Published papers

A method for quantification of new antiepileptic drugs, including lamotrigine (LTG), levetiracetam (LEV), gabapentin (GBP), and topiramate (TPM), in cellular samples, using liquid chromatography/electrospray ionization tandem mass spectrometry was developed to better understand the membrane transport mechanisms of these drugs. Cell lysate was deproteinized by methanol containing LEV-d3 as an internal standard (IS). Chromatographic separation was performed on a C18 column using gradient elution with methanol-water-formic acid (10:90:0.1, v/v/v) and methanol-formic acid (100:0.1, v/v). Analytes were detected in positive ion electrospray mode with selected reaction monitoring (SRM). This method was applicable for a linear range of 5 to 500pmol for LTG; 5 to 1000pmol for LEV; 10 to 10,000pmol for GBP; and 5 to 5000pmol for TPM. The intra-day precision, inter-day precision, and accuracy data were assessed and found to be acceptable. This developed and validated method was then successfully applied to the investigation of uptake of the new antiepileptic drugs in placental choriocarcinoma BeWo cells. The intracellular concentration of these drugs in BeWo cells, accumulating over 30min at 37°C was in the order of GBP>LTG>LEV≈TPM. Furthermore, the uptake of GBP at 4°C was much lower than that at 37°C. The uptake of GBP was saturated at high concentrations. The kinetic parameters calculated for GBP uptake in BeWo cells were determined as Km of 105.4±6.4μM and Vmax at 8153±348pmol/mg protein/min. The novel method described here should enable investigators to elucidate the transport mechanisms of these antiepileptic drugs in BeWo cells.

A column-switching liquid chromatography/electrospray ionization tandem mass spectrometry to determine paclitaxel and its metabolites, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, in human plasma was developed. The analytical system had a Shim-Pack MAYI-ODS (10 × 4.6 mm i.d.) trapping column with deproteinization ability that concentrates analytes and removes water-soluble components. This method covered a linearity range of 5-5000 ng/mL of concentrations in plasma for paclitaxel, a range of 0.87-870 ng/mL for 6α-hydroxypaclitaxel and a range of 0.87-435 ng/mL for p-3'-hydroxypaclitaxel. The intra-day precision and inter-day precision of analysis were less than 11.1%, and the accuracy was within ±14.4% at concentrations of 5, 50, 500 and 5000 ng/mL for paclitaxel, 0.87, 8.7, 87 and 870 ng/mL for 6α-hydroxypaclitaxel, and 0.87, 8.7, 87 and 435 ng/mL for p-3'-hydroxypaclitaxel. The total run time was 30 min. Our method was successfully applied to clinical pharmacokinetic investigation.

In the present study, to clarify the role of protein kinase C (PKC) in the regulation of monocarboxylate transporter 4 (MCT4) expression, we examined the regulation mechanism of MCT4 expression in human rhabdomyosarcoma (RD) cells, an in vitro skeletal muscle model. Exposure of RD cells to PMA, a PKC activator, for 24 h resulted in a two-fold increase in the amount of lactic acid in the growth medium. In parallel to an increase in lactic acid release from RD cells, the level of MCT4 mRNA and protein were also significantly increased in RD cells. A PKC inhibitory study indicated that PMA-induced stimulation of MCT4 expression can be mediated through a novel PKC isoform, especially PKCδ. Moreover, rottlerin, a selective PKCδ inhibitor, decreased PMA-induced MCT4 promoter activity. Deletion and mutational analysis suggested that the potential hypoxia-response elements (HREs) played a major role in the observed modulation of MCT4 expression by PMA. Furthermore, we found that small interfering RNA (siRNA)-mediated knockdown of hypoxia-inducible factor 1α (HIF-1α) significantly inhibited PMA-induced MCT4 promoter activity. Our results show that the effects of PMA on MCT4 expression are mediated through an indirect pathway partially involving PKCδ and HIF-1α transcription factor.

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibers also use lactic acid as a respiratory fuel. Monocarboxylate transporter (MCT) 1 has been suggested to play a major role in influx of L-lactic acid for oxidation. The regulation mechanism of MCT1 was characterized utilizing rhabdomyosarcoma cells as an in vitro skeletal muscle model. The uptake of L-lactic acid via MCT1 was studied in the presence of various intracellular regulatory pathways, including pathways mediated by protein kinases A, C and G (PKA, PKC and PKG), protein tyrosine kinase (PTK), and Ca2+/calmodulin modulators. The results showed that PKG-, PTK-, and Ca2+/calmodulin-mediated regulatory pathways play no role in the regulation of L-lactic acid uptake, but a role for PKC- and PKA-mediated pathways was apparent. Uptake of L-lactic acid appeared to be stimulated by phorbol 12-myristate 13-acetate (PMA, a PKC activator) via an increase in Vmax of transport processes with no alteration in Km. In parallel, PMA treatment also resulted in an increase in the level of MCT1 expression. On the other hand, exposure to 8-Br-cAMP, a cAMP analog, and to forskolin, an adenylyl cyclase activator, resulted in a significant decrease in L-lactic acid uptake. Additionally, 8-Br-cAMP reduced Vmax but not Km values. Parallel to the decrease in Vmax of L-lactic acid uptake, the level of MCT1 expression was decreased in response to incubation with 8-Br-cAMP. These results indicate the possible involvement of a PKC- and PKA-mediated pathway associated with expression of MCT1 and lactate transport.

Protein kinase C (PKC) modulators are very attractive therapeutic targets in cancer. Since most cancer cells display increased glycolysis, elucidations of the effects of PKC activation on glycolysis is necessary for the development of effective medicine. In the present study, to clarify the role of PKC in the regulation of glycolysis, we examined the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on the expression and activity of glucose and lactic acid metabolism-related genes in human rhabdomyosarcoma cells (RD cells). In parallel to increases in glucose uptake and mRNA levels of glucose transporters (GLUTs) induced by PMA treatment for 6 h, the hexokinase (HK) mRNA level and activity were also significantly increased in RD cells. On the other hand, a significant increase in lactate dehydrogenase (LDH) mRNA level and activity was seen when the cells were incubated with PMA for 24 h, but not for 6 or 12 h, and was associated with lactic acid production. These effects by PMA treatment were markedly suppressed by Bisindolylmaleimide (BIM), a PKC inhibitor. Furthermore, chetomin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, completely abrogated the increment of LDH mRNA level and activity as well as monocarboxylate transporter (MCT) 4, a lactic acid efflux transporter. In conclusion, we found that HK and LDH activity induced by PKC activation was associated with the glucose uptake and lactic acid level and that LDH and MCT4 are modulated by a common factor, HIF-1.

ETHNOPHARMACOLOGICAL RELEVANCE: Immunoglobulin A (IgA) secretion and alpha-defensins play a role in the innate immune system to protect against infection. Ganoderma lucidum (W.Curt.: Fr.) P. Karst. (Reishi) is a well-known mushroom in traditional Chinese medicine. This study aimed to determine the effects of Reishi on IgA secretion from Peyer's patch (PP) cells and alpha-defensin-5 (RD-5) and RD-6 expression in the rat small intestine. MATERIALS AND METHODS: The rats received an oral injection of 0.5-5mg/kg of Reishi powder (1mL/kg) by sonde. All animals were euthanized 24h after Reishi administration. We examined RD-5, RD-6, and Toll-like receptor (TLR) 4 mRNA levels in the jejunum, ileum, and in Peyer's patches (PP) through quantitative real-time PCR analysis. IgA secretion from PP was measured through enzyme-linked immunosorbent assay of the supernatant after primary culture. RESULTS: Reishi increased IgA secretion in the presence of lipopolysaccharide (LPS) and increased TLR4 mRNA levels, but had no effect on the viability of PP cells. Moreover, Reishi increased RD-5, RD-6, and TLR4 mRNA levels significantly in the ileum in a concentration-dependent manner. CONCLUSIONS: Reishi can induce IgA secretion and increase the mRNA levels of RD-5 and RD-6 in the rat small intestine, through a TLR4-dependent pathway. The present results indicate that Reishi might reduce the risk of intestinal infection.

BACKGROUND/AIM: Monocarboxylate transporters (MCTs) transport short-chain monocarboxylates, such as lactate, and have been reported to be related to poor prognosis in breast cancer. Our previous studies showed that a high glucose state altered MCT expression and changed the sensitivity of the tamoxifen active metabolite 4-hydroxytamoxifen (4-OHT) via hypoxia-inducible factor-1α (HIF-1α) protein expression. We hypothesized that MCT inhibitors affect 4-OHT-induced cytotoxicity under normal glucose conditions by decreasing HIF-1α protein expression. To test this hypothesis, we evaluated the combined effect of MCT inhibitor and 4-OHT using the estrogen receptor (ER)-positive breast cancer cell line MCF-7, under normal glucose conditions. MATERIALS AND METHODS: Expression of MCTs and oxidative stress markers was evaluated by real-time PCR. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Reactive oxygen species (ROS) were measured using the cell permeability probe 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: MCT1 expression increased under normal glucose conditions. The MCT1 substrate/inhibitor, 5-oxoproline (5-OP), enhanced 4-OHT-induced cytotoxicity. Bindarit, a selective MCT4 inhibitor, decreased 4-OHT sensitivity, similar to results of our previous study under high glucose conditions. In contrast, the combination of 5-OP and 4-OHT decreased ATP levels compared with that by 4-OHT alone in MCF-7 cells. Furthermore, 5-OP significantly increased the ROS production induced by 4-OHT. CONCLUSION: 5-OP enhances 4-OHT-induced cytotoxicity in ER-positive breast cancer cells under normal glucose conditions.

With the coronavirus disease 2019 pandemic, businesses are rapidly expanding their online practices, and the online medical care system has been established and is growing. The field of pharmacy education is also looking for ways to conduct practical online training. Hence, we developed an online role-play-based medical interview training method for fourth-year pharmacy students. The purpose of this study was to describe in detail this method and to clarify the effect of online on medical interviewing practice. The training sessions were conducted using video teleconferencing software. Two settings were used for the role-play scenarios: the pharmacy and hospital. To evaluate the effectiveness of the sessions, a questionnaire was sent to the students, and the results were analyzed using text mining. The most important requirement for successfully conducting the interviews was a stable voice connection, and we reduced audio interruptions and delays by connecting the host personal computer to a wired local area network. We also solved the problem of howling when multiple terminals were installed in the same room by muting all devices in the room. Results of the analysis of the questionnaires suggested that students were more tense online. We also found that students perceived a difference between online and face-to-face interviews in terms of eye contact and the presentation of documents. In this way, we succeeded in conducting smooth online role-playing sessions while taking countermeasures against infection. In the future, it will be necessary to devise nonverbal communication methods and digital methods of presenting the training material.

Oxaliplatin is a platinum agent that is used for treatment of colorectal cancer. A sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the quantification of oxaliplatin was developed. Human plasma ultrafiltrates were precipitated by acetonitrile containing carboplatin as an internal standard and further diluted with acetonitrile. Chromatographic separation of oxaliplatin and the internal standard was achieved with a column modified with phosphorylcholine and an isocratic mobile phase (acetonitrile/water/acetic acid=90:10:0.1, v/v/v) at the flow rate of 0.2mL/min. The lower limit of quantification for oxaliplatin was 25ng/mL. The linearity range of the method was from 25 to 5000ng/mL. The intra-day precision and inter-day precision (RSD) ranged from 0.8 to 6.1%, and the accuracy (RE) was within ±4.5%. The extraction recoveries from human plasma ultrafiltrates were 83.6-91.6%, and ion suppression caused by matrix components was 86.7-88.5% at three different levels, respectively. This method was applied to a clinical pharmacokinetic study of oxaliplatin in a cancer patient. The maximum concentration of colorectal cancer patient administered oxaliplatin was 1650ng/mL.

Docetaxel is a taxane family antineoplastic agent widely employed in cancer chemotherapy. We developed a liquid chromatography/tandem mass spectrometry method for the determination of docetaxel in human plasma. Plasma samples were deproteinized by acetonitrile containing internal standard paclitaxel. Chromatographic separation was performed on a TSKgel ODS-100 V 3 μm (50 mm × 2.0 mm i.d.) column using a mobile phase composed of acetonitrile-methanol-water-formic acid (50:5:45:0.1, v/v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. This method covered a linearity range of 5-5000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra-day precision and inter-day precision (R.S.D.) of analysis were less than 6.7%, and the accuracy (R.E.) was within ± 9.0% at the concentrations of 5, 20, 200, and 2000 ng/mL. The total run time was 5.0 min. This method was successfully applied for clinical pharmacokinetic investigation.

AIMS: The aim of this study was to determine the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, on monocarboxylate transporter 4 (MCT4) expression in rat skeletal muscle and a prototypic embryonal rhabdomyosarcoma cell line (RD cells). MAIN METHODS: We examined the alteration in Glucose transporter 4 (GLUT4) and MCT4 mRNA levels by quantitative real-time PCR. Alteration in GLUT4 and MCT4 protein levels was examined by Western blotting. KEY FINDINGS: In an in vivo study, AICAR increased MCT4 mRNA and protein levels in a fiber-type specific manner. In an in vitro study, AICAR increased MCT4 mRNA and protein levels. Moreover, AICAR-induced MCT4 expression was blocked by Compound C, an AMPK inhibitor. SIGNIFICANCE: In this study, we found that AMPK activation induced expression of MCT4 in RD cells and rat skeletal muscle in a fiber-type specific manner. These results indicate the possible involvement of an AMPK-mediated pathway associated with MCT4 expression in skeletal muscle.

OBJECTIVE: Polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are essential for adequate fetal growth. The aim of the present study was to elucidate the effects of PUFAs on the expression and function of placental transporters, which play important roles in placental functions including the supply of nutrients to the fetus, excretion of metabolites, and protection of the fetus from xenobiotics. METHODS: Human placental choriocarcinoma BeWo cells were used as a trophoblast model. PUFA-induced alteration in the gene expression of 84 transporters was investigated by a commercially available PCR array. Protein levels and the activity of transporters were assessed by western blotting and uptake experiments, respectively. The placental expression of the transporters was analyzed using pregnant Wistar rats. RESULTS: PUFAs (AA, EPA, and DHA) increased cystine/glutamate transporter xCT/SLC7A11, which mediates the cellular uptake of cystine coupled with the efflux of glutamate in human placental choriocarcinoma BeWo cells. These PUFAs also increased [14C]-cystine uptake in BeWo cells. PUFA-induced xCT/SLC7A11 mRNA expression was not blocked by nuclear factor-erythroid 2-related factor-2 (NRF2) knockdown. Reverse transcription (RT)-PCR analysis indicated that xCT/Slc7a11 mRNA was detected in rat placenta and the expression level at gestational day (GD) 12 was higher than that at GD 20. CONCLUSION: These results indicate that PUFAs promoted cystine uptake in placental cells by inducing xCT/SLC7A11 expression and NRF2 did not contribute to upregulation of xCT/SLC7A11 by PUFAs. Furthermore, xCT expression in rat placenta may change during pregnancy.

α-Defensin 5 has a particularly broad antibacterial spectrum; it eliminates pathogenic microorganisms and regulates intestinal flora. Although Caco-2 cells are similar to small intestinal cells, it is unclear whether they secrete α-defensin 5. Therefore, we investigated whether Caco-2 cells secrete α-defensin 5 and determined the secretion mechanism using cells from three cell banks (ATCC, DSMZ, and RIKEN). The Caco-2 cell proliferation rate increased with the number of culture days, irrespective of cell bank origin. On the other hand, the alkaline phosphatase activity, which affects cell differentiation and the mRNA levels of several cytokines, such as interleukin 8 (IL-8), IL-6, IL-1β, tumor necrosis factor-α (TNF-α), and IL-2, in the Caco-2 cells fluctuated with the number of culture days, and differed for each cell bank. α-Defensin 5 secretion was detected in all three cell bank Caco-2 cells; particularly, the ATCC Caco-2 cells grew linearly depending on the cell culture day as well as the levels of IL-8 and TNF-α mRNA. This suggested that α-defensin 5 secretion in the ATCC Caco-2 cells was associated with fluctuations in the mRNA levels of various cytokines, such as IL-8 and TNF-α. In conclusion, Caco-2 cells may be a simple model for screening health food components and drugs that affect α-defensin 5 secretion.

Certain non-steroidal anti-inflammatory drugs (NSAIDs) are known to have anticancer effects. However, it is unclear whether all NSAIDs have anticancer effects, and thus far, very few studies have compared the antitumor effects among multiple NSAIDs. Therefore, we aimed to identify NSAIDs that enhance the anticancer effect of cisplatin (CDDP); the effects of 17 NSAIDs in lung cancer cells and their spheroids as cancer stem cells (CSCs) were evaluated. Some of the NSAIDs showed cytotoxic effects against A549 and SBC-3 cells and their CDDP-resistant cell lines (A549/DDP and SBC-3/DDP cells, respectively). In addition, co-addition of CDDP and celecoxib, which showed cytotoxic effects, increased the resistance to CDDP by increasing SLC7A11, which is one of the CDDP resistance mechanisms, in A549/DDP and SBC-3/DDP cells. On the other hand, celecoxib also showed antitumor effects on the spheroids of A549/DDP and SBC-3/DDP cells, and enhanced the antitumor effect of CDDP while increasing the mRNA levels of SLC7A11. Moreover, diclofenac was also cytotoxic and enhanced the cytotoxic effect of CDDP in cancer cells and CSCs. In conclusion, some NSAIDs including celecoxib and diclofenac may enhance the therapeutic efficacy of CDDP.

Cisplatin (CDDP) is a highly potent anticancer drug that is widely used in the treatment of several cancers. CDDP-induced nephrotoxicity (CIN) is one of the most significant adverse effects, and oxidative stress is thought to be one of the mechanisms underlying CIN. Although there are some studies available on the variability in transporter expression in the kidney after a single CDDP dose, none have reported the change in renal transporter expression after multiple CDDP dose administrations. P-glycoprotein (P-gp), a transporter, is reported to be induced by oxidative stress. Ascorbic acid is a vitamin with antioxidant potential and therefore, may regulate the expression of P-gp transporter and affect CIN. In the present study, our aim was to assess the variability in expression of several renal transporters after multiple CDDP dose administrations and the antioxidant effect of ascorbic acid against transporter expression and CIN. Multiple doses of CDDP affected markers of kidney injury and antioxidants in the kidneys. Also, the expression of P-gp, breast cancer resistance protein, and multidrug resistance-associated protein 4 was upregulated by CDDP. Using a normal kidney cell line, we demonstrated that ascorbic acid attenuated CDDP-induced cytotoxicity due to its high superoxide scavenging ability. CDDP and ascorbic acid were injected into rats once a week for three weeks, and it was observed that co-administration of ascorbic acid attenuated CIN and regulated antioxidant marker. In addition, ascorbic acid reduced P-gp expression, which was upregulated by CDDP. In conclusion, ascorbic acid may attenuate CIN and reverse P-gp-mediated changes in drug pharmacokinetics.

Clozapine-induced sialorrhea (CIS) is a common side effect of clozapine. There is no established standard treatment of CIS since the underlying mechanism remains unknown. This study aimed to elucidate the mechanisms involved in CIS. In our clinical study, a prospective observational study evaluated the association between serum and saliva concentrations of clozapine or its metabolites and Drooling Severity and Frequency Scale (DSFS) score. In our in vivo study, we first developed a new CIS animal model; subsequently, we measured salivary secretion and concentrations of clozapine or its metabolites in the animal model. In our in vitro study, we measured the calcium ion (Ca2+) response to evaluate the effect of clozapine or its metabolites on human salivary gland cell line (HSY cells) and then examined whether their effect was inhibited by atropine. In our clinical study, serum and saliva N-desmethylclozapine concentrations were significantly correlated with nocturnal DSFS score. In our in vivo study, daily single oral administration of 100 mg/kg clozapine for 7 days significantly increased salivary secretion in rats. Furthermore, N-desmethylclozapine concentrations in serum and submandibular glands of the rats were higher than clozapine concentrations. In our in vitro study, N-desmethylclozapine only elicited an increase in the intracellular Ca2+ in HSY cells. N-desmethylclozapine-induced Ca2+ responses were inhibited by atropine. These results suggest that N-desmethylclozapine is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors. Moreover, our developed animal model that reflects CIS in clinical condition plays a key role as a bridge between basic and clinical research. SIGNIFICANCE STATEMENT: Clozapine-induced sialorrhea (CIS) is a severe and frequent adverse reaction, but the mechanism underlying CIS is less well understood. This paper reports that N-desmethylclozapine, a metabolite of clozapine, is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors and that oral administration of clozapine at 100 mg/kg once daily for 7 days to rat is the optimum method for establishing the new animal model reflecting the clinical scenario of CIS.

α-Defensin 5 is known to be secreted by Paneth cells in the small intestine and plays an important role in eliminating pathogenic microorganisms. It has been reported that a decrease in α-defensin 5 level in the human small intestine is a risk of inflammatory bowel disease (IBD). Furthermore, P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter superfamily, encoded by the ABCB1/MDR1 gene, plays an important role in the front line of host defense by protecting the gastrointestinal barrier from xenobiotic accumulation and may contribute to the development and persistence of IBD. Therefore, we examined the relationship between α-defensin 5 and the expression and function of P-gp using a human gastrointestinal model cell line (Caco-2). We found that MDR1 mRNA and P-gp protein level were increased in Caco-2 cells as well as α-defensin 5 secretion corresponded with the duration of cell culture. Exposure to α-defensin 5 peptide and recombinant tumor necrosis factor-α (TNF-α) significantly increased the expression and function P-gp. The mRNA levels of interleukin (IL)-8, IL-6, TNF-α, IL-1β, and IL-2 were also increased following exposure to TNF-α, similar to α-defensin 5 treatment. These results suggest that α-defensin 5 regulates P-gp expression and function by increasing TNF-α expression in Caco-2 cells.

BACKGROUND/AIM: Human monocarboxylate transporter 1 (hMCT1), hMCT2, and hMCT4 transport monocarboxylates, such as L-lactate and pyruvate, with pH dependency. They are often over-expressed in various cancer cells and mediate the energy balance and pH homeostasis. Therefore, hMCT inhibitors can potentially be used as anticancer drugs. However, isoform-selective inhibitors have not yet been well-characterized. In addition, several statins and 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors have been reported to inhibit hMCTs, but their selectivity has not yet been evaluated. In this study, we aimed to determine whether statins could inhibit hMCT1, hMCT2, and hMCT4. MATERIALS AND METHODS: We expressed hMCT1, hMCT2, and hMCT4 in a heterologous expression system of Xenopus oocytes and performed inhibitory experiments with various statins (fluvastatin, atorvastatin, simvastatin, rosuvastatin, pravastatin, and pitavastatin). As the three-dimensional structure of hMCT2 has been recently reported, docking simulations of statins and their structures were also performed to estimate the inhibition site. RESULTS: All statins inhibited the transport activities of hMCT1, hMCT2, and hMCT4. In addition, atorvastatin was found to be a potent isoform-selective inhibitor of hMCT2. Docking simulation indicated that atorvastatin could interact with a site surrounded by transmembrane (TM)-2, TM11, and intracellular helix in the TM6/7loop. Therefore, targeting this site may lead to the discovery of more potent hMCT2-selective inhibitors. CONCLUSION: Atorvastatin exerts selective inhibitory effects on hMCT2. These findings provide insights into the inhibitory mechanism of statins against hMCT1, hMCT2, and hMCT4 and may aid in the development of novel anticancer agents.