OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2019.11069 OL-0-0-11069 Articles Activity and mechanism of flavokawain A in inhibiting P-glycoprotein expression in paclitaxel resistance of lung cancer LiJuan 1 ZhengLei 2 YanMi 1 WuJing 1 LiuYongqing 1 TianXiaona 1 JiangWen 3 ZhangLu 3 WangRongmei 1 Department of Clinical Pharmacy, The Second Hospital of Shandong University, Jinan, Shandong 250033, P.R. China Department of Pharmacy, Shandong Provincial Third Hospital, Jinan, Shandong 250031, P.R. China Central Research Laboratory, The Second Hospital of Shandong University, Jinan, Shandong 250033, P.R. China Correspondence to: Dr Juan Li or Dr Rongmei Wang, Department of Clinical Pharmacy, The Second Hospital of Shandong University, 247 Beiyuan Road, Jinan, Shandong 250033, P.R. China, E-mail: lijuan198407@163.com, E-mail: rongmeiwang@hotmail.com 01 2020 08 11 2019 19 1 379 387 10042019 11092019 Copyright: © Li et al. 2019 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Lung cancer is one of the most common cancers, which is the leading cause of cancer-related death among various cancers worldwide. Flavokawain A (FKA), a chalcone found in the kava plant, exerts potent anticancer activity. However, the activity and mechanisms of FKA in inhibiting the viability of paclitaxel (PTX)-resistant lung cancer A549 (A549/T) have not been investigated. In the present study, the effect of FKA on the viability of A549/T and hepatotoxicity in normal liver epithelial cells was detected by Cell Counting Kit-8 assay. Flow cytometry, western blot analysis and Annexin V-FITC/PI apoptosis detection kit were used to assess cell apoptosis. The effect of FKA on permeability-glycoprotein (P-gp) expression was measured by reverse transcription-PCR and western blot analysis. The results indicated that FKA dose-dependently inhibited cell proliferation and induced cell apoptosis in PTX-resistant A549/T cells, with an IC50 value of ~21 µM, while the IC50 value of A549/T cells to PTX was 34.64 µM. FKA had no hepatic toxicity in liver epithelial cells. P-gp, which contributes to the chemoresistant phenotype, was not expressed in A549 cells but was remarkably enhanced in A549/T cells. FKA (30 µM) decreased P-gp protein expression at 24 h by 3-fold. Furthermore, FKA downregulated P-gp expression by blocking the PI3K/Akt pathway. These findings suggest FKA as a potential candidate for the treatment of PTX-resistant lung cancer.

 flavokawain A chemotherapy resistance permeability- glycoprotein PI3K/Akt signaling pathway Introduction

Lung cancer is recognized as a leading cause of cancer-associated mortality and morbidity worldwide. According to the latest report of the American Cancer Society, ~2.09 million new cases of lung cancer and ~1.76 million associated deaths were reported in the world in 2018 (1). For patients with lung cancer who cannot undergo surgery and radiotherapy, chemotherapy is still the major treatment option. However, chemotherapy only offers a modest survival advantage due to chemoresistance.

One of the molecular mechanisms that drive the development of chemoresistance is permeability-glycoprotein (P-gp)-mediated drug resistance (2). P-gp is often highly expressed in drug-resistant tumor cells. Several hydrophobic antitumor drugs, such as paclitaxel (PTX) and vinblastine, are subject to efflux out of the cells by P-gp (3). Therefore, the inhibition of P-gp-mediated drug efflux will presumably resensitize resistant cancer cells to chemotherapy drugs, which may lead to favorable chemotherapeutic outcomes. Various P-gp inhibitors, such as verapamil and cyclosporine, have been identified to reverse chemoresistance and sensitize tumor cells to chemotherapeutic agents (4). However, the clinical use of first-generation reversal agents (verapamil and cyclosporine) and second-generation inhibitors (dexverapamil and PSC-833) failed, due to undesired side effects and toxicity issues (5). Thus, no P-gp inhibitor has yet been applied for clinical use.

Natural compounds have great potential in inhibiting tumor growth and reversing chemotherapy resistance (6,7). Chalcones are the most common group of flavonoid/isoflavonoid compounds found in vegetables and fruits. Earlier studies have demonstrated that numerous chalcones and their derivatives exhibit antitumor potential (8–10) with almost no toxic side effects to normal cells (11). Flavokawain A (FKA), a novel chalcone from the kava plant, induced apoptosis and G2/M arrest in different tumor cells (12–14). However, the potential therapeutic effect and the underlying molecular mechanism of FKA in paclitaxel (PTX)-resistant A549 cells are yet to be elucidated.

In the present study, the PTX-resistant cell line A549/T was established. The efficacy of FKA in the inhibition of A549/T cell viability in vitro was assessed. Additionally, the capacity of FKA in reversing P-gp-mediated PTX resistance and the potential underlying mechanisms were also investigated.

 Materials and methods <sec> <title>Reagents

FKA of ≥99% purity was purchased from Sigma-Aldrich (Merck KGaA). FKA was dissolved in dimethyl sulfoxide (DMSO) to form a 30 mM stock solution. Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies, Inc. PTX, LY294002 and DAPI were all obtained from Sigma-Aldrich (Merck KGaA). Insulin-like factor-1 (IGF-1) was purchased from Abcam (cat. no. 128524). Monoclonal rabbit anti-human P-gp (cat. no. 13342), monoclonal rabbit anti-human Akt (cat. no. 4691), polyclonal rabbit anti-human phosphorylated (p)-Akt (Ser 473; cat. no. 9271), monoclonal rabbit anti-human PARP (46D11; cat. no. 9532) and polyclonal rabbit anti-human β-actin (cat. no. 4970) were obtained from Cell Signaling Technology, Inc. The monoclonal mouse anti-human GAPDH antibody (cat. no. 60004-1-Ig) was obtained from ProteinTech Group, Inc. Horseradish peroxidase (HRP)-labelled goat anti-rabbit immunoglobulin G (cat. no. TA130023) and HRP-labelled goat anti-mouse immunoglobulin G (cat. no. TA130003) were obtained from OriGene Technologies, Inc.

 Cell culture

Human lung adenocarcinoma cells A549 and PTX-resistant A549 (A549/T) cells were kindly gifted by the Central Research Laboratory of the Second Hospital of Shandong University (Jinan, China). Human hepatic epithelial cells THLE-3 were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cells were cultured in RPMI-1640 (HyClone; GE Healthcare Life Sciences) containing 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), penicillin-streptomycin (100 U/ml) and 2 mM glutamine. The cells were cultured at 37°C in an incubator with 5% CO2. The A549/T cells were maintained in medium with 3 nM PTX to maintain PTX resistance in this cell line. Before the experiment, cells were cultured in drug-free medium for ≥2 weeks.

 Cell viability assay

The effect of PTX or FKA on the viability of A549 and A549/T cells was evaluated by Cell Counting Kit-8 assay. The toxicity effect of FKA was also evaluated in human hepatic epithelial THLE-3 cells. A549, A549/T and THLE-3 cells were cultured in 96-well plates (4×103 cells/well) and incubated overnight. Subsequently, the cells were stimulated for 48 h with increasing concentrations of PTX or FKA. The controls were treated with equal volume of DMSO. Cell proliferation inhibition was assayed by the Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc.) and the methods used were performed according to manufacturer's protocol. The absorbance was measured at 450 nm using a microplate reader.

 Cell apoptosis assay

Cells were plated at a density of 2×105 cells/2 ml medium on 6-well plates for 24 h. Following treatment with various concentrations of FKA (0, 5, 10 and 30 µM) for 24 h, cell apoptosis was detected using DAPI staining. Cells were fixed with 90% ethanol/5% acetic acid for 1 h at room temperature. Following 2 washes with PBS, cells were incubated with DAPI solution (1.5 mg/ml in PBS) for 30 min at room temperature. Images of DAPI fluorescence were captured using a fluorescence microscope (magnification, ×200; Nikon Corporation). After treated by different concentrations of FKA (0, 5, 10 and 30 µM) for 24 h at 37°C, cells were digested with trypsin and centrifuged at 120 × g for 5 min at 4°C. Following 2 washes with PBS, levels of apoptosis were analyzed using an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (BD Biosciences), according to the manufacturer's protocol. Quantification of fluorescence was determined using flow cytometry (FACSCalibur™; BD Biosciences), and the data were analyzed using WinMDI software v2.8 (Purdue University Cytometry Laboratories).

 Reverse transcription-semi quantitative PCR (RT-PCR)

Cells were treated with 0, 3 and 30 µM FKA for 24 h at 37°C. Total mRNA was subsequently extracted with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Complementary (c)DNA was generated using a reverse transcription kit reagent kit (Promega Corporation). The mixture was incubated at 42°C for 10 min. The resultant cDNA was then amplified using primers specific for P-gp (forward primer, 5′-TGACCCGCACTTCAGCTACAT-3′; and reverse primer, 5′-ACTGGGCTTCCCGATGATGTA-3′). As an internal control, GAPDH expression was detected (forward primer, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse primer, 5′-CACCCTGTTGCTGTAGCCAA-3′). The PCR conditions were as follows: 94°C for 5 min; 20 cycles at 94°C for 60 sec, 60°C for 30 sec and 72°C for 60 sec; and 94°C for 5 min. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel and stained using ethidium bromide, then quantified using the ChemiImager 400 software (ProteinSimple). Experiments were conducted in triplicate and normalized to the expression of GAPDH.

 Western blot analysis

Briefly, A549/T cells (3×105) were incubated with different concentrations of FKA (0, 2.5, 5, 10 and 30 µM) for 24 h. Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer according to the manufacturer's protocol (Beyotime Institute of Biotechnology, Inc.). Total protein was quantified using the BCA protein assay (Beyotime Institute of Biotechnology, Inc.). Samples containing equal amounts of protein (50 µg) from the lysates were separated using 8 and 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore; Merck KGaA). The blot was incubated with 5% skimmed milk for 1 h at room temperature. Next, the membrane was probed with monoclonal rabbit anti-human antibodies against P-gp, Akt and a polyclonal rabbit anti-human antibody against p-Akt (Ser 473) (all 1:1,000) overnight at 4°C. Following washes with TBS/Tween-20 buffer, the membranes were incubated with either the HRP-labelled goat anti-rabbit secondary antibody (1:10,000) or the HRP-labelled goat anti-mouse secondary antibody (1:10,000) for 2 h at room temperature. GAPDH served as a protein loading control. For poly (ADP-ribose) polymerase (PARP) expression detection, the western blot method used is aforementioned however monoclonal rabbit anti-human PARP (cat. no. 46D11; 1:1,000) was used. β-actin served as a protein loading control. The ECL detection system (GE Healthcare Life Sciences) was used to detect the signal. Protein levels were quantified using densitometry using ImageJ v1.6 softwate (National Institutes of Health).

 Inhibition or activation of PI3K/Akt signaling

The PI3K inhibitor LY294002 and the PI3K-specific agonist IGF-1 were used to suppress or activate the PI3K/Akt signaling pathway, respectively, in A549/T cells. Cells (1×104 cells/well) were incubated with the following: DMSO; 30 µM FKA for 24 h; LY294002 (10 µM) for 1 h; IGF-1 (12.5 nM) (15) for 1 h; 30 µM FKA for 24 h, followed by LY294002 for 1 h; or 30 µM FKA for 24 h, followed by IGF-1 for 1 h. The protein expression levels of p-Akt (Ser 473) and P-gp were measured by western blot analysis. Each experiment was conducted in triplicates to determine the means and standard deviations (SDs).

 Statistical analyses

The data are presented as the mean ± SD of ≥3 independent experiments and analyzed by GraphPad Prism 8 (GraphPad Software, Inc.). Comparisons of cell viability of human PTX-resistant A549/T cells and parental A549 cells following PTX or FKA treatment were evaluated by one-way analysis of variance (ANOVA) with Tukey's post-hoc test. Other multiple group comparisons were performed with one-way ANOVA, followed by Dunnett's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>FKA significantly inhibits A549/T cell viability

As shown in Fig. 1A, PTX dose-dependently inhibited cell proliferation in A549 cells, with an IC50 value of 0.3 µM. However, the cell viability of PTX-resistant A549/T cells was 79% following treatment with 30 µM PTX, and the IC50 was 34.64 µM. As shown in Fig. 1B, A549/T cells were sensitive to FKA, with a marginally decreased IC50 value of ~21 µM compared with ~26 µM in A549 cells. FKA at increasing concentrations of 2.5, 5, 10, 20 and 30 µM resulted in decreased cell viability in a dose-dependent manner, inhibiting A549/T cell growth by ~6.2, 11.4, 25.0, 28.3 and 59.4%, while inhibiting A549 cell growth by ~7.3, 20.7, 28.3, 30.5 and 56.8%, respectively (Fig. 1B). In addition, FKA at 30 µM exhibited almost no toxic effects on normal human hepatic epithelial THLE-3 cells (Fig. 1C). Moreover, morphological analysis revealed that cell shrinkage was observed following FKA treatment in A549/T cells, and cell shrinkage became more noticeable as the time and dose of FKA increased, suggesting that apoptosis was induced by FKA (Fig. 1D). The above data demonstrated that FKA was capable of inhibiting A549/T cell proliferation.

 FKA induces apoptosis in A549/T cells

Flow cytometry was used to analyze apoptosis in A549/T cells treated with FKA. The results revealed that FKA caused a markedly dose-dependent increase in the proportion of apoptotic cells at 24 h, with ~9.2, 17.9 and 21.4% early and late apoptotic cells following treatment with 5, 10 and 30 µM FKA, respectively (Fig. 2A). In addition, increased cleavage of PARP, a hallmark of apoptosis, was observed in A549/T cells exposed to 30 µM FKA, (Fig. 2B). The nucleic morphological changes associated with apoptosis were assessed by staining nuclear DNA with DAPI. FKA treatment (30 µM) at 48 h resulted in a marked increase in the number of apoptotic cells (indicated using arrows) with condensed and fragmented DNA (Fig. 2Ca) compared with A549/T cells treated with 30 µM FKA for 24 h (Fig. 2Cb). Together, these results suggest that FKA induced apoptosis in A549/T cells.

 FKA inhibits the protein expression of P-gp

RT-semi quantitative PCR and western blot analysis were performed to evaluate the effects of FKA on P-gp expression at the mRNA and protein level. The results demonstrated that the mRNA expression of P-gp slightly decreased to 90 and 80% compared with the control cells, following treatment with 3 and 30 µM FKA, respectively (Fig. 3). However, the differences were not statistically significant. The results from the western blot analysis revealed that P-gp was not expressed in A549 cells but it was highly expressed in A549/T cells (P<0.01; Fig. 4A). Subsequently, the protein levels of P-gp in A549/T cells were measured in response to various concentration of FKA. The expression of P-gp was significantly decreased, ~3.0-fold, following treatment with 30 µM FKA for 24 h (Fig. 4B). Subsequently, the changes in P-gp expression with respect to treatment duration were investigated. As indicated in Fig. 4C, the level of P-gp expression decreased from 16 to 24 h (~2.5-fold) following treatment with 30 µM FKA. Thus, treatment with FKA decreased the protein expression of P-gp in A549/T cells.

 FKA downregulates P-gp expression via inhibition of the PI3K/Akt signaling pathway

The involvement of PI3K/Akt signaling pathway in P-gp-mediated multidrug resistance has been reported (16). High protein expression of P-gp, Akt and p-Akt (Ser 473) was detected in A549/T cells. FKA downregulated the protein expression of P-gp, Akt and p-Akt (Ser 473). Notably, the levels of P-gp, Akt and p-Akt (Ser 473) were significantly decreased following treatment with 30 µM FKA, as compared with the control group (P<0.05; Fig. 5). To confirm whether the suppression or activation of the PI3K/Akt pathway influenced the levels of P-gp, the effect of the PI3K inhibitor LY294002 and the PI3K agonist IGF-1 on P-gp and PI3K/Akt signaling was investigated. The expression of p-Akt (Ser 473) was decreased following treatment with LY294002 for 1 h compared with the untreated control (P<0.01; Fig. 6A and B). A549/T cells treated with 30 µM FKA for 24 h exhibited decreased expression of P-gp and p-Akt (Ser 473). In addition, the combined treatment of FKA and LY294002 (pretreatment with 30 µM FKA for 24 h, followed by treatment with 10 µM LY294002 for 1 h) exerted additive inhibitory effects on P-gp and p-Akt (Ser 473) expression. As illustrated in Fig. 6B, the expression levels of P-gp and p-Akt (Ser 473) were not influenced by PI3K agonist IGF-1 but inhibited by 30 µM FKA. The combined treatment of FKA and IGF-1 (pretreatment with 30 µM FKA for 24 h, followed by treatment with IGF-1 for 1 h) also inhibited the expression of p-Akt (Ser 473). Thus, 30 µM FKA could reverse IGF-1-induced activation of the PI3K/Akt signaling pathway. Overall, FKA was demonstrated to inhibit P-gp expression via the PI3K/Akt signaling pathway.

 Discussion

Drug resistance is the main obstacle in cancer chemotherapy and results in poor therapeutic outcome (17). P-gp overexpression is one of the main mechanisms underlying drug resistance. Inhibiting the expression or function of P-gp is a key step towards improved treatment of patients with cancer. Inhibitors of the drug-efflux pump have been reported as effective in increasing the sensitivity to anticancer drugs (18). However, no potential modulators are currently licensed for clinical application, due to the associated toxicities or unacceptable side effects.

Chalcones and their derivatives have great advantages in overcoming chemotherapeutic drug resistance by inhibiting the protein expression or functions of ATP-binding cassette (ABC) transporters, including P-gp and multidrug resistance (MDR)-associated protein 1 (MRP1) (19). 2′-Hydroxy-2,4,6′-trimethoxychalcone was shown to significantly inhibit the protein expression of P-gp in MES-SA/DX5 cells and overcome P-gp-mediated MDR in drug-resistant uterine sarcoma cells (20). Komoto et al (21) found that the chalcone licochalcone A decreased the protein expression of P-gp in BT20 breast cancer cells. The present study demonstrated that FKA did not block the efflux of Rh-123 out of cells (data not shown), which demonstrated that FKA had no influence on the efflux function of ABC transporters. Furthermore, the mRNA expression of P-gp was unaffected following treatment with 30 µM FKA. Conversely, the protein level of P-gp was significantly inhibited by 30 µM FKA. In summary, to the best of our knowledge, the present study is the first to report that FKA decreased the viability of A549/T cells by inhibiting the protein expression of P-gp.

The PI3K/Akt pathway is a commonly activated signaling pathway in cancer, which plays an important role in inhibiting cancer cell apoptosis and promoting cancer cell proliferation, invasion and angiogenesis (22). As the central node of the PI3K/Akt signaling cascade, Akt activates multiple oncogenic signaling pathways to promote cancer, and Akt hyperactivation has been associated with poor differentiation and worse prognosis (23). Akt also regulates the expression of the MDR1 gene, while P-gp is one of the downstream factors of the PI3K/Akt pathway (24). Increased Akt phosphorylation has been reported to be associated with chemotherapeutic resistance (25). However, the association between the inhibition of P-gp expression and the PI3K/Akt pathway by FKA is still unclear. In the present study, the effect of FKA on P-gp expression via the PI3K/Akt pathway was investigated. PTX-resistant A549/T cells displayed high expression levels of p-Akt and P-gp, which represented high PI3K/Akt activity, as well as an association between PI3K signaling and the resistant phenotype. Following treatment with 30 µM FKA, the expression levels of P-gp, Akt and p-Akt (Ser 473) decreased significantly. In addition, it was found that LY294002 and IGF-1 promoted and inhibited Akt phosphorylation, respectively. These results indicated that FKA inhibited P-gp expression by inhibiting the PI3K/Akt pathway.

Numerous small molecule Akt inhibitors have been developed and tested in preclinical or clinical models. Ipatasertib, a novel selective ATP competitive small-molecule inhibitor of Akt, preferentially targets active p-Akt and exerts anti-tumor activity (26). LY294002, a flavonoid-derived PI3K/Akt inhibitor, inhibits PI3K by competing with ATP for binding to the PI3K active site (27). LY294002 also inhibits Akt phosphorylation (28). Furthermore, it was reported that LY294002 had inhibitory effects on ABC transporters, including breast cancer resistance protein (BCRP), MRP1 and P-gp. LY294002 competitively inhibited the transport activity of BCRP, while exerting inhibitory effects on MRP1 function via competitive inhibition of substrate transport and modulation of expression (29). LY294002 also antagonized the transport activity of P-gp without influencing its expression (29). Since FKA belongs to flavonoid/isoflavonoid compounds, its chemical structure is similar to that of LY294002. The present study demonstrated the inhibition of P-gp protein expression, rather than its pumping function, by FKA. The inhibitory effect of FKA on BCRP and MRP1 requires further investigation.

The kinase function of Akt is activated following the phosphorylation of two residues, threonine 308 (Thr 308) and serine 473 (Ser 473). It was reported that FKA downregulates Akt activation by suppressing Akt phosphorylation in HER2-overexpressing breast cancer cells (14). In the present study, FKA suppressed Akt activation by decreasing p-Akt (Ser 473) expression level. The Akt (pan) (C67E7) monoclonal anitbody (cat. no. 4691; Cell Signaling Technology, Inc.) detects endogenous levels of total (both phosphorylated and non-phosphorylated) Akt1, Akt2 and Akt3 protein. The target undergoes a wide range of post-translational modifications (PTMs). These PTMs can lead to a slight shift in signal. Each Akt isoform can have a unique expression pattern depending on the specific treatment and specific cells. The existing two bands of Akt (in Fig. 5A and Fig. 6) are close in size to 60 kDa is common in A549 cells. The same double band pattern was also observed in A549 cells in other publications detecting total Akt (30,31).

A previous report claimed that kava extracts may cause hepatotoxicity (32). Interestingly, it was reported that liver toxicity cases occurred in the South Pacific region only, and this may be due to the tropical humidity and temperature as the kava plants may have been contaminated by mould hepatotoxins instead (33). A previous study also confirmed that kava alone did not affect mouse development and induced no signs of hepatotoxicity (34). In order to detect whether FKA has hepatotoxicity, the effect of FKA on human normal liver cells was investigated. The result demonstrated that FKA exerted no obvious hepatotoxicity to normal liver epithelial cells. Overall, there is a need to determine the acute and subchronic toxicity of FKA in vivo.

The effect of FKA on the expression of other ABC transporters such as MRP1 and BCRP has not been studied. Thus, further investigation is required in order to fully understand the mechanism by which FKA inhibits the function of ABC transporters and to determine the signaling pathway that inhibits P-gp expression. In addition, the inhibitory activity of FKA alone or combined with chemotherapy drugs in resistant tumor cells requires investigation in future in vivo studies.

In summary, the present study demonstrated the capacity of FKA to decrease the viability in PTX-resistance cells and inhibit P-gp expression. Furthermore, FKA inhibits the protein expression of P-gp via inhibition of the PI3K/Akt pathway. The findings of the present study may provide a foundation for the development of better strategies for reversing chemotherapy resistance.

 Acknowledgements

The authors would like to thank Dr Wen Jiang for flow cytometry data analysis and Dr Lu Zhang for providing the cells (both Central Research Laboratory at the Second Hospital of Shandong University, Shandong, China).

 Funding

The present study was funded by the Shandong Natural Science Foundation (grant no. ZR2017BH014), the Shandong Key Research and Development Program (grant nos. 2018GSF118010 and 2018GSF118075) and the Shandong Province Medical and Health Technology Development Plan (grant no. 2016WS0334).

 Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

 Authors' contributions

JL and RW designed the study. JL, MY, YL, JW and XT performed the experiments. JL and RW contributed new reagents and analytic tools. LeZ WJ and LuZ analyzed the data. JL wrote the paper. JL and RW revised the paper.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References BrayFFerlayJSoerjomataramISiegelRLTorreLAJemalAGlobal cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countriesCA Cancer J Clin68394424201810.3322/caac.2149230207593 ChakravartyGMathurAMalladePGerlachSWillisJDattaASrivastavSAbdel-MageedABMondalDNelfinavir targets multiple drug resistance mechanisms to increase the efficacy of doxorubicin in MCF-7/Dox breast cancer cellsBiochimie1245364201610.1016/j.biochi.2016.01.01426844637 Podolski-RenićABankovićJDinićJRíos-LuciCFernandesMXOrtegaNKovačević-GrujičićNMartínVSPadrónJMPešićMDTA0100, dual topoisomerase II and microtubule inhibitor, evades paclitaxel resistance in P-glycoprotein overexpressing cancer cellsEur J Pharm Sci105159168201710.1016/j.ejps.2017.05.01128502672 AbrahamJSalamaNNAzabAKThe role of P-glycoprotein in drug resistance in multiple myelomaLeuk Lymphoma562633201510.3109/10428194.2014.90789024678978 JoshiPVishwakarmaRABharateSBNatural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancerEur J Med Chem138273292201710.1016/j.ejmech.2017.06.04728675836 GuoQCaoHQiXLiHYePWangZWangDSunMResearch progress in reversal of tumor multi-drug resistance via natural productsAnticancer Agents Med Chem1714661476201710.2174/187152061766617101610570429034843 LiuYYueCLiJWuJWangSSunDGuoYLinZZhangDWangREnhancement of cisplatin cytotoxicity by retigeric acid B involves blocking DNA repair and activating DR5 in prostate cancer cellsOncol Lett1528712880201829435013 CabralBLSda SilvaACGde ÁvilaRICortezAPLuzinRMLiãoLMde Souza GilESanzGVazBGSabinoJRA novel chalcone derivative, LQFM064, induces breast cancer cells death via p53, p21, KIT and PDGFRAEur J Pharm Sci107115201710.1016/j.ejps.2017.06.01828627468 SantosMBPinhanelliVCGarciaMARSilvaGBaekSJFrançaSCFachinALMarinsMRegasiniLOAntiproliferative and pro-apoptotic activities of 2-and 4-aminochalcones against tumor canine cellsEur J Med Chem138884889201710.1016/j.ejmech.2017.06.04928738308 LeeDHJung JungYKohDLimYLeeYHShinSYA synthetic chalcone, 2-hydroxy-2,3,5-trimethoxychalcone triggers unfolded protein response-mediated apoptosis in breast cancer cellsCancer Lett37219201610.1016/j.canlet.2015.12.01726742460 JandialDDBlairCAZhangSKrillLSZhangYBZiXMolecular targeted approaches to cancer therapy and prevention using chalconesCurr Cancer Drug Targets14181200201410.2174/156800961466614012216051524467530 AbuNAkhtarMNYeapSKLimKLHoWYZulfadliAJOmarARSulaimanMRAbdullahMPAlitheenNBFlavokawain A induces apoptosis in MCF-7 and MDA-MB231 and inhibits the metastatic process in vitroPLoS One9e105244201410.1371/journal.pone.010524425286005 AbuNMohamedaNETangarajooNYeapSKAkhtarMNAbdullahMPOmarARAlitheenNBIn vitro toxicity and in vivo immunomodulatory effects of flavokawain A and flavokawain B in Balb/C miceNat Prod Commun1011991202201526411010 JandialDDKrillLSChenLWuCKeYXieJHoangBHZiXInduction of G2M arrest by flavokawain A, a kava chalcone, increases the responsiveness of HER2-overexpressing breast cancer cells to herceptinMolecules22E462201710.3390/molecules2203046228335434 ZhangSKuangGZhaoGWuXZhangCLeiRXiaTChenJWangZMaRInvolvement of the mitochondrial p53 pathway in PBDE-47-induced SH-SY5Y cells apoptosis andits underlying activation mechanismFood Chem Toxicol62699706201310.1016/j.fct.2013.10.00824140468 MuthusamyGGunaseelanSPrasadNRFerulic acid reverses P-glycoprotein-mediated multidrug resistance via inhibition of PI3K/Akt/NF-κB signaling pathwayJ Nutr Biochem636271201910.1016/j.jnutbio.2018.09.02230342318 MaHChengLHaoKLiYSongXZhouHJiaLReversal effect of ST6GAL 1 on multidrug resistancein human leukemia by regulating the PI3K/Akt pathway and the expression of P-gp and MRP1PLoS One9e85113201410.1371/journal.pone.008511324454800 CalatozzoloCGelatiMCiusaniESciaccaFLPolloBCajolaLMarrasCSilvaniAVitellaro-ZuccarelloLCrociDExpression of drug resistance proteins Pgp, MRP1, MRP3, MRP5 and GST-pi in human gliomaJ Neurooncol74113121200510.1007/s11060-004-6152-716193381 LindamulageIKVuHYKarthikeyanCKnocklebyJLeeYFTrivediPLeeHNovel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 functionSci Rep710298201710.1038/s41598-017-10972-028860494 ShinSYLeeMSLeeDHLeeDHKohDLeeYHThe synthetic compound 2-hydroxy-2,4,6-trimethoxychalcone overcomes P-glycoprotein-mediated multi-drug resistance in drug-resistant uterine sarcoma MES-SA/DX5 cellsJ Korean Soc Appl Bi58105109201510.1007/s13765-015-0017-y KomotoTTBernardesTMMesquitaTBBortolottoLFBSilvaGBitencourtTABaekSJMarinsMFachinALChalcones repressed the AURKA and MDR proteinsinvolved in metastasis and multiple drug resistance in breast cancer cell linesMolecules232018201810.3390/molecules23082018 WangHJiaXHChenJRWangJYLiYJOsthole shows the potential to overcome P-glycoprotein-mediated multidrug resistance in human myelogenous leukemia K562/ADM cells by inhibiting the PI3K/Akt signaling pathwayOncol Rep3536593668201610.3892/or.2016.473027109742 CaoZLiaoQSuMHuangKJinJCaoDAKT and ERK dual inhibitors: The way forward?Cancer Lett4593040201910.1016/j.canlet.2019.05.02531128213 HanZHongLHanYWuKHanSShenHLiCYaoLQiaoTFanDPhospho Akt mediates multidrug resistance of gastric cancer cells through regulation of P-gp, Bcl-2 and BaxJ Exp Clin Cancer Res26261268200717725107 MorishitaNTsukaharaHChayamaKIshidaTWashioKMiyamuraTYamashitaNOdaMMorishimaTActivation of Akt is associated with poor prognosis and chemotherapeutic resistance in pediatric B-precursor acute lymphoblastic leukemiaPediatr Blood Cancer598389201210.1002/pbc.2403422183914 SauraCRodaDRosellóSOliveiraMMacarullaTPérez-FidalgoJAMorales-BarreraRSanchis-GarcíaJMMusibLBudhaNA first-in-human phase I study of the ATP-competitive AKT inhibitor ipatasertib demonstrates robust and safe targeting of AKT in patients with solid tumorsCancer Discov7102113201710.1158/2159-8290.CD-16-051227872130 ToledoLMLydonNBElbaumDThe structure-based design of ATP-site directed protein kinase inhibitorsCurr Med Chem6775805199910495352 OhnishiKYasumotoJTakahashiAOhnishiTLY294002, an inhibitor of PI-3K, enhances heat sensitivity independently of p53 status in human lung cancer cellsInt J Oncol29249253200616773206 ImaiYYamagishiHOnoYUedaYVersatile inhibitory effects of the flavonoid-derived PI3K/Akt inhibitor, LY294002, on ATP-binding cassette transporters that characterize stem cellsClin Transl Med124201210.1186/2001-1326-1-2423369170 CaoPLiuBDuFLiDWangYYanXLiXLiYScutellarin suppresses proliferation and promotes apoptosis in A549 lung adenocarcinoma cells via AKT/mTOR/4EBP1 and STAT3 pathwaysThorac Cancer10492500201910.1111/1759-7714.1296230666790 JiangWChenYSongXShaoYNingZGuWPim-1 inhibitor SMI-4a suppresses tumor growth in non-small cell lung cancer via PI3K/AKT/mTOR pathwayOnco Targets Ther1230433050201910.2147/OTT.S20314231114247 TeschkeRKava hepatotoxicity-a clinical reviewAnn Hepatol9251265201010.1016/S1665-2681(19)31634-520720265 TeschkeRQiuSXLebotVHerbal hepatotoxicity by kava: Update on pipermethystine, flavokavain B, and mould hepatotoxins as primarily assumed culpritsDig Liver Dis43676681201110.1016/j.dld.2011.01.01821377431 NarayanapillaiSCLeitzmanPO'SullivanMGXingCFlavokawains a and B in kava, not dihydromethysticin, potentiate acetaminophen-induced hepatotoxicity in C57BL/6 miceChem Res Toxicol2718711876201410.1021/tx500319425185080

Effect of FKA on the viability of PTX-resistant A549 cells and normal human hepatic epithelial cells. (A) The in vitro effect of PTX on established human PTX-resistant A549/T and parental A549 cells. *P<0.05 vs. 30 µM FKA treated A549/T cells. (B) Cell viability of A549 cells and A549/T cells following treatment with FKA for 48 h, as determined by MTT assay. *P<0.05 vs. 20 µM FKA. **P<0.01 vs. 0 µM FKA. (C) The effect of FKA on the viability of THLE-3 cells. (D) Morphological changes in A549/T cells following 48 h-treatment with 0, 5, 10 and 30 µM FKA. Data are presented as the mean ± standard deviation of 3 independent experiments. FKA, flavokawain A; PTX, paclitaxel.

Effect of FKA on cell apoptosis in resistant A549/T cells. (A) FKA-induced cell apoptosis was detected by Annexin V/PI staining and flow cytometry analysis. (B) The expression of PARP in cells treated with FKA at various concentrations was examined by western blot analysis. β-actin served as the loading control. *P<0.05 vs. treated with 0 µM FKA. (C) Apoptosis in A549/T cells was visualized using nuclear DAPI staining. Cells were exposed for 24 or 48 h at 37°C, following the indicated treatments, prior to staining with DAPI for 30 min. Arrow pointing to apoptotic cells. FKA, flavokawain A; PARP, poly (ADP-ribose) polymerase.

FKA inhibits P-gp mRNA expression. (A) Image of agarose ethidium bromide gel and (B) P-gp mRNA expression in A549/T cells treated with 0, 3 and 30 µM FKA. FKA, flavokawain A; P-gp, permeability-glycoprotein.

FKA inhibits P-gp protein expression. (A) Western blot analysis of P-gp expression in A549 and A549/T cells. The expression of P-gp in A549/T cells treated with (B) FKA for 24 h at various concentrations and (C) 30 µM FKA at various times. **P<0.01. FKA, flavokawain A; P-gp, permeability-glycoprotein.

FKA inhibits the PI3K/Akt signaling pathway in A549/T cells. Western blot analysis of the (A) protein expression levels of P-gp, Akt and p-Akt (Ser 473) in A549/T cells treated with various concentrations of FKA for 24 h. GAPDH served as an internal control for P-gp and total Akt was used for p-Akt, The ratios of p-Akt (Ser 473) to Akt after treated with 0, 2.5, 5, 10 and 30 µM FKA were 0.32, 0.3, 0.28, 0.27, 0.18 respectively. (B) The relative protein level of P-gp, Akt and p-Akt (Ser 473) was quantified using densitometry. *P<0.05 vs. 0 µM FKA. FKA, flavokawain A; P-gp, permeability-glycoprotein, p-, phosphorylated.

FKA downregulates P-gp expression via inhibition of the PI3K/Akt signaling pathway. A549/T cells were either untreated or treated with (A) FKA, LY294002 or co-treated with FKA and LY294002 or (B) FKA, IGF-1 or co-treated with FKA and IGF-1 using western blot analysis. GAPDH served as the internal control for P-GP and total AKT served as an internal control for p-AKT. The ratios of p-Akt (Ser 473) to Akt treated with control, FKA, LY294002 or FKA combined with LY294002 were 0.35, 0.33, 0.15, 0.17 respectively. The ratios of p-Akt (Ser 473) to Akt treated with control, FKA, IGF-1 or FKA combined with IGF-1 were 0.45, 0.16, 0.4, 0.13 respectively. The relative level of P-gp, Akt and p-Akt (Ser 473) after treatment with (C) FKA, LY294002 or FKA combined with LY294002 and (D) FKA, IGF-1 or FKA combined with IGF-1 treatment. *P<0.05. **P<0.01. FKA, flavokawain A; P-gp, permeability-glycoprotein, p-, phosphorylated; IGF-1, insulin-like factor-1.