Acteoside from Conandron ramondioides Reduces Microcystin-LR Cytotoxicity by Inhibiting Intracellular Uptake Mediated by OATP1B3

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Reagents
• Cell culture
• Cell survival determination
• Intracellular accumulation of microcystin-LR
• Uptake study of microcystin-LR
• Interaction of microcystin-LR with intracellular proteins
• Immunoblot analysis for the phosphorylation of ERK, p38, and p53
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 µg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 µM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.

Introduction

The cyclic heptapeptide microcystin-LR is one of the hepatotoxins produced and released by toxic cyanobacteria, such as Microcystis, Anabaena, Nostoc, and Oscillatoria [1], and it is toxic only to animals and human hepatocytes [2]. Microcystin-LR is recognized by human hepatocyte uptake transporters organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 and is thus taken up into the hepatocytes as substrates of the transporters and causes cytotoxicity [3]. When microcystin-LR enters hepatocytes, it covalently and irreversibly binds to serine/threonine protein phosphatase 1 (PP1) and PP2A. Inhibiting PP1 and PP2A causes excessive phosphorylation of intracellular proteins, resulting in various functions such as cell death, cell proliferation, and cell-cycle arrest [3], [4]. Understanding the molecular mechanism underlying toxicity and detoxification of microcystin-LR is required to establish the chemoprevention of microcystin-LR toxicity to maintain human health.

Naringin, which is a flavonoid, has previously been shown to reduce microcystin-LR cytotoxicity by inhibiting the uptake of microcystin-LR into OATP1B3-expressing cells [5]. Other compounds in the crude drugs that can reduce microcystin-LR cytotoxicity were also screened. Crude drugs are usually more effective than food ingredients in treating and preventing diseases and have fewer side effects than drugs. Liver-specific or liver-preferential toxic compounds such as nodularin [6], okadaic acid [7], amanitin [8], and phalloidin [9] are recognized by OATP1B1 and OATP1B3 and are thus taken up into the hepatocytes as transporter substrates and initiate cytotoxicity, as observed in the case of microcystin-LR. Therefore, the compound found in this study is expected to be used for the chemoprevention of these toxic compounds.

The crude drug iwajisha, derived from the herb plant Conandron ramondioides and containing acteoside as one of its major components, is utilized as a folk remedy for the treatment of stomach ache, stomach cancer, liver injury, liver cancer, menstrual disorder, uterus cancer, tuberculosis, and kidney dysfunction [10]. Acteoside is a phenylethanoid glycoside found in various medicinal plants, such as the Verbenaceae and Gesneriaceae families [11], and has been shown to exert antiviral, hepatoprotective, anti-inflammatory, and possibly other beneficial effects [12], [13].

In this study, iwajisha was found to reduce microcystin-LR cytotoxicity to OATP1B3-expressing cells. Furthermore, the effect of acteoside, which is the main component of iwajisha [14], on microcystin-LR cytotoxicity and its uptake by OATP1B3-expressing cells were investigated.

Results

The onset of microcystin-LR cytotoxicity is dependent on its selective uptake via OATP1B1 and OATP1B3. Hence, we used OATP1B3-expressing HEK293 cells in this study. HEK293-OATP1B3 cells were sensitive to microcystin-LR exposure with an IC₅₀ value of approximately 8.7 nM ([Fig. 1]). HEK293-CV cells, in contrast, could survive exposure to up to 200 nM microcystin-LR (data not shown). These results were consistent with those reported in our previous study [3].

Next, we evaluated the effect of several types of crude drugs on the onset of microcystin-LR cytotoxicity in HEK293-OATP1B3 cells. Iwajisha reduced the cytotoxicity of microcystin-LR ([Fig. 1]) in a concentration-dependent manner. Iwajisha concentrations of 10 and 20 µg/mL reduced cell susceptibility to microcystin-LR by approximately 3.2 and 6.0 times its IC₅₀ values, respectively.

Following that, we tested the effect of acteoside, one of the main components of iwajisha, on the onset of microcystin-LR cytotoxicity. Acetoside inhibited microcystin-LR cytotoxicity in a concentration-dependent manner ([Fig. 2 a]). Acteoside concentrations of 5, 10, and 20 µM reduced cell susceptibility to microcystin-LR by approximately 3.1, 3.6, and 7.4 times its IC₅₀-values, respectively.

Furthermore, we investigated the effect of acteoside on the cytotoxicity of nodularin and okadaic acid, both of which are substrates of OATP1B3, and microcystin-LR. Acteoside reduced the cytotoxicity of nodularin in a concentration-dependent manner ([Fig. 2 b]). Acteoside concentrations of 5, 10, and 20 µM reduced cell susceptibility to nodularin by approximately 2.2, 3.9, and 7.5 times its IC₅₀ values, respectively ([Fig. 2 b]). Acteoside concentrations of 5, 10, and 20 µM also significantly reduced cell susceptibility to okadaic acid. Acteoside exposure increased the IC₅₀ values in these conditions by 1.5-, 1.5-, and 2.0-fold, respectively ([Fig. 2 c]).

To evaluate the effect of acteoside on the accumulation of microcystin-LR and its binding proteins, microcystin-LR immunoblot analysis was performed. Under nonreduced and no-heating conditions, at least three microcystin-binding proteins with different molecular masses (approximately 22, 29, and 31 kDa) were detected 24 h after exposure to 50 nM microcystin-LR. When microcystin-LR was combined with acteoside, the detected signal intensity of their microcystin-binding proteins was reduced in an acteoside concentration-dependent manner compared with only microcystin-LR exposure ([Fig. 3]).

For kinetic analysis of acteoside-induced inhibition of microcystin-LR uptake mediated by OATP1B3, we analyzed the uptake property of microcystin-LR into the cells expressing the transporter carrier protein, OATP1B3. After incubating HEK293-OATP1B3 cells with 1 µM microcystin-LR for 15 min at 37 °C in the presence of 5, 10, or 20 µM acteoside, microcystin-LR uptake was measured. Co-exposure to acteoside inhibited microcystin-LR uptake in a concentration-dependent manner. An acteoside concentration of 12.3 µM was found to effectively inhibit the uptake of 1 µM microcystin-LR by 50% ([Fig. 4 a]).

To understand the pattern and mechanism of acteoside-induced inhibition of microcystin-LR uptake (v) mediated by OATP1B3, HEK293-OATP1B3 cells were incubated with different concentration combinations of acteoside and microcystin-LR. A Dixon plot of 1/v versus acteoside concentrations was plotted, and the result was consistent with a noncompetitive inhibition with an apparent Ki value of 12.7 µM ([Fig. 4 b]).

We aimed to investigate whether acteoside affected not only OATP1B3 function but also intracellular proteins. HEK293-OATP1B3 cell lysates were incubated with various concentrations of microcystin-LR for 2 h at 4, 25, or 37 °C. Among these three temperature conditions, the signal intensity of the main band, with a molecular mass of approximately 22 kDa was the strongest at 25 °C among the three temperature conditions (data not shown). In the cases where the temperature was 37 °C, the signal intensity of the 22 kDa band was lower than that of cases where the temperature was 4 °C and 25 °C (data not shown). The strongest band signal was detected at concentrations greater than 0.25 nM microcystin-LR and it increased in a concentration-dependent manner ([Fig. 5 a]). Cell lysates were pretreated with various concentrations of acteoside for 1 h at 25 °C, before being incubated for 2 h at 25 °C with 4.3 pg/µg protein (5.4 nM). Pretreatment with 50 µM acteoside caused this compound to inhibit the interaction of microcystin-LR with its binding cellular protein(s) ([Fig. 5 b]). In contrast, pretreatment with 20 µM acteoside or less had no effect. The signal intensity of the main band, which had a molecular mass of approximately 22 kDa, was nearly the same as that of one of the main bands in [Fig. 3], having the same molecular mass ([Fig. 5 c]).

To investigate the effect of acteoside on microcystin-LR-induced hyper-phosphorylation of intracellular proteins, immunoblot analysis for extracellular signal-regulated kinase (ERK), p38, and p53 was performed because some previous reports [3], [4] have indicated that the phosphorylation of these molecules was enhanced after exposure to microcystin-LR. As a result, we investigated the effect of acteoside on the hyper-phosphorylation of ERK, p38, and p53. Cell lysates were immunoblotted for determining the phosphorylation status of these molecules after co-exposure to 50 nM microcystin-LR and a maximum of 20 µM acteoside for 24 h. ERK was expressed in HEK293-OATP1B3 cells under normal culture conditions, but its phosphorylation was minimal. Notably, acteoside was found to induce the phosphorylation of ERK. Following exposure to 50 nM microcystin-LR, ERK phosphorylation increased, as did the protein level, as observed in our previous study [3]. Furthermore, co-exposure to 20 µM acteoside increased the phosphorylation of ERK induced by 50 nM microcystin-LR ([Fig. 6 a]).

Although p38 was expressed in HEK293-OATP1B3 cells under normal and 20 µM acteoside exposure conditions, its phosphorylation was negligible. The phosphorylation of p38 was enhanced after exposure to microcystin-LR. Co-exposure to 20 µM acteoside reduced the 50 nM microcystin-LR-induced phosphorylation of p38 ([Fig. 6 b]).

The expression and phosphorylation of p53 were detected only to a slight extent under control conditions. When cells were exposed to 20 µM acteoside, the expression of p53 was stabilized without phosphorylation. Phosphorylation was used to stabilize the expression of p53 after exposure to 50 nM microcystin-LR treatment. Co-exposure to 20 µM acteoside reduced the 50 nM microcystin-LR-induced phosphorylation of p53 ([Fig. 6 c]).

Discussion

Treatment with crude drugs is effective for the prevention of several diseases because these drugs have high efficacy and few side effects. In this study, we demonstrated that iwajisha, which is a common herbal medicine, had the ability to reduce the cytotoxicity of microcystin-LR ([Fig. 1]). This finding prompted us to attempt to identify the functional component in iwajisha that helps in attenuating the microcystin-LR cytotoxicity and to understand the mechanism of attenuation.

One of the main components of iwajisha is acteoside, a phenylethanoid glycoside found in many medicinal plants, including the Verbenaceae and Gesneriaceae families [11]. Owing primarily to its potent antioxidant properties, acteoside has broad-spectrum pharmacological activities [15]. Acteoside, according to Lee et al. [16], induced the downregulation of cytochrome P450-2E1 (CYP2E1) protein expression in mice, protecting the mice liver against substance toxicity following toxicological activation mediated by CYP2E1. Our previous study, in contrast, found that microcystin-LR induced the up-regulation of CYP2E1 mRNA expression in HepG2 cells [17]. CYP2E1, as well as CYP1A1 and CYP1A2, are found primarily in the liver and are capable of not only hydrolyzing several substances as phase I enzymes but also producing reactive oxygen species (ROS) [18]. However, we recently demonstrated that ROS production was not involved in the onset of the cytotoxicity of microcystin-LR using HEK293-OATP1B3 cells [5]. We investigated the mechanism by which acteoside attenuates microcystin-LR cytotoxicity.

First, we determined whether acteoside can attenuate microcystin-LR cytotoxicity. Thus, acteoside was found to be one of the functional components in iwajisha that helps in inhibiting microcystin-LR cytotoxicity. Furthermore, acteoside attenuated the cytotoxicity of nodularin and okadaic acid, which are both OATP1B3 substrates and ligands for PP1 and PP2A, as well as microcystin-LR ([Fig. 2]). These results suggested that acteoside could attenuate the cytotoxicity of OATP1B3 substrates by inhibiting the OATP1B3-mediated uptake of substances into the cells.

Furthermore, we used immunoblot analysis for microcystin-LR to determine the effect of acteoside on the accumulation of microcystin-LR and their interaction with intracellular proteins. Microcystin-LR-binding proteins had molecular masses of approximately 22, 29, and 31 kDa, respectively ([Fig. 3]). Catalase [19], glutathione S-transferase [20], L-3-hydroxyacyl coenzyme A dehydrogenase [20], aldehyde dehydrogenase 2 [21], ATP synthase [22], proteasome β2 subunit [23], carboxylesterase 2 [24], PP1, and PP2A have been shown to interact with microcystin-LR. In terms of their molecular mass, PP2A catalytic C subunit α isoform (36 kDa), β isoform (38 kDa), glutathione S-transferase (26 kDa), and proteasome β2 subunit (23 kDa) appear to be promising candidates for the results of this study. Further research will be required to identify the microcystin-LR-binding proteins found in this study. In any case, co-exposure to acteoside reduced intracellular accumulation of microcystin-LR as the complex of microcystin-LR and its binding proteins were reduced in a concentration-dependent manner, which suggested that acteoside could reduce or enhance cellular uptake or efflux of microcystin-LR, respectively. Acteoside also inhibited the interaction between microcystin-LR and its binding proteins. However, free-microcystin-LR and microcystin-LR conjugated with small molecules, such as glutathione, remained undetected.

We attempted to determine if acteoside inhibits the uptake of microcystin-LR into HEK293-OATP1B3 cells to confirm whether acteoside inhibits the OATP1B3-mediated uptake of substances. We investigated the inhibitory kinetic analysis of acteoside on the transport property of microcystin-LR. We previously showed that the transport property of microcystin-LR mediated by OATP1B3 was saturable, with Km values of 1.2 µM [3] and 1.37 µM [7]. The uptake of microcystin-LR into the HEK293-OATP1B3 cells was reduced in a concentration-dependent manner after co-exposure to 1 µM microcystin-LR and various concentrations of acteoside, with an IC₅₀ value of 12.3 µM ([Fig. 4 a]). These results indicated that acteoside attenuated the cytotoxicity of microcystin-LR at least partially by inhibiting its uptake into the cells mediated by OATP1B3 in a noncompetitive manner. Apart from OATP1B3 inhibition, other functions, such as inhibition of the interaction between microcystin-LR and its cellular binding proteins, may also be involved in the attenuation of microcystin-LR cytotoxicity.

Phosphorylation of ERK, p38, and p53 significantly increased in our previous study [3], [4] after incubation with microcystin-LR, and it may be closely associated with cell survival and death. As a result, we analyzed ERK, p38, and p53 phosphorylation in HEK293-OATP1B3 cells after their co-exposure to microcystin-LR with acteoside. Of note, acteoside could induce ERK phosphorylation when exposed alone, implying that acteoside activated the survival signaling cascade. Son et al. [25] found that acteoside causes ERK phosphorylation in B16F10 cells, which is consistent with this finding. Acteoside has been shown to exert antiviral, hepatoprotective, anti-inflammatory, and other beneficial effects [11]. Because ERK activation is linked to several cellular events such as proliferation, differentiation, and survival [3], acteoside may intracellularly activate ERK regardless of microcystin-LR. In contrast, acteoside did not affect the phosphorylation of p38 and p53 when exposed alone. Various cellular stresses, including hypoxia, proinflammatory cytokines, UV, ROS, and heat shock, activate p38, and their activation is regulated by PP2A [3]. The aforementioned stresses also activate p53 [4]. ERK, p38, and p53 phosphorylation were significantly increased 24 h after incubation with 50 nM microcystin-LR, as previously reported [3], [4]. We demonstrated that acteoside could reduce the magnitude of phosphorylation of p38 and p53.

In conclusion, the current study found that iwajisha extract attenuated the microcystin-LR cytotoxicity in OATP1B3-expressing HEK293 cells. Acteoside may be one of the most effective compounds in iwajisha extracts for attenuating microcystin-LR cytotoxicity. Acteoside reduced the intracellular microcystin-LR level and inhibited the interaction of microcystin with its binding proteins. Furthermore, acteoside, with or without microcystin-LR, induced the phosphorylation of ERK, which is one of the molecules associated with survival signaling. The attenuation of microcystin-LR cytotoxicity by acteoside could be attributed to ERK activation. However, further research is required to determine how acteoside activates the ERK signaling cascade and how ERK contributes to the attenuation of microcystin-LR cytotoxicity. Acteoside also inhibited the activation of stress-response signaling, including p38 phosphorylation and p53 phosphorylation, which was accompanied by the activation of survival signaling. Finally, acteoside was shown to attenuate the cytotoxicity of okadaic acid and nodularin, as well as microcystin-LR, implying that acteoside is useful in the chemoprevention of these OATP1B3-related toxic compounds.

Materials and Methods

Reagents

Microcystin-LR, nodularin, okadaic acid, and Eagleʼs minimum essential medium (MEM) were purchased from Wako. Fetal calf serum (FCS) was obtained from Cancera International. The dried leaf of Conandron ramondioides was purchased from Tochimoto Tenkaido Co. Ltd. The powdered leaf (1.0 g) of C. ramondioides was extracted using absolute methanol (80 g) at room temperature for 24 h and the solvent was evaporated under reduced pressure. The crude extract obtained was used for the MTT assay. Acteoside (purity > 98%) was obtained from Wako. Monoclonal mouse antibodies against human phospho-ERK, ERK, phospho-p38, p38, phospho-p53, and p53, as well as polyclonal rabbit antibodies against human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin, were purchased from Cell Signaling Technology and Gene Tex. Monoclonal antibody against microcystin-LR was purchased from Enzo Life Sciences. Secondary antibodies against mouse IgG and rabbit IgG were supplied by GE Healthcare Science. A secondary antibody against goat IgG was supplied by Jackson Immuno Research Laboratories.

Cell culture

HEK293-OATP1B3 cells were previously generated by stable transfection of HEK293 cells [8], [26] with the plasmid pcDNA3.1(+)-SLCO1B3. Stable transfection of the empty plasmid pcDNA3.1(+) resulted in the generation of HEK293-CV cells. Cells were cultured in MEM supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin (MEM-10% FCS), and 400 µg/mL G418 at 37 °C, 100% humidity, and 5% CO₂.

Cell survival determination

HEK293-OATP1B3 and HEK293-CV cells were exposed to various concentrations of microcystin-LR and incubated in a CO₂ incubator for 72 h. Following incubation with microcystin-LR, a colorimetric assay using MTT was used to assess the susceptibility of the cells to their reagents in vitro, as described before [27]. Exponentially growing cells were trypsinized and harvested, and equal numbers (1.6 × 10⁴/well) of cells were inoculated into each well of a 96-well microplate in 180 µL of MEM-10% FCS. After overnight incubation, cells were treated with 20 µL of microcystin-LR and incubated for 3 days. Next, 50 µL of 1 mg/mL MTT solution was added to each well, and the plates were incubated at 37 °C in a CO₂ incubator for 3 h. The resulting formazan was dissolved with dimethyl sulfoxide after aspirating the culture medium. Plates were shaken for 5 min before being read at 570 nm with a microplate reader, SUNRISE (TECAN), and cell viability was determined.

For attenuation studies, cells in 170 µL of MEM-10% FCS were pretreated for 2 min with 10 µL of iwajisha-water extract or acteoside, then immediately treated with 20 µL of microcystin-LR, nodularin, or okadaic acid solution and incubated for another 3 days. The IC₅₀ value was calculated using Excel software as the concentration of microcystin-LR, nodularin, or okadaic acid that reduced cell viability to 50% of that in the control medium.

Intracellular accumulation of microcystin-LR

In a CO₂ incubator, HEK293-OATP1B3 cells were exposed to 50 nM microcystin-LR for 24 h. Following incubation, cell lysates were prepared and immunoblotted for microcystin-LR-binding proteins. Under nonreducing conditions, protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heat treatment. Transfer to polyvinylidene fluoride (PVDF) membranes was performed electrophoretically for 30 min at 15 V (constant voltage) using a semi-dry blotting system after electrophoresis (Transblot SD apparatus, Bio-Rad). The PVDF membrane was blocked with 5% BSA in 10 mM Tris-Cl buffer (pH 8.0) containing 0.35 M NaCl and 0.05% Tween 20 (TBS-T) for 1 h at room temperature and incubated overnight at 4 °C with the primary antibody against microcystin-LR in Can Get Signal (Toyobo). The PVDF membrane was washed three times with TBS-T before being incubated in Can Get Signal (Toyobo) for 60 min with horseradish peroxidase-conjugated mouse antibody. The PVDF membrane was rinsed twice and then washed four times with TBS-T for 5 min. The ECL Western blotting detection reagents (GE Healthcare) were then applied evenly for 1 min. The ECL signals were detected using the C-Digit (LI-CORE) scanning detector.

HEK293-OATP1B3 cells were co-exposed to 50 nM microcystin-LR with 5, 10, or 20 µM acteoside for the inhibition study. Microcystin-LR immunoblot analysis was performed on cell lysates.

Uptake study of microcystin-LR

Microcystin-LR uptake in HEK293-OATP1B3 cells was assessed using an ELISA assay, as previously described [5]. Cells were seeded in six-well plates at a density of 2 × 10⁶ cells/well and cultured in a CO₂ incubator for 24 h with 10 mM sodium butyrate, a histone deacetylases inhibitor. Cells were washed with prewarmed (37 °C) uptake buffer before the uptake experiments. Transport studies were conducted by incubating cells in 1 mL of prewarmed uptake buffer with various concentrations of microcystin-LR coupled with or without various concentrations of acteoside for 15 min at 37 °C. Following aspiration of the microcystin-LR containing the uptake buffer, transport of microcystin-LR was stopped by washing cells twice with ice-cold 0.5% BSA in uptake buffer, followed by three washes with 1 mL of ice-cold uptake buffer, and finally with 1 mL of ice-cold PBS. Cells were harvested and placed into 1 mL of ice-cold PBS. The harvested cells were centrifuged at 1000 × g for 3 min at 4 °C. The pellet (cells) was placed in 250 µL of hypotonic lysis buffer and left at room temperature for 30 min. The cells were homogenized and centrifuged at 16 000 × g for 15 min at room temperature after being heated for 10 min at 95 °C to denature the intracellular microcystin-binding protein. The supernatants (25 µL) were tested for microcystin-LR using an ELISA. The microcystin-LR ELISA analysis was conducted following the manufacturerʼs instructions. Protein concentrations were determined using the bicinchoninic acid assay kit (Thermo).

Interaction of microcystin-LR with intracellular proteins

Cell lysates from HEK293-OATP1B3 cells were pretreated for 1 h with various concentrations of acteoside or vehicle at 25 °C before being treated with various concentrations of microcystin-LR or vehicle. These cell lysate samples were then incubated for 2 h at 25 °C. Following incubation, cell lysates were immunoblotted for microcystin-LR-binding proteins as described above.

Immunoblot analysis for the phosphorylation of ERK, p38, and p53

Cell lysates were prepared from cultured HEK293-OATP1B3 cells and immunoblotting was performed as previously described [3]. Protein samples were incubated at 95 °C for 5 min before being separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. After electrophoresis, the separated proteins were transferred to PVDF membranes and immunoblotted for p-ERK, ERK, p-p38, p38, p-p53, p53, or GAPDH. The ECL signals were detected using the C-Digit ECL scanning detector (LI-CORE).

Statistical analysis

The Wilcoxon–Mann–Whitney test was used to compare group differences. p values of < 0.05 were considered significant.

Contributorsʼ Statement

Data collection: S. Takumi, K. Hashimoto, M. Tomioka, M. Sato, W. He, Y. Komatsu. Design of the study: M. Komatsu, S. Aoki, R. Ikeda, T. Furukawa. Statistical analysis: K. Hashimoto, M. Tomioka, M. Sato, W. He, Y. Komatsu, S. Takumi, M. Komatsu. Analysis and interpretation of the data: S. Takumi, K. Shiozaki, M. Komatsu. Drafting the manuscript: S. Takumi, M. Komatsu. Critical revision of the manuscript: K. Shiozaki, S. Aoki, R. Ikeda, T. Furukawa, S. Takumi, M. Komatsu.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

We thank Prof. Dietrich Keppler (German Cancer Research Center) for kindly providing the HEK293-OATP1B3 cells. We also thank Narumi Tokunaga for her excellent technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research (C) 16K07875 and 19K06222 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

• References

• 1 van Apeldoorn ME, van Egmond HP, Speijers GJ, Bakker GJ. Toxins of cyanobacteria. Mol Nutr Food Res 2007; 51: 7-60
  CrossrefPubMedSearch in Google Scholar
• 2 Gehringer MM. Microcystin-LR and okadaic acid-induced cellular effects: A dualistic response. FEBS Lett 2004; 557: 1-8
  CrossrefPubMedSearch in Google Scholar
• 3 Komatsu M, Furukawa T, Ikeda R, Takumi S, Nong Q, Aoyama K, Akiyama S, Keppler D, Takeuchi T. Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3. Toxicol Sci 2007; 97: 407-416
  CrossrefPubMedSearch in Google Scholar
• 4 Takumi S, Komatsu M, Furukawa T, Ikeda R, Sumizawa T, Akenaga H, Maeda Y, Aoyama K, Arizono K, Ando S, Takeuchi T. p53 plays an important role in cell fate determination after exposure to Microcystin-LR. Environ Health Perspect 2010; 118: 1292-1298
  CrossrefPubMedSearch in Google Scholar
• 5 Takumi S, Ikema S, Hanyu T, Shima Y, Kurimoto T, Shiozaki K, Sugiyama Y, Park HD, Ando S, Furukawa T, Komatsu M. Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells. Environ Toxicol Pharmacol 2015; 39: 974-981
  CrossrefPubMedSearch in Google Scholar
• 6 Herfindal L, Myhren L, Kleppe R, Krakstad C, Selheim F, Jokela J, Sivonen K, Døskeland SO. Nostocyclopeptide-M1: A potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and OATP1B1. Mol Pharm 2011; 8: 360-367
  CrossrefPubMedSearch in Google Scholar
• 7 Ikema S, Takumi S, Maeda Y, Kurimoto T, Bohda S, Chigwechokha PK, Sugiyama Y, Shiozaki K, Furukawa T, Komatsu M. Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3. Food Chem Toxicol 2015; 83: 229-236
  CrossrefPubMedSearch in Google Scholar
• 8 Letschert K, Faulstich H, Keller D. Molecular characterization and inhibition of amanitin uptake into human hepatocytes. Toxicol Sci 2006; 91: 140-149
  CrossrefPubMedSearch in Google Scholar
• 9 Fehrenbach T, Cui Y, Faulstich H, Keppler D. Characterization of the transport of the bicyclic peptide phalloidin by human hepatic transport proteins. Naunyn Schmiedebergs Arch Pharmacol 2003; 368: 415-420
  CrossrefPubMedSearch in Google Scholar
• 10 Miyaichi Y, Ohichi M, Yaguchi K, Kawata Y, Kizu H. Studies on the constituents of the leaves of Conandron ramondioides. J Nat Med 2006; 60: 159-160
  CrossrefPubMedSearch in Google Scholar
• 11 Dai X, Su S, Cai H, Wei D, Zheng T, Zhu Z, Yan H, Shang E, Guo S, Qian D, Duan J. Comparative pharmacokinetics of acteoside from total glycoside extracted from leaves of Rehmannia and Dihuangye total glycoside capsule in normal and diabetic nephropathy rats. Biomed Chromatogr 2017; 31: e4013
  CrossrefPubMedSearch in Google Scholar
• 12 Xiang Q, Kadota S, Tani T, Namba T. Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol Pharm Bull 1996; 19: 1580-1585
  CrossrefPubMedSearch in Google Scholar
• 13 Chathuranga K, Kim MS, Lee H, Kim T, Kim J, Chathuranga WAG, Ekanayaka P, Wijerathne HMSM, Cho W, Kim HI, Ma JY, Lee J. Anti-respiratory syncytial virus activity of Plantago asiatica and Clerodendrum trichotomum extracts in vitro and in vivo . Viruses 2019; 11: 604
  CrossrefPubMedSearch in Google Scholar
• 14 Nonaka G, Nishioka I. Bitter phenylpropanoid glycosides from Conandron ramoidioides . Phytochemistry 1977; 16: 1265-1267
  CrossrefPubMedSearch in Google Scholar
• 15 Xiufen W, Hiramatsu N, Matsubara M. The antioxidative activity of traditional Japanese herbs. Biofactors 2004; 21: 281-284
  CrossrefPubMedSearch in Google Scholar
• 16 Lee KJ, Woo E, Choi CY, Shin DW, Lee DG, You HJ, Jeong HG. Protecive effect of acteoside on carbon tetrachloride-induced hepatotoxicity. Life Sci 2004; 74: 1051-1064
  CrossrefPubMedSearch in Google Scholar
• 17 Nong Q, Komatsu M, Izumo K, Indo HP, Xu B, Aoyama K, Majima HJ, Horiuchi M, Morimoto K, Takeuchi T. Involvement of reactive oxygen species in microcystin-LR-induced cytogenotoxicity. Free Radic Res 2007; 41: 1326-1337
  CrossrefPubMedSearch in Google Scholar
• 18 Gonzalez FJ. Role of cytochrome P450 in chemical toxicity and oxidative stress: Studies with CYP2E1. Mutat Res 2005; 569: 101-110
  CrossrefPubMedSearch in Google Scholar
• 19 Hu Y, Da L. Insights into the selective binding and toxic mechanism of microcystin to catalase. Spectrochim Acta A Mol Biomol Spectrosc 2014; 121: 230-237
  CrossrefPubMedSearch in Google Scholar
• 20 Mori T, Kubo T, Kaya K, Hosoya K. Comprehensive study of proteins that interact with microcystin-LR. Anal Bioanal Chem 2012; 402: 1137-1147
  CrossrefPubMedSearch in Google Scholar
• 21 Chen T, Cui J, Liang Y, Xin X, Young DO, Chen C, Shen P. Identification of human liver mitochondrial aldehyde dehydrogenase as a potential target for microcystin-LR. Toxicology 2006; 220: 71-80
  CrossrefPubMedSearch in Google Scholar
• 22 Mikhailov A, Härmälä-Braskén A, Hellman J, Meriluoto J, Eriksson JE. Identification of ATP-synthase as a novel intracellular target for microcystin-LR. Chem Biol Interact 2003; 142: 223-237
  CrossrefPubMedSearch in Google Scholar
• 23 Zhu Z, Zhang L, Shi G. Proteasome as a molecular target of Microcystin-LR. Toxins (Basel) 2015; 7: 2221-2231
  CrossrefPubMedSearch in Google Scholar
• 24 Takumi S, Shimono T, Ikema S, Hotta Y, Chigwechokha PK, Shiozaki K, Sugiyama Y, Hashimoto M, Furukawa T, Komatsu M. Overexpression of carboxylesterase contributes to the attenuation of cyanotoxin microcystin-LR toxicity. Comp Biochem Physiol C Toxicol Pharmacol 2017; 194: 22-27
  CrossrefPubMedSearch in Google Scholar
• 25 Son Y, Lee S, Kim S, Jang Y, Chun J, Lee J. Acteoside inhibits melanogenesis in B16F10 cells through ERK activation and tyrosinase down-regulation. J Pharm Pharmacol 2011; 63: 1309-1319
  CrossrefPubMedSearch in Google Scholar
• 26 König J, Cui Y, Nies AT, Keppler D. A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol 2000; 278: 156-164
  CrossrefPubMedSearch in Google Scholar
• 27 Komatsu M, Sumizawa T, Mutoh M, Chen Z, Terada K, Furukawa T, Yang X, Gao H, Miura N, Sugiyama T, Akiyama S. Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance. Cancer Res 2000; 60: 1312-1316
  PubMedSearch in Google Scholar

Correspondence

Publication History

Received: 27 April 2022

Accepted after revision: 28 October 2022

Article published online:
10 January 2023

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 van Apeldoorn ME, van Egmond HP, Speijers GJ, Bakker GJ. Toxins of cyanobacteria. Mol Nutr Food Res 2007; 51: 7-60
  CrossrefPubMedSearch in Google Scholar
• 2 Gehringer MM. Microcystin-LR and okadaic acid-induced cellular effects: A dualistic response. FEBS Lett 2004; 557: 1-8
  CrossrefPubMedSearch in Google Scholar
• 3 Komatsu M, Furukawa T, Ikeda R, Takumi S, Nong Q, Aoyama K, Akiyama S, Keppler D, Takeuchi T. Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3. Toxicol Sci 2007; 97: 407-416
  CrossrefPubMedSearch in Google Scholar
• 4 Takumi S, Komatsu M, Furukawa T, Ikeda R, Sumizawa T, Akenaga H, Maeda Y, Aoyama K, Arizono K, Ando S, Takeuchi T. p53 plays an important role in cell fate determination after exposure to Microcystin-LR. Environ Health Perspect 2010; 118: 1292-1298
  CrossrefPubMedSearch in Google Scholar
• 5 Takumi S, Ikema S, Hanyu T, Shima Y, Kurimoto T, Shiozaki K, Sugiyama Y, Park HD, Ando S, Furukawa T, Komatsu M. Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells. Environ Toxicol Pharmacol 2015; 39: 974-981
  CrossrefPubMedSearch in Google Scholar
• 6 Herfindal L, Myhren L, Kleppe R, Krakstad C, Selheim F, Jokela J, Sivonen K, Døskeland SO. Nostocyclopeptide-M1: A potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and OATP1B1. Mol Pharm 2011; 8: 360-367
  CrossrefPubMedSearch in Google Scholar
• 7 Ikema S, Takumi S, Maeda Y, Kurimoto T, Bohda S, Chigwechokha PK, Sugiyama Y, Shiozaki K, Furukawa T, Komatsu M. Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3. Food Chem Toxicol 2015; 83: 229-236
  CrossrefPubMedSearch in Google Scholar
• 8 Letschert K, Faulstich H, Keller D. Molecular characterization and inhibition of amanitin uptake into human hepatocytes. Toxicol Sci 2006; 91: 140-149
  CrossrefPubMedSearch in Google Scholar
• 9 Fehrenbach T, Cui Y, Faulstich H, Keppler D. Characterization of the transport of the bicyclic peptide phalloidin by human hepatic transport proteins. Naunyn Schmiedebergs Arch Pharmacol 2003; 368: 415-420
  CrossrefPubMedSearch in Google Scholar
• 10 Miyaichi Y, Ohichi M, Yaguchi K, Kawata Y, Kizu H. Studies on the constituents of the leaves of Conandron ramondioides. J Nat Med 2006; 60: 159-160
  CrossrefPubMedSearch in Google Scholar
• 11 Dai X, Su S, Cai H, Wei D, Zheng T, Zhu Z, Yan H, Shang E, Guo S, Qian D, Duan J. Comparative pharmacokinetics of acteoside from total glycoside extracted from leaves of Rehmannia and Dihuangye total glycoside capsule in normal and diabetic nephropathy rats. Biomed Chromatogr 2017; 31: e4013
  CrossrefPubMedSearch in Google Scholar
• 12 Xiang Q, Kadota S, Tani T, Namba T. Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol Pharm Bull 1996; 19: 1580-1585
  CrossrefPubMedSearch in Google Scholar
• 13 Chathuranga K, Kim MS, Lee H, Kim T, Kim J, Chathuranga WAG, Ekanayaka P, Wijerathne HMSM, Cho W, Kim HI, Ma JY, Lee J. Anti-respiratory syncytial virus activity of Plantago asiatica and Clerodendrum trichotomum extracts in vitro and in vivo . Viruses 2019; 11: 604
  CrossrefPubMedSearch in Google Scholar
• 14 Nonaka G, Nishioka I. Bitter phenylpropanoid glycosides from Conandron ramoidioides . Phytochemistry 1977; 16: 1265-1267
  CrossrefPubMedSearch in Google Scholar
• 15 Xiufen W, Hiramatsu N, Matsubara M. The antioxidative activity of traditional Japanese herbs. Biofactors 2004; 21: 281-284
  CrossrefPubMedSearch in Google Scholar
• 16 Lee KJ, Woo E, Choi CY, Shin DW, Lee DG, You HJ, Jeong HG. Protecive effect of acteoside on carbon tetrachloride-induced hepatotoxicity. Life Sci 2004; 74: 1051-1064
  CrossrefPubMedSearch in Google Scholar
• 17 Nong Q, Komatsu M, Izumo K, Indo HP, Xu B, Aoyama K, Majima HJ, Horiuchi M, Morimoto K, Takeuchi T. Involvement of reactive oxygen species in microcystin-LR-induced cytogenotoxicity. Free Radic Res 2007; 41: 1326-1337
  CrossrefPubMedSearch in Google Scholar
• 18 Gonzalez FJ. Role of cytochrome P450 in chemical toxicity and oxidative stress: Studies with CYP2E1. Mutat Res 2005; 569: 101-110
  CrossrefPubMedSearch in Google Scholar
• 19 Hu Y, Da L. Insights into the selective binding and toxic mechanism of microcystin to catalase. Spectrochim Acta A Mol Biomol Spectrosc 2014; 121: 230-237
  CrossrefPubMedSearch in Google Scholar
• 20 Mori T, Kubo T, Kaya K, Hosoya K. Comprehensive study of proteins that interact with microcystin-LR. Anal Bioanal Chem 2012; 402: 1137-1147
  CrossrefPubMedSearch in Google Scholar
• 21 Chen T, Cui J, Liang Y, Xin X, Young DO, Chen C, Shen P. Identification of human liver mitochondrial aldehyde dehydrogenase as a potential target for microcystin-LR. Toxicology 2006; 220: 71-80
  CrossrefPubMedSearch in Google Scholar
• 22 Mikhailov A, Härmälä-Braskén A, Hellman J, Meriluoto J, Eriksson JE. Identification of ATP-synthase as a novel intracellular target for microcystin-LR. Chem Biol Interact 2003; 142: 223-237
  CrossrefPubMedSearch in Google Scholar
• 23 Zhu Z, Zhang L, Shi G. Proteasome as a molecular target of Microcystin-LR. Toxins (Basel) 2015; 7: 2221-2231
  CrossrefPubMedSearch in Google Scholar
• 24 Takumi S, Shimono T, Ikema S, Hotta Y, Chigwechokha PK, Shiozaki K, Sugiyama Y, Hashimoto M, Furukawa T, Komatsu M. Overexpression of carboxylesterase contributes to the attenuation of cyanotoxin microcystin-LR toxicity. Comp Biochem Physiol C Toxicol Pharmacol 2017; 194: 22-27
  CrossrefPubMedSearch in Google Scholar
• 25 Son Y, Lee S, Kim S, Jang Y, Chun J, Lee J. Acteoside inhibits melanogenesis in B16F10 cells through ERK activation and tyrosinase down-regulation. J Pharm Pharmacol 2011; 63: 1309-1319
  CrossrefPubMedSearch in Google Scholar
• 26 König J, Cui Y, Nies AT, Keppler D. A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol 2000; 278: 156-164
  CrossrefPubMedSearch in Google Scholar
• 27 Komatsu M, Sumizawa T, Mutoh M, Chen Z, Terada K, Furukawa T, Yang X, Gao H, Miura N, Sugiyama T, Akiyama S. Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance. Cancer Res 2000; 60: 1312-1316
  PubMedSearch in Google Scholar