overexpression of P-gp through the coordinate inhibitory action on MEK/ERK and PI3K/Akt signaling pathways.
(–)-Epigallocatechin gallate (EGCG) is the most abundant component of tea polyphenols having various profitable properties, including anti-oxidative activity and cancer-preventive effect (1). In addition, concerning antitumor drug resistance, EGCG has been shown to decrease doxorubicin (DOX) resistance in a human carcinoma xenograft model mice (2) and inhibit the transport function of P-glycoprotein (P-gp), an export drug transporter causing antitumor multidrug resistance (MDR) in tumor cells (3-5).
Human P-gp is a plasma membrane protein of 170 kDa encoded by the MDR1 (ABCB1) gene. The expression of MDR1 gene and P-gp is affected by various chemical substances and physico-chemical stress (5-7). We have previously suggested that the P-gp level was up-regulated through the activation of protein kinase C/nuclear factor-ĸB (PKC/NF-ĸB) in rats with streptozotocin-induced diabetes (8). Further, the expression of MDR1 gene has been also shown to be induced by PI3K/Akt/NF-ĸB signaling (9, 10).
EGCG has been shown to activate ERK and PI3K/Akt signaling and induce antioxidant enzymes in human mammary epithelial cells (11). Contrarily, it has been shown that EGCG inhibits the PI3K/Akt system in fibroblast cells (12) and the phosphorylation of ERK and Akt kinases in epidermal growth factor (EGF)-stimulated cells (13). More recently, accumulating evidence indicates that EGCG can inhibit PI3K/Akt and MAPK signaling systems (14-17).
From these backgrounds, in the present study, we aimed to clarify whether EGCG could suppress MDR and inhibit the induction of P-gp overexpression by DOX through inhibition of PI3K/Akt and ERK/MAPK signal transduction systems.
Materials and Methods
Chemicals. EGCG, DOX, rhodamine 123 (Rho123), LY294002 and SB202190 were purchased from Sigma Japan (Tokyo, Japan). SP600125 was purchased from Biomol (Plymouth Meeting, PA, USA). U0126 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Rapamycin was purchased from LC Laboratories (Woburn, MA, USA).
Cell culture. Human hepatoma HepG2 cells (RIKEN, Tsukuba, Japan) were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% (v/v) heat-inactivated fetal calf serum (BioWest, Nauille, France), 100 U/ml of penicillin (Invitrogen Japan, Tokyo, Japan), 100 μg/ml of streptomycin (Invitrogen) and 0.25 μg/ml of amphotericin B (Invitrogen) in 35-mm plastic dishes under 5% CO2 at 37°C until semi-confluent. Then, varying concentrations of agents were added to the culture medium and the cells cultured for the designated periods. DOX and EGCG were dissolved in dimethyl sulfoxide (DMSO; Sigma) at an appropriate concentration and added to the culture medium. The concentration of DMSO was adjusted to 0.1% (v/v) of the culture medium in each group.
Rho 123 accumulation assay. Rho123 was dissolved in DMSO and added to the cell culture medium at 3 μM. Cells were incubated for 30 min at 37°C and washed 3 times with phosphate-buffered saline (PBS, pH 7.4). Then, intracellular Rho123 was extracted with 1 ml of ethanol and the fluorescence intensity was measured with ARVO MX multilabel reader (PerkinElmer, Waltham, MA, USA) with excitation and emission wave-lengths of 485 nm and 535 nm, respectively.
Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated using TRIzol Reagent (Invitrogen). Then, cDNA was prepared by incubation of 0.5-1.0 μg of the RNA with RNA reverse transcriptase (ReverTra Ace; TOYOBO, Tokyo, Japan) in 20 μl of reaction buffer according to the ReverTra Ace data sheet. PCR was carried out with 0.5 units of Taq DNA polymerase (Blend Taq; TOYOBO) in 25 μl of PCR solution using Thermal Cycler PxE (Thermo Fisher Scientific, Waltham, MA, USA). PCR primers for MDR1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by Hokkaido System Science. A primer set of MDR1 and GAPDH genes were as follows; MDR1 forward: 5’-AAGCTTAGTACCAAAGAGGCTCTC-3’, MDR1 reverse: 5’-GGCTAGAAACAATAGTGAAAACAA-3’, GAPDH forward: 5’-ACCACAGTCCATGCCATCAC-3’, GAPDH reverse: 5’-TCCACCACCCTGTTGCTGTA-3’. The optimum cycle numbers of PCR were 30 to 35 cycles for MDR1 and 20 to 25 cycles for GAPDH. The PCR products (10 μl) were electrophoresed on a 1.5% agarose gel and visualized with ultraviolet light and ethidium bromide. Densitometry analysis of the images was performed using ImageJ for Windows supplied by National Institutes of Health (Bethesda, MD, USA).
Western blot analysis of P-gp, ERK1/2, p38 MAPK, JNK and Akt. Cells (1×104) were solubilized in 20 μl loading dye containing 125 mM Tris (pH6.8, Sigma), 5% sodium dodecyl sulfate (Sigma), 40% urea (Sigma), 0.1 mM sodium orthovanadate (Sigma), 0.2 M dithiothreitol (Sigma) and the sample was isolated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (8, 10% gel) and transferred onto a Hybond enhanced chemiluminescence (ECL) Nitrocellulose membrane (GE Healthcare, Tokyo, Japan). After blocking with skim milk, the membrane was treated with primary antibodies.
P-gp was detected with the C219 anti-MDR1 P-gp mouse monoclonal antibody (Gene Tex, Irvine, CA, USA) as a primary antibody and horseradish-labeled goat anti-mouse IgG antibody (Santa Cruz Biotechnology, Dallas, CA, USA) as a secondary antibody. Total and phosphorylated ERK1/2, as well as p38 MAPK were detected with anti-ERK1/2, anti-phospho-ERK1/2, anti-38 MAPK and anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA, USA), respectively, as primary antibodies and horseradish-labeled goat anti-rabbit IgG antibody (Cell Signaling Technology) as a secondary antibody.
Western blot analysis of Akt was conducted according to previous reports (18).
Specific antibodies for GAPDH (Sigma) and β-actin (Medical & Biological Laboratories, Aichi, Japan) were used to indicate loading standards.
The specific immunoreactive band was detected using Immobilon Western Detection Reagents (Merk Millipore, Bedford, MA, USA) and a luminescence imager (Light-Capture II; ATTO, Tokyo, Japan). The cellular protein concentration was determined using a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).
Statistical analysis. Data are expressed as the mean±standard deviation (S.D.). Statistical significance was determined by a one-way analysis of variance (ANOVA) and Holm's multiple-comparison test. All p-values <0.05 were considered statistically significant. All statistical analyses were performed using R Commander Plug-in for the EZR (Easy R) Package (RcmdrPlugin.EZR) (19).
Results
Preventive effect of EGCG on DOX-induced overexpression of P-gp and intracellular Rho123 accumulation. Rho123 is a useful substrate of P-gp for the evaluation of export function of P-gp, although it is not completely specific for P-gp (20-22). Since the expression of P-gp and MDR1 gene is induced by DOX as we have previously reported (22), we investigated the preventive action of EGCG against DOX-induced overexpression of P-gp and Rho123 accumulation in the cells. As shown in Figure 1, DOX-induced P-gp overexpression was apparently decreased by EGCG treatment. In relation to the induction of P-gp, the uptake of Rho123 was significantly decreased by DOX pretreatment. The uptake was significantly recovered in the cells pretreated with EGCG and DOX compared with the cells pretreated with DOX alone. These results indicated that EGCG had a potential of preventing DOX-induced overexpression of P-gp and recovering intracellular accumulation of P-gp substrate.
Effects of cell signaling inhibitors on DOX-induced overexpression of MDR1 mRNA. To clarify the mechanism of DOX-induced MDR1 expression, we investigated the effects of some cell signaling inhibitors for MDR1 mRNA expression in HepG2 cells. First, we examined whether DOX-induced MDR1 up-regulation is inhibited by LY294002 (a PI3K inhibitor). As shown in Figure 2A, LY294002 partially inhibited DOX-induced increase in the MDR1 mRNA level, while LY294002 did not affect the basal expression level.
There is contradictory evidence regarding the involvement of the p38 MAPK pathway in MDR1 gene expression. Namely, Barancik et al. (23) and Katayama et al. (24) have reported that SB202190 (a p38 MAPK inhibitor) does not influence MDR1 gene expression; Lu et al. (15), however, have recently indicated that SB203580 down-regulates both the P-gp protein level and MDR1 mRNA level in A2780/Taxol cells. In the present study, SB202190 (10 μM) significantly suppressed the DOX-induced up-regulation of the MDR1 gene, while SB202190 did not affect the basal expression level (Figure 2B). This indicated that the p38 MAPK pathway influenced DOX-induced MDR1 gene expression.
We also investigated the effects of two other inhibitors on MAPK signaling. As shown in Figure 2C, U0126 (a MEK1/2 inhibitor) slightly decreased DOX-induced expressions of MDR1 mRNA but SP600125 (a c-Jun N-terminal kinase (JNK) inhibitor) did not influence its level (Figure 2D).
From these results, it was suggested that DOX induced MDR1 mRNA overexpression through PI3K signaling pathway and p38 MAPK signaling pathway in HepG2 cells. MEK/ERK signaling pathway may also contribute to the DOX-induced overexpression of MDR1 mRNA; however, JNK signaling pathway does not seem to concern the induction.
Effects of EGCG and DOX on the phosphorylation of Akt, ERK1/2 and p38 MAPK. To clarify the mechanism of the inhibitory action of EGCG against DOX-induced overexpression of MDR1 mRNA, we examined the effect of EGCG on the phosphorylation of Akt, ERK1/2 (p44/p42 MAPK) and p38 MAPK. As shown in Figure 3A, DOX significantly increased the phosphorylation of Akt at Ser-473, as was shown previously (25, 26), and EGCG strongly inhibited the phosphorylation of Akt without affecting the total Akt level. As for ERK1/2, DOX significantly increased the phosphorylation of ERK1/2, as previously demonstrated (27). EGCG strongly inhibited the DOX-induced phosphorylation of p42 molecule and had a tendency to suppress DOX-induced phosphorylation of p44 without affecting the total ERK1/2 levels (Figure 3B). As for p38 MAPK, DOX induced the phosphorylation of p38 MAPK (at Thr180/Tyr182) as reported previously (27, 28) but EGCG did not affect the basal and DOX-induced phosphorylation of p38 MAPK and the total p38 MAPK level in HepG2 cells (Figure 3C). From these results, it was indicated that the inhibition of the PI3K/Akt and ERK signaling pathways by EGCG contributed to prevent the DOX-induced up-regulation of MDR1 mRNA level.
Effect of rapamycin on P-gp expression in HepG2 cells exposed to DOX. Recently, it has been indicated that EGCG has an inhibitory property against mTOR, as well as PI3K (17). Therefore, we investigated the effect of rapamycin on P-gp level as this agent inactivates mammalian target of rapamycin complex 1 (mTORC1). In the present study, rapamycin significantly inhibited the induction of P-gp by DOX (Figure 4). Since mTORC1 mediates a signal of PI3K/Akt pathway (29), we speculated that the activation of PI3K/Akt signaling by DOX and the inhibition of it by EGCG, seen in Figure 3A, might contribute to the regulation of P-gp overexpression.
Effects of the inhibitors for PI3K/Akt and MEK/ERK signaling pathways on P-gp expression in HepG2 cells exposed to DOX. As shown in Figures 2 and 3, the inhibitors for PI3K/Akt and MEK/ERK signaling pathways decreased the induction of MDR1 mRNA and EGCG blocked DOX-induced activation of PI3K/Akt and MEK/ERK signaling. Further, rapamycin inhibited DOX-induced P-gp expression (Figure 4). Then we examined the effect of inhibitors for PI3K/Akt and MEK/ERK signaling pathways on DOX-induced overexpression of P-gp (Figure 5). U0126 significantly inhibited DOX-induced P-gp overexpression, as well as MDR1 mRNA. However, LY294002 did not affect the expression of P-gp, while wortmannin also did not affect the DOX-induced overexpression of P-gp (data not shown). Interestingly, however, P-gp level was synergistically inhibited by the co-application of the inhibitors for PI3K/Akt and MEK/ERK signaling pathways. From these results, it was indicated that the dual inhibition of PI3K/Akt and MEK/ERK signaling pathways can effectively regulate the overexpression of P-gp and that PI3K/Akt signaling and MEK/ERK signaling pathways would coordinately regulate the expression of P-gp.
Discussion
Green tea polyphenol, containing more than 65% EGCG, has been shown to sensitize MDR of KB-A-1 cells when inoculated to nude mice (30). EGCG has been known to inhibit export function of P-gp and to reverse DOX resistance in vivo (2, 3, 31), while a recent study by Cheng et al. (32) showed EGCG-based complex micelles with DOX effectively reducing cardiotoxicity in mice.
In this study, we showed that EGCG selectively prevented DOX-induced P-gp overexpression at a dosage without influencing the basal expression of P-gp. Further, we showed that EGCG recovered Rho123 accumulation along with the suppression of P-gp overexpression when the cells were co-pretreated with DOX and EGCG. Thus, our results indicated that EGCG prevented the overexpression of P-gp and, thereby, inhibited enhancement of P-gp-mediated extrusion of its substrate.
In the present study, to clarify the signaling pathway concerning the inhibitory effect of EGCG on DOX-induced MDR1 gene expression, we first evaluated the effects of specific kinase inhibitors on DOX-induced MDR1 mRNA expression in HepG2 cells. Regarding the role of PI3K/Akt signaling pathway, our results indicated that MDR1 gene overexpression by DOX was partially inhibited by a PI3K/Akt inhibitor (Figure 2A). However, a PI3K/Akt inhibitor did not affect P-gp overexpression (Figure 5). Although EGCG inhibits DOX-induced Akt phosphorylation (Figure3A), the inhibition itself may scarcely contribute to the inhibition of P-gp overexpression in DOX-treated HepG2 cells. This phenomenon may indicate that MDR1 mRNA overexpression is not enough for inducing P-gp overexpression through the activation of PI3K/Akt.
It has already been reported an increase in phospho-ERK levels in DOX-treated HepG2 cells (33). In this study, we also showed that DOX induced activation of MEK/ERK pathway in HepG2 cells and that U0126, a MEK/ERK inhibitor, inhibited P-gp overexpression (Figure 5) and MDR1 mRNA (Figure 2C). Further, EGCG significantly inhibited DOX-induced phosphorylation of p42 ERK molecule (Figure 3B). These findings may suggest that MEK/ERK pathway significantly contributes to the overexpression of P-gp induced by DOX. Further, it may be suggested that DOX-stimulated MEK/ERK signaling may positively and post-transcriptionally control P-gp expression. In addition, it was indicated that the inhibitory action of EGCG against MEK/ERK pathway was implicated in the inhibition of P-gp overexpression.
P-gp expression was synergistically inhibited by the combined treatment with PI3K/Akt and ERK inhibitors (Figure 5). This evidence may indicate that DOX increases P-gp expression through co-activation of these kinases. Further, these signaling pathways may coordinately regulate the expression of P-gp. As EGCG inhibits PI3K/Akt and MEK/ERK signaling, EGCG would negatively regulate P-gp overexpression through dual inhibition of these signaling pathways in HepG2 cells, although further potential mechanisms remain to be clarified. As rapamycin, which is an inhibitor of mTOR, inhibited DOX-induced overexpression of P-gp (Figure 4) and EGCG reportedly has an inhibitory effect on mTOR (14), which is a down-stream effector of PI3K/Akt signaling system, cross-talk of PI3K/Akt and ERK signaling pathways (29) may be involved in the control of P-gp expression.
Conclusion
EGCG inhibited DOX-induced overexpression of P-gp. According to the evidence of DOX-induced overexpression of P-gp accompanied by co-activation of MEK/ERK and PI3K/Akt signaling cascades and the inhibition by EGCG of DOX-induced activation of these pathways, it can be suggested that EGCG has a profitable efficacy of regulating DOX-induced P-gp overexpression through the coordinate inhibition of PI3K/Akt and MEK/ERK signaling pathways.
Footnotes
This article is freely accessible online.
Conflicts of Interest
All Authors declare that there are no conflicts of interest.
Funding
This research did not receive any specific grant from any funding agency of the public, commercial or not-for-profit sector.
- Received April 12, 2017.
- Revision received April 26, 2017.
- Accepted April 27, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved