Trousseau's Syndrome With Severe Visual Loss As the Initial Symptom.

There are limited reports on patients with Trousseau syndrome, a condition characterized by hypercoagulability associated with malignant tumors, initially manifesting with reduced visual function. We present a case of a patient who experienced bilateral vision loss and was subsequently diagnosed with Trousseau's syndrome following examination and investigations. A 70-year-old man, undergoing chemotherapy for advanced pancreatic cancer, reported decreased visual acuity in both eyes. A dilated fundus examination revealed retinal pigment epithelial atrophy in the posterior pole and cotton-wool spots. Optical coherence tomography exhibited partial disruption of the ellipsoid zone in the parafoveal region, and full-field electroretinogram results were subnormal, although the macular retinal structure was preserved. Brain magnetic resonance imaging (MRI) detected occipital lobe infarction. Elevated coagulability markers, including D-dimer (5.5µg/mL), led to the diagnosis of Trousseau's syndrome. In cases where patients with malignant tumors present with profound visual loss, considering the possibility of Trousseau's syndrome and conducting assessments of brain function and coagulability is crucial for accurate diagnosis and appropriate management.

Comparison of Pregnane X Receptor Antagonists for Enhancing the Antitumor Effect of Cisplatin.

BACKGROUND: Cisplatin is a platinum compound capable of inducing apoptosis of cancer cells. However, cancer cells can become cisplatin-resistant. A recent study showed that a pregnane X receptor (PXR) antagonist, leflunomide, can enhance the antitumor activity of cisplatin and overcome such resistance. This study determined whether PXR antagonists ketoconazole and phenethyl isothiocyanate (PEITC) enhance the antitumor activity of platinum compounds and by which mechanism(s) of action. MATERIALS AND METHODS: Caspase-3 activity, intracellular platinum level, and expression of ATP-binding cassette subfamily C member 2 (ABCC2; previously named multidrug resistance-associated protein 2) were assessed in HepG2 human hepatocellular carcinoma cells exposed to carboplatin or cisplatin with and without PXR antagonist. RESULTS: In combination with platinum compounds, PEITC increased the intracellular platinum level, while ketoconazole induced higher caspase-3 activity. Additionally, PEITC suppressed ABCC2 protein expression. CONCLUSION: These results suggested that ketoconazole and PEITC enhance the antitumor activity of platinum compounds by different and complex mechanisms.

The Involvement of Pregnane X Receptor-regulated Pathways in the Antitumor Activity of Cisplatin.

BACKGROUND/AIM: Nuclear receptors regulate the expression of cellular transporters, which may be contributing factors for cisplatin (CDDP) resistance. This study aimed to clarify whether nuclear receptor ligands could be potentially used as drugs to overcome CDDP resistance. MATERIALS AND METHODS: Caspase-3 activity was measured using a fluorogenic substrate. mRNA levels were determined using real-time polymerase chain reaction. RESULTS: Pregnane X receptor (PXR) showed an expression level change dependent on caspase-3 activation by CDDP in HepG2. Rifampicin, a PXR agonist, reduced the accumulation of CDDP and suppressed growth inhibition and caspase-3 activation in HepG2 after CDDP exposure. Leflunomide, a PXR antagonist, significantly enhanced caspase-3 activation by CDDP in HepG2 and CDDP-resistant HepG2/R. CONCLUSION: These results suggest that PXR can modify the antitumor activity of CDDP, presumably through regulating the expression of transporters, which control intracellular CDDP concentration. Thus, PXR antagonists can be further investigated as potential drugs capable of overcoming CDDP resistance.

Angiographic Evaluation of Vascular Damage in Rat Liver After Administration of Epirubicin or Miriplatin.

BACKGROUND: Maintaining the function of blood vessels is important for the control of hepatocellular carcinoma (HCC) during treatment with repeated transcatheter arterial chemoembolization (TACE). This study was designed to compare the vascular damage caused by miriplatin (MPT), which has been commonly used for TACE, with the damage caused by epirubicin (EPI). MATERIALS AND METHODS: We used the portal vein of healthy rats for the administration of the drug (MPT or EPI) and/or soybean oil as vehicle. After 2 days, angiography was performed by X-ray computer tomography. RESULTS: The influence of soybean oil on blood vessel function was volume-dependent. EPI showed dose-dependent effects on angiography, and 0.5 mg EPI led to severe (grade 4) blood flow disturbance in all animals. The effect of 1 mg MPT on blood vessels was mild (grade 1) in all animals and not different from that of soybean oil alone. CONCLUSION: Less vascular damage is caused by MPT than by EPI, suggesting that MPT is a useful drug for TACE in HCC.

Cisplatin-loaded, Sialyl Lewis X-Modified Liposomes: Drug Release, Biodistribution and Antitumor Efficacy.

BACKGROUND/AIM: The aim of this study was to evaluate the characteristics of two cisplatin (CDDP)-loaded, sialyl Lewis X-modified liposomes (CDDP-SLX-LIP) with different particle size. Each had the same lipid composition and CDDP concentration, but a different drug-to-lipid ratio. MATERIALS AND METHODS: The amount of platinum in the filtrates was determined by the ICP method. The antitumor effect was assessed using rats bearing MRMT-1 tumor. RESULTS: CDDP release was faster from the larger liposome formulation (L-LIP) than from the smaller one (S-LIP). Treatment with the two CDDP-SLX-LIPs resulted in comparable antitumor effects without obvious side-effects, but treatment with CDDP solution resulted in significant weight loss and an elevated BUN. Tumor accumulation of CDDP-SLX-LIP was clearly greater than that of CDDP solution and accumulation of S-LIP was greater than that of L-LIP. CONCLUSION: CDDP-SLX-LIP exhibited antitumor effects depending on their release behavior and tumor accumulation.

Changes in the Expression of Various Transporters as Influencing Factors of Resistance to Cisplatin.

BACKGROUND: Changes in the expression of transporters have been reported as factors in resistance to cisplatin (CDDP). This study was designed to clarify whether CDDP-resistant strains isolated from a cell line had the same characteristics, and whether these characteristics could be therapeutic targets. MATERIALS AND METHODS: Intracellular platinum levels were determined by the inductively-coupled plasma method. mRNA expression levels were determined using the real-time polymerase chain reaction. RESULTS: Some CDDP-resistant HepG2 cell lines exhibited changes in the expression of copper transporter 1, multidrug resistant protein (MRP)2, and/or MRP3, resulting in decreased intracellular platinum amounts, while others showed no change in platinum accumulation. Expression of these transporters was not necessarily maintained in a constant direction within the cell population isolated from the same origin. CONCLUSION: These results suggest that the CDDP-resistant tumors caused by a decrease in intracellular platinum content consist of a heterogeneous cell population showing expression changes of several transporters.

Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells.

A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH3)2}2(µ-OH)(µ-methyl-pyrazolate)]2+ (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na+/K+-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.

Population Pharmacokinetic-Toxicodynamic Modeling and Simulation of Cisplatin-Induced Acute Renal Injury in Rats: Effect of Dosing Rate on Nephrotoxicity.

Nephrotoxicity is the major dose-limiting toxicity of cisplatin (CDDP). The aim of this study was to develop a pharmacokinetic (PK)/toxicodynamic (TD) model of CDDP-induced acute renal injury in rats and to simulate nephrotoxicity at various dosing rates. CDDP was administered to rats by a 30-s bolus or a 2-h infusion (1.0, 2.5, 5.0, and 7.5 mg/kg). Unbound CDDP concentrations in plasma and urine were determined up to 2 h after administration in the PK study, and plasma creatinine (Cr) levels were monitored for up to 7 days as an index of nephrotoxicity in the TD study. The PK was linear and was fitted with a traditional 2-compartment model. The TD was nonlinear and differed between dosing rates. The creatinine concentration profiles were fitted with a signal transduction-indirect response model. Population analysis using a nonlinear mixed-effect model was adapted to the developed PK/TD model and was well-validated. Dosing simulations from the developed population PK/TD model indicated that CDDP-induced nephrotoxicity was due to not only Cmax but also the time above the toxic concentration of CDDP. Prolongation of infusion time will not necessarily attenuate acute nephrotoxicity. This study demonstrated the potential utility of PK/TD modeling for preventing nephrotoxicity.

[Improvement of the viscosity and the intrahepatic distribution of miriplatin-lipiodol suspension].

The effects of warming or emulsification with the water-soluble contrast medium, Iomeron (IOM), on reducing the viscosity of miriplatin-lipiodol (MPT-LPD) suspension were studied. Reduction in the viscosity of MPT-LPD suspension was ob- served upon increasing the temperature. Although the O/W MPT-LPD emulsion with a low ratio of MPT-LPD to IOM reduced the viscosity, the effect was lesser than that achieved with the warming treatment. Radiographic images of the liver obtained after administration of the emulsion into the rat portal vein showed that warming resulted in improved intrahepatic distribution of the formulation, which was dependent on the reduction of viscosity. Emulsification also led to better intrahepatic distribution, but this distribution did not depend on the viscosity of the formulation. The MPT-LPD emulsion showed different distribution properties from the MPT-LPD suspension, and it was difficult to estimate the intrahepatic distribution property from the viscosity of the emulsion. Thus, we suggest that emulsification and warming of MPT-LPD are effective methods for improving the intrahepatic distribution of the MPT formulation.

[Basic studies on the lipiodolization of miriplatin in combination with CDDP].

Platinum release and initial hepatic toxicity of a formulation containing both miriplatin (MPT) and cisplatin (CDDP), prepared to improve the weak initial effect of MPT-Lipiodol (LPD) suspension, were evaluated. No difference in platinum release from CDDP was found between CDDP-LPD and MPT·CDDP-LPD, which suggested that platinum release was not affected by the viscosity of MPT-LPD. On the day following administration into rat portal vein, drugs suspended in LPD increased liver function values, and these values returned to the previous levels 3 days after administration. Both the CDDP-LPD and MPT· CDDP-LPD groups showed higher liver function values than the MPT-LPD group, and there was little difference in liver function values between the CDDP-LPD and MPT·CDDP-LPD groups. Thus, MPT·CDDP-LPD retains the characteristics of MPTLPD and CDDP-LPD without reducing the effects of either drug or enhancing their side effects.

Effects of insulin on CYP3A activity and nicardipine disposition in streptozotocin-induced diabetic rats.

OBJECTIVES: The aim of the study was to clarify the effect of insulin treatment on drug metabolism and disposition. METHODS: We investigated the mRNA expression and activity of cytochrome P450 (CYP) 3A, which is involved in the metabolism of several drugs, by using a rat model of diabetes and insulin-treated diabetes. In addition, we investigated the mRNA expression of the nuclear receptors reported to regulate the transcription of CYP3A, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). We also assessed the disposition of nicardipine, which is mainly metabolised by CYP3A, using both rat models to evaluate the influence of insulin treatment on drug disposition. KEY FINDINGS: We noted that alterations in the serum bile acid concentration in both rat groups were related to the changes in CAR mRNA expression, CYP3A mRNA expression and CYP3A activity. Furthermore, although the enhanced CYP3A activity in the diabetic rat accelerated the elimination of nicardipine, insulin administration decreased the enhanced CYP3A activity in the diabetic group and delayed the elimination of nicardipine to the same level as that in the control group. However, the steady-state volume of distribution was increased in the insulin-treated diabetic group as compared to the control and diabetic groups. We further noted that although the CYP3A activity in the diabetic group returned to the same level as in that in the non-diabetic group by insulin treatment, other values, such as the distribution volume of nicardipine, did not show a similar return. CONCLUSIONS: Based on our results, we suggest that alterations in the drug disposition in diabetes and insulin-treated diabetes should be taken into consideration in order to provide safe and effective drug therapy.

The pharmacokinetics of morphine and its glucuronide conjugate in a rat model of streptozotocin-induced diabetes and the expression of MRP2, MRP3 and UGT2B1 in the liver.

OBJECTIVES: The aim was to investigate the pharmacokinetics of morphine and its metabolite, morphine-3-glucuronide (M3G), in a rat model of streptozotocin (STZ)-induced diabetes. METHODS: Morphine (15 mg/kg) was administered intravenously, and the concentrations of morphine and M3G in the plasma, urine and bile were measured by HPLC. Changes in the expression of multidrug resistance-associated proteins (MRP2 and MRP3) and UDP-glucuronosyltransferase 2B1 (UGT2B1) mRNA in the liver were also estimated by reverse-transcriptase PCR. KEY FINDINGS: Plasma morphine concentrations were lower in the STZ-diabetic rats than controls although the elimination half-life of morphine was similar in the two groups (47.9 +/- 10.7 min and 47.2 +/- 8.6 min, respectively). The concentration of M3G in plasma was higher in STZ-diabetic than control rats, and the biliary excretion of M3G was lower in the STZ-diabetic rats (7.4 +/- 2.3% vs 13.3 +/- 2.0%). The urinary excretion of M3G was similar in the two groups (10.1 +/- 6.8% vs 10.9 +/- 4.9%). The expression of MRP3 and UGT2B1 mRNA was increased in STZ-diabetic rats, whereas expression of MRP2 mRNA was decreased. CONCLUSIONS: In STZ-diabetic rats, the distribution volume of morphine increased, the glucuronidation rate and M3G transportation into the blood were enhanced, and the excretion of M3G was decreased, leading to an increase in the plasma M3G concentration.

The disposition of pravastatin in a rat model of streptozotocin-induced diabetes and organic anion transporting polypeptide 2 and multidrug resistance-associated protein 2 expression in the liver.

The combination of diabetes and hyperlipidemia promotes the development of atherosclerosis. Therefore, it is important for diabetic patients to control blood fat. 3-Hydroxy-3-methylglutaryl enzyme A (HMG-CoA) reductase inhibitors (statins), like pravastatin, are frequently administered to diabetic patients for this purpose. Although the alterations of metabolic enzymes and transporters in the diabetic liver maybe change the disposition of pravastatin, the effect has not been fully investigated. In the present study, we investigated the disposition of pravastatin and the mRNA expression of transporters in the liver. Pravastatin (5 mg.kg(-1) body weight) was administered intravenously to diabetic rats, and the pravastatin concentrations in the plasma, urine, and bile were measured by high-performance liquid chromatography. Changes in the mRNA expressions of multidrug resistance-associated protein 2 (MRP2) and organic anion transporting polypeptide 2 (OATP2) in the liver were also estimated using reverse transcriptase-polymerase chain reaction (RT-PCR). We found that the plasma pravastatin concentration was lower in the diabetic rat because the transportation of pravastatin into hepatocytes was promoted along with increased expression of OATP2. The biliary excretion ratio of pravastatin was significantly lower in the diabetic rat because the pravastatin transportation into bile was reduced along with the decreased expression of MRP2. To clarify these phenomena, the analysis of mRNA expression using real-time PCR and the measurement of the amount and the activity of proteins are necessary in future study.

Altered expression of nuclear receptors affects the expression of metabolic enzymes and transporters in a rat model of cholestasis.

Hepatic metabolism is altered in some clinical conditions owing to the changes in the expression of metabolic enzymes and transporters. Therefore, we think that investigating the altered expression of metabolic enzymes and transporters is of particular significance to studies on drug disposition in some clinical conditions. We also believe that a simultaneous in vivo investigation of all factors affecting nuclear receptors and regulated genes is important to understand the relationship between nuclear receptors and their target genes. In this study, we induced cholestasis in rats by bile duct ligation (BDL), and investigated the changes in the mRNA expression of metabolic enzymes, transporters, and nuclear receptors and the protein levels of nuclear receptors in the nucleus by reverse transcriptase-polymerase chain reaction and Western blotting. In the liver of the rats subjected to BDL, the mRNA expression levels of cytochrome P450, conjugation enzymes, and transporters were concomitantly altered. The altered mRNA and protein levels of constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor alpha (PPARalpha) in the nucleus were consistent with the changes in the plasma concentrations of total and conjugated bilirubin and fatty acid, respectively. The mRNA expression of CAR and PPARalpha was linearly associated with the expression of the corresponding target genes. These results suggested that the increase in the levels of bilirubin and fatty acid on the BDL groups altered the mRNA and protein levels of CAR and PPARalpha, respectively in the nucleus, and this in turn altered the mRNA expression of metabolic enzymes and transporters as a hepatoprotective mechanism.

Altered expression of MRP2, MRP3 and UGT2B1 in the liver affects the disposition of morphine and its glucuronide conjugate in a rat model of cholestasis.

OBJECTIVES: The aim was to investigate the disposition of morphine and morphine-3-glucuronide (M3G) in a rat model of cholestasis induced by bile duct ligation (BDL). METHODS: Morphine (15 mg/kg) was administered intravenously, and morphine and M3G concentrations in the plasma and urine measured by HPLC. Changes in the mRNA expression of multidrug resistance-associated protein (MRP)2, MRP3 and UDP-glucuronosyltransferase (UGT)2B1 in the liver were estimated using RT-PCR. KEY FINDINGS: Although the plasma morphine concentrations declined exponentially, the elimination was delayed 3 and 5 days after BDL. Plasma M3G concentrations on day 1 after BDL were similar to those in the untreated control group, but were increased 3 and 5 days after BDL. Expression of MRP3 and UGT2B1 mRNA increased after BDL. The urinary excretion of M3G was increased significantly after BDL. CONCLUSIONS: Enhanced glucuronidation of morphine and transportation of M3G into the blood increased the plasma M3G concentration in the BDL groups. However, M3G disposition 1 day after BDL was similar to that in the untreated control group because urinary excretion of M3G increased.

Effect of absorption rate on pharmacokinetics of ibuprofen in relation to chiral inversion in humans.

The effect of absorption rate on the pharmacokinetics of ibuprofen enantiomers was investigated in 12 healthy Han Chinese male volunteers following oral administration of immediate-release (IR) and sustained-release (SR) preparations containing racemic ibuprofen (rac-ibuprofen). The area under the curve of the plasma concentration-time curve (AUC; (mean+/-s.d.) values for rac-ibuprofen were 192.90+/-43.47 for the SR preparation and 195.90+/-31.69 microg h mL(-1) for the IR preparation. AUC values for the enantiomers after administration of the SR formulation were 55.38+/-17.79 and 92.51+/-30.68 microg h mL(-1) for R- and S-ibuprofen, respectively, and were 65.94+/-20.06 and 100.81+/-32.28 microg h mL(-1) for R- and S-ibuprofen after administration of the IR preparation. These values did not differ significantly. C(max) values were significantly decreased with the SR preparation: 25.11+/-5.71, 12.24+/-3.79 and 12.38+/-3.55 microg h mL(-1) for rac-, R-, and S-ibuprofen, respectively, after administration of the SR preparation, vs 46.21+/-8.20, 20.82+/-5.90 and 23.46+/-7.30 microg h mL(-1) for rac-, R-, and S-ibuprofen, respectively, after administration of the IR preparation. Mean residence time was significantly increased: 7.01+/-1.29, 5.52+/-1.25 and 7.04+/-1.30 h for rac-, R-, and S-ibuprofen, respectively, after administration of the SR preparation vs 4.34+/-0.89, 3.43+/-0.64 and 4.51+/-0.79 h for rac-, R-, and S-ibuprofen, respectively, after administration of the IR preparation. AUC values for S-ibuprofen were significantly larger than those for R-ibuprofen in both preparations, indicating unidirectional chiral inversion. The S/R ratio of serum concentrations of enantiomers was 1.78-fold higher at 6 h after administration of the SR preparation compared with the IR preparation (P<0.01). These results indicate that ibuprofen undergoes pre-systemic chiral inversion in parallel with a systemic process and that the clinical effects of rac-ibuprofen in humans depend on the absorption rate.

Role of macrophage migration inhibitory factor in corneal neovascularization.

PURPOSE: To determine the role of macrophage migration inhibitory factor (MIF) in inflammatory corneal neovascularization. METHODS: Corneal neovascularization was induced by suturing 10-0 nylon 1 mm away from limbal vessel or limbal scraping after 0.15 M NaOH application in BALB/c mice. MIF expression was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemistry. To investigate the function of MIF in inflammatory corneal neovascularization, the neovascularized area and number of infiltrating F4/80-positive cells (monocytes/macrophages) were compared between wild-type mice and homozygous MIF-deficient mice. RESULTS: MIF mRNA and protein markedly increased in the neovascularized corneas compared with normal corneas by RT-PCR and Western blot analysis, respectively. MIF expression was upregulated immunohistochemically, not only in the corneal epithelium but also in the stromal infiltrating cells of neovascularized corneas. Neovascularized area in corneas of MIF(-/-) mice was significantly small compared with that in wild-type mice on day 7 after corneal suture and on day 14 after limbal scrape, and MIF(-/-) cornea had approximately 30% less neovascularized area than did wild-type cornea in both models. Neovascularized corneas in MIF-deficient mice had significantly fewer monocytes/macrophages than those in wild-type control mice. CONCLUSIONS: These findings indicate that MIF, abundantly expressed in neovascularized corneas, has an angiogenic role in inflammatory corneal neovascularization and may be a therapeutic target for suppression of corneal neovascularization.

Novel mechanisms of platinum drug resistance identified in cells selected for resistance to JM118 the active metabolite of satraplatin.

PURPOSE: The goal of this study was to identify molecular determinants of sensitivity and resistance to JM118, the active metabolite of satraplatin, an orally bioavailable cisplatin analog that has activity in prostate cancer. EXPERIMENTAL DESIGN: Human ovarian carcinoma 2008/JM118 cells were derived from parental 2008 cells by repeated exposure to JM118; the revertant 2008/JM118/REV subline was isolated from the 2008/JM118 cells by growth in the absence of drug. Drug sensitivity was determined by clonogenic assay and Pt levels were measured by ICP-MS. RESULTS: Eight sequential rounds of selection yielded the 2008/JM118 subline that was 4.9-fold resistant to JM118 and cross-resistant at varying levels to satraplatin, cisplatin, carboplatin, and oxaliplatin. Cross-resistance to the other Pt drugs was lost as resistance to JM118 waned. The same parental 2008 cells selected for resistance to cisplatin were partially cross-resistant to JM118. The 2008/JM118 cells accumulated significantly more Pt than the 2008 cells when exposed to low concentrations of either JM118 or cisplatin indicating a detoxification process that involves intracellular sequestration. In contrast, 2008 cells selected for cisplatin resistance accumulated less cisplatin and less JM118 reflecting a mechanism involving reduced accumulation. The 2008 and 2008/JM118 cells did not differ in their uptake or efflux of 64Cu, expression of Cu efflux transporters ATP7A or ATP7B or their glutathione content. The 2008/JM118 cells exhibited 3.0-7.7-fold hypersensitivity to docetaxel, paclitaxel and doxorubicin. Expression profiling identified 4 genes that were significantly up-regulated and 19 that were down-regulated in the 2008/JM118 cells at a false discovery rate of 1 gene. CONCLUSIONS: While the cellular defense mechanisms that protect cells against JM118 also mediate resistance to the other Pt drugs, these mechanisms are quite different from those commonly found in cells selected for resistance to cisplatin. JM118-resistant cells accumulate more rather than less Pt and rely on an intracellular detoxification mechanism different from that involved in cisplatin resistance. This is consistent with clinical evidence suggesting that satraplatin has activity in diseases in which cisplatin does not. In this model, JM118 resistance is associated with substantial collateral hypersensitivity to docetaxel, paclitaxel, and doxorubicin.

Role of Na+, K+-ATPase alpha1 subunit in the intracellular accumulation of cisplatin.

The present study was undertaken to identify what regulates intracellular cisplatin (CDDP) accumulation and what changes in membrane fraction of CDDDP-resistant cell line. The CDDP-resistant rat hepatoma cell line, H4-II-E/CDDP, shows a significant decrease in intracellular platinum accumulation compared with parental H4-II-E cells, although there was no difference in the efflux of CDDP between these two cell lines. In this study, we examined the contribution of functional change in active transport to the CDDP resistance of H4-II-E/CDDP cells. Compared with the resistant cells, platinum accumulation in the parental cells was clearly decreased by low temperature or ATP depletion. In addition, the Na+, K+-ATPase inhibitor ouabain and the K+ channel inhibitor tetraethylammonium decreased platinum accumulation in parental cells but did not change the accumulation in resistant cells. Amphotericin B, an antifungal agent, increased the intracellular platinum accumulation in resistant cells to the same level as in parent cells. Western blot analysis demonstrated that the Na+, K+-ATPase alpha1 subunit was reduced in resistant cells compared with the parental cells, although there was no difference in the expression of the beta1 subunit between the two cell lines. Furthermore, the Na+, K+-ATPase alpha1 subunit of H4-II-E was decreased following a 24-h exposure to CDDP. These results suggest that Na+, K+-ATPase-dependent active transport of CDDP does not occur in resistant cells, and, furthermore, our findings provide the first evidence that the Na+, K+-ATPase alpha1 subunit plays an important role in the transport of CDDP.

Continuous exposure to low-dose cisplatin and apoptosis.

A high concentration of cisplatin induces apoptosis in many tumor cell lines. Whether cisplatin induces apoptosis even in a controlled release formulation has not been determined. We therefore studied the relationship between the dosing regimen of cisplatin and the induction of apoptosis in rat hepatoma AH-109A cells. A colorimetric assay was used to quantify cell proliferation and viability, and caspase activity was determined using an exogenous fluorogenic peptide substrate. When delivered as a single dose, cisplatin caused a dose-dependent inhibition of AH-109A growth and enhancement of caspase-3 activity. Also, DNA laddering was detected in cells that had elevated caspase-3 activity. However, caspase-3 activity was low and DNA laddering and a sub-G1 population were not detected when cells were treated with a combination of cisplatin and the caspase inhibitor Z-VAD-FMK. These results suggest that cisplatin is cytotoxic in AH-109A cells because it induces apoptosis. We next examined intermittent exposure to cisplatin to estimate the effects of continuous exposure by a controlled release formulation. Cisplatin was divided into equal parts and was added intermittently into the medium resulting in the same final concentration as the single dose. The individual additions alone were not cytotoxic, but all of the doses together had a similar cytotoxic effect as a single exposure of cisplatin. The intermittent exposure resulted caspase-3 activity even higher than a single dose. These findings indicate that cisplatin induces apoptosis in AH-109A cells when delivered continuously even at the concentration that alone have no activity.