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Open Access

Peer-reviewed

Research Article

Development of multi-drug resistance to anticancer drugs in HepG2 cells due to MRP2 upregulation on exposure to menthol

  • Katsuhito Nagai ,

    Roles Writing – original draft

    nagaika@osaka-ohtani.ac.jp

    Affiliation Laboratory of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Japan

    ⨯
  • Mayuko Tamura,

    Roles Investigation

    Affiliation Laboratory of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Japan

    ⨯
  • Ryuga Murayama,

    Roles Investigation

    Affiliation Laboratory of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Japan

    ⨯
  • Shuhei Fukuno,

    Roles Investigation

    Affiliation Laboratory of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Japan

    ⨯
  • Takuya Ito,

    Roles Conceptualization

    Affiliation Laboratory of Natural Medicines, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Japan

    ⨯
  • Hiroki Konishi

    Roles Writing – review & editing

    Affiliation Laboratory of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Japan

    ⨯

Abstract

Background

Menthol exerts relaxing, antibacterial, and anti-inflammatory activities, and is marketed as a functional food and therapeutic drug.

Aim

In the present study, the effects of menthol on the expression of multidrug resistance associated protein 2 (MRP2) and its association with the cytotoxicity of epirubicin (EPI) and cisplatin (CIS) were examined using HepG2 cells.

Methods

The expression levels of target genes were examined by real-time PCR. The intracellular concentration of incorporated EPI was measured by high-performance liquid chromatography. Cell viability was evaluated by MTT analysis.

Results

The expression of MRP2 mRNA was increased by exposing HepG2 cells to menthol for 24 hr. Consistent with a previous report suggesting an inverse correlation between MRP2 and Akt behavior, increased expression of MRP2 was also observed on suppression of the Akt function. Intracellular accumulation of EPI was significantly decreased by exposure of HepG2 cells to menthol, and a significant decrease in the intracellular concentration of EPI remaining was observed in HepG2 cells exposed to menthol. The decreased intracellular accumulation of EPI was significantly suppressed by treatment with MK-571, but not verapamil. Both EPI and CIS exerted cytocidal effects on HepG2 cells, but the decrease in cell viability was significantly attenuated by 24-hr menthol pre-exposure.

Conclusion

These results demonstrate that menthol causes hepatocellular carcinoma to acquire resistance to anticancer drugs such as EPI and CIS by MRP2 induction.

Citation: Nagai K, Tamura M, Murayama R, Fukuno S, Ito T, Konishi H (2023) Development of multi-drug resistance to anticancer drugs in HepG2 cells due to MRP2 upregulation on exposure to menthol. PLoS ONE 18(9): e0291822. https://doi.org/10.1371/journal.pone.0291822

Editor: Miquel Vall-llosera Camps, PLOS ONE, UNITED KINGDOM

Received: December 13, 2022; Accepted: September 6, 2023; Published: September 21, 2023

Copyright: © 2023 Nagai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grants JP20K07118) and a grant from the Kobayashi Foundation (No. 13, 2018, T.I.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Hepatocellular carcinoma (HCC) is a serious malignancy worldwide, with increasing incidence and mortality over time [1]. Currently, approximately 80% of HCC patients are diagnosed at an advanced stage and are not amenable to surgical resection. Transarterial chemoembolization is currently believed to be the effective treatment for patients with advanced HCC, and epirubicin (EPI) and cisplatin (CIS) are widely utilized as anti-HCC agents [2, 3]. However, patients undergoing chemotherapy may increasingly develop multi-drug resistance (MDR) against anticancer agents, which is responsible for a major obstacle to HCC treatment. In addition, the intake of specific supplements can lead to MDR to anticancer agents [4]. There are a variety of mechanisms underlying MDR, one of which is the reduction in intracellular concentration associated with increased efflux of anticancer agents [5, 6]. ABC transporters such as the P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) families play essential roles in this type of MDR [7, 8].

Menthol is a naturally occurring compound found in plants of the Mentha genus, commonly known as mint. Menthol is commercially available as a supplement and therapeutic drug because of several beneficial biological properties [9, 10]. Our previous study demonstrated that menthol causes hepatocellular carcinoma HepG2 cells to acquire resistance to doxorubicin (DOX) by increasing its extracellular efflux through the upregulation of P-gp [11]. However, whether the expression of other ABC transporters is affected by menthol exposure remains unclear. Recently, it was reported that suppression of the Akt signal following menthol exposure contributes to neuroprotection from inflammatory damage [12]. In light of experimental evidence that Calculus Bovis, a traditional Chinese medicine, could alleviate liver injury and up-regulate the expression of MRP2 in 17α-ethynylestradiol-induced cholestasis by a mechanism involving inhibition of the Akt signaling pathway [13], it is conceivable that MRP2 is induced following menthol exposure.

In the present study, using HepG2 cells, we examined the effects of menthol on the expression of MRP2 which markedly contributes to MDR of HCC [14]. Furthermore, the change in cytotoxicity of EPI and CIS was investigated following exposure to menthol.

Materials and methods

Chemicals

EPI hydrochloride and CIS were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Menthol was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All other reagents were of commercial or analytical grade and required no further purification.

Cell culture

HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (Fujifilm Wako Pure Chemical Co.) containing 10% fetal bovine serum (Biowest USA, Riverside, MO, USA) at 37°C under a humidified atmosphere of 5% CO2 in air.

Experimental design

For real-time PCR, HepG2 cells were exposed to either menthol or an Akt inhibitor for 24 hr. For the transport assay, HepG2 cells were exposed to menthol for 24 hr. The reagents used were dissolved in DMSO. Control cells were exposed to vehicle alone for the same time.

For the evaluation of cell viability, HepG2 cells were divided into four experimental groups, designated as: anticancer drug (EPI or CIS), menthol, anticancer drug plus menthol, and control groups. In the anticancer drug group, the cells were exposed to vehicle alone for 24 hr, followed by exposure to anticancer drugs for 24 hr. In the menthol group, the cells were consecutively incubated with menthol for 48 hr. In the anticancer drug plus menthol group, 24-hr simultaneous exposure to anticancer drugs and menthol was conducted 24 hr after addition of menthol. Control cells were exposed to vehicle alone for the same time.

Real-time quantitative PCR

The total RNA fraction was extracted with ISOGEN (NIPPON GENE Co., LTD, Tokyo, Japan) and the GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich Co.). Real-time quantitative PCR was performed using SYBR green as the fluorescent dye (Toyobo Co., Ltd., Osaka, Japan), following reverse transcription. The conditions for denaturation, annealing, and extension reactions in the PCR process were 95°C for 10 sec, 57°C for 10 sec, and 72°C for 30 sec, respectively. 18S-ribosome was used as an internal control. The base sequences of the MRP2 primer were 5’-CCTAGACAACGGGAAGATT-3’ (5’ primer) and 5’-CCATAAAGTAAAAGGGTCCAG-3’ (3’ primer). The base sequences of the 18S-ribosome primer were 5’-CGTCTGCCCTATCAACTTTCG-3’ (5’ primer) and 5’-CGTTTCTCAGGCTCCCTCT-3’ (3’ primer). The mRNA levels were quantified based on standard curves, and the results are expressed relative to control values, which were given an arbitrary value of 1.

Transport assay

After pre-incubation of HepG2 cells with Hanks’ balanced salt solution (HBSS) for 10 min in the presence or absence of 100 μM MK-571 or verapamil, the cells were exposed to EPI at a final concentration of 5 μM for certain amounts of times. The transport reaction was stopped by the addition of ice-cooled excess phosphate-buffered saline (PBS). The cells were lysated using ultrapure water. In the efflux experiment, HepG2 cells had been loaded with EPI in HBSS for 30 min, and then were incubated in drug-free HBSS for 3 min following washing twice with ice-cold PBS. The procedure after the reaction stop was performed in the same manner as described above.

Assay procedure

After the addition of 50 μL of 2 μg/mL daunorubicin (Sigma-Aldrich Co.), an internal standard (IS), 75 μL of 4% zinc sulfate, and 75 μL of methanol to a 100-μL lysis sample, the mixture was vortexed and centrifuged at 5,000 g for 10 min. One hundred microliters of supernatant were injected into the high-performance liquid chromatography (HPLC) system. The analytes were separated on an RP-18 GP II column (150 × 4.6 mm; Kanto Chemical Co., Inc., Tokyo, Japan) using a mixture of 15 mM acetate buffer (pH 2.5) and acetonitrile (v/v, 2:3) as the mobile phase. The mobile phase was delivered at 0.7 mL/min, and the column effluent was monitored with an excitation wavelength of 470 nm and an emission wavelength of 550 nm. The concentration of EPI was quantified using the peak area ratio compared with the IS. The protein concentration was measured based on the Bradford method and applied to the estimation of the intracellular amount of EPI.

Cell viability

On the 3rd day, cell viability was evaluated by measuring mitochondrial enzyme activity for reducing MTT to corresponding formazan dye according to our previous report [11]. Cell viability is expressed relative to control values, given an arbitrary value of 100.

Statistical analysis

Data are expressed as means ± SD. Differences between the means of two groups were evaluated using Student’s unpaired t-test. The significance of differences between control and test values was determined using Dunnett’s test or the two-tailed multiple t-test with Bonferroni correction following one-way analysis of variance. Differences with a p-value of 0.05 or less were considered significant.

Results

Effect of menthol and Akt inhibitor on the expression of mRNA for MRP2

The effect of menthol and Akt inhibitors on gene expression of MRP2 was examined (Fig 1). While 10 μM menthol had no significant effect on expression of MRP2, significantly higher expression of MRP2 was observed in HepG2 cells exposed to menthol at concentrations of 50 and 100 μM (Fig 1A). The expression of MRP2 mRNA was significantly increased by exposure of HepG2 cells to 5 μM MK-2206, 40 μM perifosine, or 40 μM ipatasertib, which are Akt inhibitors (Fig 1B).

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Fig 1. Effects of menthol and Akt inhibitors on the gene expression of MRP2.

HepG2 cells were exposed to 10–100 μM menthol, 5 μM MK-2206, 40 μM perifosine, or 40 μM ipatasertib for 24 hr. Total RNA was extracted and purified from HepG2 cells, and the expression level of MRP2 was measured by real-time PCR. The results represent the mean ± SD of three independent experiments. ***: p<0.001 (ver. Control group).

https://doi.org/10.1371/journal.pone.0291822.g001

Effects of menthol on the activity of EPI transport

In the examination of EPI transport, its intracellular amount transported into HepG2 cells in the menthol-exposed group was significantly lower than that in the control group (Fig 2A). In the efflux experiment, the intracellular level of EPI remaining in HepG2 cells was significantly decreased by menthol exposure (Fig 2B). As shown in Fig 3, the decreased intracellular EPI accumulation of EPI in HepG2 cells exposed to menthol was almost cancelled by treatment with MK-571, but not verapamil.

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Fig 2. Effect of menthol on intracellular accumulation and efflux of EPI.

HepG2 cells were exposed to 100 μM menthol for 24 hr. (A) Cells were preincubated for 10 min at 37°C and then incubated with 5 μM EPI for 10 or 30 min. (B) After the cells had been loaded with 5 μM EPI for 30 min at 37°C, they were incubated in drug-free HBSS for 3 min at 37°C. The results represent the mean ± SD of three independent experiments. **: p<0.01 (ver. Control group), ***: p<0.001 (ver. Control group).

https://doi.org/10.1371/journal.pone.0291822.g002

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Fig 3. Effect of MK-571 and verapamil on decreased intracellular accumulation of EPI after menthol exposure.

HepG2 cells were exposed to 100 μM menthol for 24 hr. Cells were preincubated for 10 min at 37°C with or without 100 μM MK-571, and 100 μM verapamil, and then incubated with 5 μM EPI for 30 min under the same conditions as for preincubation. The results represent the mean ± SD of three independent experiments. **: p<0.01, ***: p<0.001.

https://doi.org/10.1371/journal.pone.0291822.g003

Effects of menthol on the cytotoxicity of EPI and CIS

The viability of HepG2 cells was decreased in the presence of EPI or CIS, but the decreases were significantly attenuated by exposure to menthol in advance (Fig 4). Menthol alone had no effect on viability of HepG2 cells (Fig 4).

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Fig 4. Effects of menthol on cytotoxicity of EPI and CIS.

The cells were exposed to 5 μM EPI or 80 μM CIS 24 hr after treatment with 100 μM menthol, and cell viability was then evaluated by the MTT method 24 hr later. The results are shown as means ± SD of three independent experiments. **: p<0.01, ***: p<0.001, NS: not significant.

https://doi.org/10.1371/journal.pone.0291822.g004

Discussion

Induction of some MRP families is responsible for MDR to anticancer drugs and is a major obstacle to the conduction of cancer chemotherapy [15, 16]. MRP1 (ABCC1), the first cloned member of this family, confers resistance to a variety of drugs such as anthracyclines and vinca alkaloids [17]. However, no significant expression of MRP1 mRNA was detected in human liver [18]. Resistance to CIS and anthracyclines was shown in MRP2-overexpressing cells constructed by molecular techniques [19]. MRP2 was reported to be expressed on the plasma membrane of all HCCs used in this study, while MRP1 was expressed only in some HCCs and localized to the intracellular membrane [14]. Therefore, it is likely that MRP2 rather than MRP1 is much deeply implicated in HCC resistance. In the present study, we investigated using HepG2 cells the effects of menthol on MRP2 expression and the cytotoxicity of anticancer drugs serving as substrates of MRP2.

It was confirmed that gene expression of MRP2 in HepG2 cells was increased by exposure to menthol as was when the Akt function was suppressed in the presence of its inhibitor. These results support experimental observation in studies using animals that neuronal Akt pathway was suppressed after menthol administration and whereby the expression of MRP2 was inversely related to the signal response to the Akt pathway [12, 13]. In HepG2 cells exposed to menthol, the intracellular accumulation of EPI was significantly reduced, indicating the marked contribution of menthol to enhanced efflux of the drug. The previous study demonstrated that higher expression of P-gp, MRP1, and MRP2 was observed in Hela cells exposed to EPI [20]. The gene expression of these transporters in cancer cells can be decreased by various molecular techniques, which causes an increase in intracellular EPI accumulation [21–25]. Furthermore, the cytotoxic effect of EPI on diffuse large B-cell lymphoma cells was enhanced by NF-κB pathway-mediated control of P-gp expression [26]. These findings suggested the possible involvement of P-gp, MRP1, and MRP2 in efflux of EPI. However, our recent study demonstrated that MRP families were markedly involved in EPI efflux rather than P-gp [27]. Furthermore, in the present study, the enhanced function of MRP2 by menthol exposure can be explained by the increased intracellular EPI level in the presence of MK-571 (MRP2 inhibitor), but not verapamil (P-gp inhibitor), in menthol-exposed HepG2 cells. Based on these findings, we strongly consider that MRP2 plays a central role as a determinant of the intracellular EPI concentration by acting as an efflux pump, although it is susceptible to induction by menthol.

HepG2 cells were shown to be sensitive to EPI, but decreased viability was significantly attenuated by pre-exposure to menthol. As the cytocidal efficacy of anthracyclines was likely to be correlated with the intracellular accumulation of the drugs [27], it is strongly suggested that induction of MRP2 following menthol exposure is a cause of the reduced cytotoxicity of EPI. To confirm that menthol is generally capable of producing MRP2-mediated MDR, it was necessary to evaluate whether the cytotoxic effect of other anticancer drugs would also be attenuated by menthol. In previous studies, the observed resistance to CIS could not be explained by induction of P-gp expression [28]. Active efflux of glutathione-conjugated CIS was reported to be mediated by MRP2 [29], which was supported by evidence that intracellular glutathione levels were related to CIS cytotoxicity [30]. The observation is suggestive of the possible involvement of efflux mediated by MRP2 in a CIS resistance. Furthermore, the previous studies have clarified that reduction in intracellular accumulation of CIS is associated with increased levels of MRP2 mRNA, which resulted in resistance to CIS [31]. In the present study, cytocidal potency of CIS was also attenuated by pre-exposing HepG2 cells to menthol, suggesting that the development of MDR attributed to menthol exposure is common across anticancer drugs serving as substrates of MRP2, leading to serious issues associated with therapeutic failure.

Peppermint oil, which contains menthol as principal ingredient, has been shown to be useful in treating gastroesophageal reflux and irritable bowel syndrome (IBS) [10, 32, 33]. The usual daily dose of peppermint oil for the treatment of IBS is about 1.1 g. The serum concentrations of menthol have been determined to reach 10 μM when 36 mg peppermint oil capsules were orally administered to healthy male volunteers [34]. In this regard, when peppermint oil capsules are used for the treatment of IBS, serum levels of menthol can reach 100 μM due to efficient intestinal absorption with over 70% bioavailability [35], which can lead to the failure of HCC treatment through upregulation of MRP2 in clinical settings. In addition to EPI and CIS, MRP2 contributes to the efflux of many structurally unrelated chemotherapeutic drugs such as vincristine [7]. Therefore, medical staff should advise HCC patients receiving anticancer drugs not to take menthol-rich supplements to reduce the risk of MDR.

Conclusion

The present study demonstrated that menthol exposure carries a risk of developing MDR in HepG2 cells due to the upregulation of MRP2. Our study will help to promote the successful treatment of HCC using anticancer drugs.

Acknowledgments

We acknowledge Akari Kokado and Kiyoka Nagano for their technical assistance in the experiments.

References

  1. 1. Finn RS. Current and future treatment strategies for patients with advanced hepatocellular carcinoma: role of mTOR inhibition. Liver Cancer. 2012, 1(3–4): 247–56. pmid:24159589
  2. 2. Chu TH, Chan HH, Hu TH, Wang EM, Ma YL, Huang SC, et al. Celecoxib enhances the therapeutic efficacy of epirubicin for Novikoff hepatoma in rats. Cancer Med. 2018, 7(6): 2567–80. pmid:29683262
  3. 3. Niizeki T, Iwamoto H, Shirono T, Shimose S, Nakano M, Okamura S, et al. Clinical importance of regimens in hepatic arterial infusion chemotherapy for advanced hepatocellular carcinoma with macrovascular invasion. Cancers (Basel). 2021, 13(17): 4450. pmid:34503259
  4. 4. Holtzman CW, Wiggins BS, Spinler SA. Role of P-glycoprotein in statin drug interactions. Pharmacotherapy. 2006, 26(11): 1601–7. pmid:17064205
  5. 5. Zhu H, Liu Z, Tang L, Liu J, Zhou M, Xie F, et al. Reversal of P-gp and MRP1-mediated multidrug resistance by H6, a gypenoside aglycon from Gynostemma pentaphyllum, in vincristine-resistant human oral cancer (KB/VCR) cells. Eur J Pharmacol. 2012, 696(1–3): 43–53. pmid:23051672
  6. 6. Tsuji K, Wang YH, Takanashi M, Odajima T, Lee GA, Sugimori H, et al. Overexpression of lung resistance-related protein and P-glycoprotein and response to induction chemotherapy in acute myelogenous leukemia. Hematol Rep. 2012, 4(3): e18. pmid:23087807
  7. 7. Borst P, Evers R, Kool M, Wijnholds J. The multidrug resistance protein family. Biochim Biophys Acta. 1999, 1461(2): 347–57. pmid:10581366
  8. 8. Fletcher JI, Williams RT, Henderson MJ, Norris MD, Haber M. ABC transporters as mediators of drug resistance and contributors to cancer cell biology. Drug Resist Updat. 2016, 26(5): 1–9. pmid:27180306
  9. 9. Juergens UR, Stober M, Vetter H. The anti-inflammatory activity of L-menthol compared to mint oil in human monocytes in vitro: a novel perspective for its therapeutic use in inflammatory disease. Eur J Med Res. 1998, 3(12): 539–45. pmid:9889172
  10. 10. Pirotta M. Irritable bowel syndrome- The role of complementary medicines in treatment. Aust Fam Physician. 2009, 38(12): 966–8. pmid:20369148
  11. 11. Nagai K, Fukuno S, Miura T, Uchino Y, Sehara N, Konishi H. Reduced cytotoxicity in doxorubicin-exposed HepG2 cells pretreated with menthol due to upregulation of P-glycoprotein. Pharmazie. 2020, 75(10): 510–511. pmid:33305727
  12. 12. Du J, Liu D, Zhang X, Zhou A, Su Y, He D, et al. Menthol protects dopaminergic neurons against inflammation-mediated damage in lipopolysaccharide (LPS)-Evoked model of Parkinson’s disease. Int Immunopharmacol. 2020, 85(8): 106679. pmid:32559722
  13. 13. Wu T, Zhang Q, Li J, Chen H, Wu J, Song H. Up-regulation of BSEP and MRP2 by Calculus Bovis administration in 17α-ethynylestradiol-induced cholestasis: Involvement of PI3K/Akt signaling pathway. J Ethnopharmacol. 2016, 190(8): 22–32. pmid:27237619
  14. 14. Nies AT, König J, Pfannschmidt M, Klar E, Hofmann WJ, Keppler D. Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma. Int J Cancer. 2001, 94(4): 492–9. pmid:11745434
  15. 15. Hipfner DR, Deeley RG, Cole SP. Structural, mechanistic and clinical aspects of MRP1. Biochim Biophys Acta. 1999, 1461(2): 359–76. pmid:10581367
  16. 16. König J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta. 1999, 1461(2): 377–94. pmid:10581368
  17. 17. Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole SP, Deeley RG. Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs. Cancer Res. 1994, 54(2): 357–61. pmid:8275468
  18. 18. Kool M, de Haas M, Scheffer GL, Scheper RJ, van Eijk MJ, Juijn JA, et al. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res. 1997, 57(16): 3537–47. pmid:9270026
  19. 19. Cui Y, König J, Buchholz JK, Spring H, Leier I, Keppler D. Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol. 1999, 55(5): 929–37. pmid:10220572
  20. 20. Lo YL, Liu Y. Reversing multidrug resistance in Caco-2 by silencing MDR1, MRP1, MRP2, and BCL-2/BCL-xL using liposomal antisense oligonucleotides. PLoS One. 2014, 9(3): e90180. pmid:24637737
  21. 21. Lee YK, Lin TH, Chang CF, Lo YL. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma. PLoS One. 2013, 8(11): e82478. pmid:24303084
  22. 22. Juang V, Lee HP, Lin AMY, Lo YL. Cationic PEGylated liposomes incorporating an antimicrobial peptide tilapia hepcidin 2–3: an adjuvant of epirubicin to overcome multidrug resistance in cervical cancer cells. Int J Nanomedicine. 2016, 11(11): 6047–64. pmid:27895479
  23. 23. Lin GL, Ting HJ, Tseng TC, Juang V, Lo YL. Modulation of the mRNA-binding protein HuR as a novel reversal mechanism of epirubicin-triggered multidrug resistance in colorectal cancer cells. PLoS One. 2017, 12(10): e0185625. pmid:28968471
  24. 24. Lo YL, Wang W. Formononetin potentiates epirubicin-induced apoptosis via ROS production in HeLa cells in vitro. Chem Biol Interact. 2013, 205(3): 188–97. pmid:23867903
  25. 25. Zhang LH, Yang AJ, Wang M, Liu W, Wang CY, Xie XF, et al. Enhanced autophagy reveals vulnerability of P-gp mediated epirubicin resistance in triple negative breast cancer cells. Apoptosis. 2016, 21(4): 473–88. pmid:26767845
  26. 26. Liu K, Song J, Yan Y, Zou K, Che Y, Wang B, et al. Melatonin increases the chemosensitivity of diffuse large B-cell lymphoma cells to epirubicin by inhibiting P-glycoprotein expression via the NF-κB pathway. Transl Oncol. 2021, 14(1): 100876. pmid:33007707
  27. 27. Nagai K, Fukuno S, Shiota M, Tamura M, Yabumoto S, Konishi H. Differences in transport characteristics and cytotoxicity of epirubicin and doxorubicin in HepG2 and A549 cells. Anticancer Res. 2021, 41(12): 6105–12. pmid:34848465
  28. 28. Borst P, Evers R, Kool M, Wijnholds J. A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst. 2000, 92(16): 1295–302. pmid:10944550
  29. 29. Taniguchi K, Wada M, Kohno K, Nakamura T, Kawabe T, Kawakami M, et al. A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation. Cancer Res. 1996. 56(18): 4124–9. pmid:8797578
  30. 30. Ozols RF. Pharmacologic reversal of drug resistance in ovarian cancer. Semin Oncol. 1985, 12(3 Suppl 4): 7–11. pmid:4048979
  31. 31. Liedert B, Materna V, Schadendorf D, Thomale J, Lage H. Overexpression of cMOAT (MRP2/ABCC2) is associated with decreased formation of platinum-DNA adducts and decreased G2-arrest in melanoma cells resistant to cisplatin. J Invest Dermatol. 2003, 121(1): 172–6. pmid:12839578
  32. 32. Amjadi MA, Mojab F, Kamranpour SB. The effect of peppermint oil and symptomatic treatment of pruritus in pregnant women. Iran J Pharm Res. 2012, 11(4): 1073–7. pmid:24250539
  33. 33. Kline RM, Kline JJ, Di Palma J, Barbero GJ. Enteric-coated, pH-dependent peppermint oil capsules for the treatment of irritable bowel syndrome in children. J Pediatr. 2001, 138(1): 125–8. pmid:11148527
  34. 34. Mascher H, Kikuta C, Schiel H. Pharmacokinetics of menthol and carvone after administration of an enteric coated formulation containing peppermint oil and caraway oil. Arzneimittelforschung. 2001, 51(6): 465–9. pmid:11455677
  35. 35. Hiki N, Kaminishi M, Hasunuma T, Nakamura M, Nomura S, Yahagi N, et al. A phase I study evaluating tolerability, pharmacokinetics, and preliminary efficacy of L-menthol in upper gastrointestinal endoscopy. Clin Pharmacol Ther. 2011, 90(2): 221–8. pmid:21544078