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Development of Multi-drug Resistance to Anticancer Drugs in HepG2 Cells Due to MRP2 Upregulation on Exposure to Menthol

Katsuhito Nagai, Mayuko Tamura, Ryuga Murayama, Shuhei Fukuno, Takuya Ito, Hiroki Konishi


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

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.

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.


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].

Materials and methods


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.


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).


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.


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.


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

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.

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.


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