Im-Sook Song

Transcription Factor Sp1 Plays an Important Role in the Regulation of Copper Homeostasis in Mammalian Cells

Copper is an essential metal nutrient, yet copper overload is toxic. Here, we report that human copper transporter (hCtr) 1 plays an important role in the maintenance of copper homeostasis by demonstrating that expression of hCtr1 mRNA was up-regulated under copper-depleted conditions and down-regulated under copper-replete conditions. Overexpression of full-length hCtr1 by transfection with a recombinant hCtr1 cDNA clone reduced endogenous hCtr1 mRNA levels, whereas overexpression of N terminus-deleted hCtr1 did not change endogenous hCtr1 mRNA levels, suggesting that increased functional hCtr1 transporter, which leads to increased intracellular copper content, down-regulates the endogenous hCtr1 mRNA. A luciferase assay using reporter constructs containing the hCtr1 promoter sequences revealed that three Sp1 binding sites are involved in the basal and copper concentration-dependent regulation of hCtr1 expression. Modulation of Sp1 levels affected the expression of hCtr1. We further demonstrated that the zinc-finger domain of Sp1 functions as a sensor of copper that regulates hCtr1 up and down in response to copper concentration variations. Our results demonstrate that mammalian copper homeostasis is maintained at the hCtr1 mRNA level, which is regulated by the Sp1 transcription factor.

Elevated glutathione levels confer cellular sensitization to cisplatin toxicity by up-regulation of copper transporter hCtr1

Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDP-resistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to up-regulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinum-based antitumor agents.

Galangin Suppresses the Proliferation of β-Catenin Response Transcription-Positive Cancer Cells by Promoting Adenomatous Polyposis Coli/Axin/Glycogen Synthase Kinase-3β-Independent β-Catenin Degradation

Galangin is a naturally occurring bioflavonoid with anticancer activity against certain human cancers, yet little is known about its mechanism of action. Here, we used a chemical biology approach to reveal that galangin suppresses β-catenin response transcription (CRT), which is aberrantly up-regulated in colorectal and liver cancers, by promoting the degradation of intracellular β-catenin. Inhibition of glycogen synthase kinase-3β (GSK-3β) activity or mutation of the GSK-3β-targeted sequence from β-catenin was unable to abrogate the galangin-mediated degradation of β-catenin. In addition, galangin down-regulated the intracellular β-catenin levels in cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or Axin, which are components of the β-catenin destruction complex. Galangin repressed the expression of β-catenin/T-cell factor-dependent genes, such as cyclin D1 and c-myc, and thus inhibited the proliferation of CRT-positive cancer cells. Structure-activity data indicated that the major structural requirements for galangin-mediated β-catenin degradation are hydroxyl groups at positions 3, 5, and 7. Our findings suggest that galangin exerts its anticancer activity by promoting APC/Axin/GSK-3β-independent proteasomal degradation of β-catenin.

Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide

We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1), OAT3, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 1 (OCT1), OCT2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3) and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3). The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (Km) =41.5 μM, maximum uptake rate (Vmax) =46.2 pmol/minute, and intrinsic clearance (CLint) =1.11 μL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CLint values of 0.035 and 0.034 μL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 μM, respectively. However, catalposide did not significantly inhibit the transport activities of OCT1, OCT2, OAT1, P-gp, or BCRP. In conclusion, OAT3, OATP1B1, and OATP1B3 are major transporters that may regulate the pharmacokinetic properties and may cause herb–drug interactions of catalposide, although their clinical relevance awaits further evaluation.

Pharmacokinetics of Jaspine B and Enhancement of Intestinal Absorption of Jaspine B in the Presence of Bile Acid in Rats

We aimed to investigate the pharmacokinetics and the underlying mechanisms of the intestinal absorption, distribution, metabolism, and excretion of Jaspine B in rats. The oral bioavailability of Jaspine B was 6.2%, but it decreased to 1.6% in bile-depleted rats and increased to 41.2% (normal) and 23.5% (bile-depleted) with taurocholate supplementation (60 mg/kg). Consistent with the increased absorption in the presence of bile salts, rat intestinal permeability of Jaspine B also increased in the presence of 10 mM taurocholate or 20% bile. Further studies demonstrated that the enhanced intestinal permeability with bile salts was due to increased lipophilicity and decreased membrane integrity. Jaspine B was designated as a highly tissue-distributed compound, because it showed large tissue to plasma ratios in the brain, kidney, heart, and spleen. Moreover, the recovery of Jaspine B from the feces and urine after an intravenous administration was about 6.3%, suggesting a substantial metabolism of Jaspine B. Consistent with this observation, 80% of the administered Jaspine B was degraded after 1 h incubation with rat liver microsomes. In conclusion, the facilitated intestinal permeability in the presence of bile salts could significantly increase the bioavailability of Jaspine B and could lead to the development of oral formulations of Jaspine B with bile salts. Moreover, the highly distributed features of Jaspine B in the brain, kidney, heart, and spleen should be carefully considered in the therapeutic effect and toxicity of this compound.

Preparation and Characterization of Quercetin-Loaded Solid Dispersion by Solvent Evaporation and Freeze-Drying Method

We prepared solid dispersion formulations of quercetin to enhance its solubility and dissolution rate. Various quercetinloaded solid dispersion were tested with quercetin, poloxamer 407, and carrier such as hydroxypropyl methyl cellulose (HPMC), polyethylene glycol 8000 (PEG 8000), and polyvinylpyrrolidone K40 (PVP K40) using solvent evaporation and freeze drying methods in terms of both the aqueous solubility and the dissolution rates of quercetin. The solubility of quercetin as its solid dispersion formulations was markedly improved compared with that of quercetin powder. Especially, highest solubility of quercetin was observed when HPMC was used as a carrier. The cumulative dissolution of quercetin within 360 min from solid dispersion composed of quercetin, poloxamer 407, and HPMC was 8.8-fold higher than the dissolution of pure quercetin. The results of powder Xray diffraction (XRD) and scanning electron microscope (SEM) indicated that quercetin transformed from a crystalline to an amorphous form through the solid dispersion formulation process. These results suggest that the solid dispersion formulation of quercetin with poloxamer 407 and HPMC could be a promising option for enhancing the solubility and dissolution rate of quercetin.

Differential Cytotoxic Effects of Jaspine B in Various Cancer Cells

Jaspine B is an anhydrophytosphingosine that is isolated from a marine sponge. Because of its structural similarity to sphingosine, it shows anti-cancer effects in human carcinomas. Therefore, this study aims to investigate its anti-proliferative effect on various cancer cells and to correlate its association with the intracellular accumulation of Jaspine B in relevant cancer cells. The anti-proliferative effect of Jaspine B in various cancer cells was determined by a cell viability test, and the intracellular concentration of Jaspine B in relevant cancer cells was determined using mass spectrometry coupled with liquid chromatography. The correlation coefficient and p value between the cytotoxicity and the cell accumulation of Jaspine B were determined using SPSS 16.1. The cytotoxicity of Jaspine B varied depending on the type of cancer cell when compared the EC 50 values of Jaspine B. Breast and melanoma cancer cells were susceptible to Jaspine B, whereas renal carcinoma cells were resistant. The intracellular concentrations of Jaspine B had a reciprocal correlation with the EC 50 values in the same cells (r = 0.838). The results suggested that the anti-proliferative effect of Jaspine B was associated with the cellular accumulation of this compound. However, Jaspine B was not a substrate for P-glycoprotein and breast cancer resistance protein, as major efflux pumps caused multidrug resistance. The maintenance of a high intracellular concentration is crucial for the cytotoxic effect of Jaspine B; however, efflux pumps may not be a controlling factor for Jaspine B-related resistance in cancer cells.

Effect of Red Ginseng Extract on the Pharmacokinetics and Efficacy of Metformin in Streptozotocin-Induced Diabetic Rats

The purpose of this study was to investigate the effect of red ginseng extract on the pharmacokinetics (PK) and efficacy of metformin in streptozotocin-induced diabetic rats. The diabetes mellitus rat model was established by intraperitoneally administering multiple doses of streptozotocin (30 mg/kg, twice on day 1 and 8), and diabetic rats received metformin 50 mg/kg with or without single or multiple administration of Korean red ginseng extract (RGE, 2 g/kg/day, once or for 1 week). RGE administration did not affect the plasma concentration and renal excretion of metformin. Further, diabetic rats were administered metformin (50 mg/kg) and RGE (2 g/kg) alone or concomitantly for 5 weeks, and both regimens decreased the fasting blood glucose and glycated hemoglobin (Hb-A1c) levels. Furthermore, fasting blood glucose levels were reduced by metformin or RGE administered alone but recovered to the control level following co-administration, suggesting that the effect was additive. However, triglyceride and free fatty acid levels were not different with metformin and RGE treatment alone or in combination. Biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol levels were not different among the three treatment groups. In conclusion, RGE and metformin showed an additive effect in glycemic control. However, the co-administration of RGE and metformin did not cause PK interactions or affect biochemical parameters including the free fatty acid, triglyceride, AST, ALT, or cholesterol levels.

Pharmacokinetic Drug-Drug Interaction and Responsible Mechanism between Memantine and Cimetidine

A sensitive and simple chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to evaluate memantine in rat plasma. Memantine and propranolol (internal standard) in rat plasma was extracted using a methanol precipitation method. The standard curve value was 0.2–1000 ng/mL and selectivity, linearity, inter-day and intra-day accuracy and precision were within acceptance criteria. Using this validated method, drug-drug interactions between memantine and cimetidine was measured following co-administration of memantine and cimetidine intravenously and orally. Plasma exposure of memantine was increased by 1.6- and 3.0-fold by co-medication with cimetidine intravenously and orally, respectively. It suggested that the drug interaction occurred during the gut absorption process, which was consistent with the results showing that the intestinal permeability of memantine in the presence of cimetidine was 3.2-fold greater than that of memantine alone. Inhibition of cimetidine on hepatic elimination of memantine rather than renal excretion was also attributed to the drug-drug interaction between memantine and cimetidine, which explained the decreased clearance of memantine by co-medication with cimetidine. In conclusion, the newly developed simple and sensitive LC-MS/MS analytical method was applied to investigate the pharmacokinetic drug-drug interactions of memantine. Plasma exposure of memantine by co-administration with cimetidine was increased because of its enhanced intestinal permeability and the decreased metabolic activity of memantine.

Tolerability and pharmacokinetics of ginsenosides Rb1, Rb2, Rc, Rd, and compound K after single or multiple administration of Red ginseng extract in human subjects

Background We investigated the tolerability and pharmacokinetic properties of various ginsenosides, including Rb1, Rb2, Rc, Rd, and compound K, after single or multiple administrations of red ginseng extract in human beings. Methods Red ginseng extract (dried ginseng > 60%) was administered once and repeatedly for 15 days to 15 healthy Korean people. After single and repeated administration of red ginsengextract, blood sample collection, measurement of blood pressure and body temperature, and routine laboratory test were conducted over 48-h test periods. Results Repeated administration of high-dose red ginseng for 15 days was well tolerated and did not produce significant changes in body temperature or blood pressure. The plasma concentrations of Rb1, Rb2, and Rc were stable and showed similar area under the plasma concentration-time curve (AUC) values after 15 days of repeated administration. Their AUC values after repeated administration of red ginseng extract for 15 days accumulated 4.5- to 6.7-fold compared with single-dose AUC. However, the plasma concentrations of Rd and compound K showed large interindividual variations but correlated well between AUC of Rd and compound K. Compound K did not accumulate after 15 days of repeated administration of red ginseng extract. Conclusion A good correlation between the AUC values of Rd and compound K might be the result of intestinal biotransformation of Rb1, Rb2, and Rc to Rd and subsequently to compound K, rather than the intestinal permeability of these ginsenosides. A strategy to increase biotransformation or reduce metabolic intersubject variability may increase the plasma concentrations of Rd and compound K.