Impact of bodyweight-based starting doses on the safety and efficacy of lenvatinib in primarily Japanese patients with hepatocellular carcinoma.

AIM: The phase III REFLECT study utilized bodyweight-based lenvatinib dosing in patients with unresectable hepatocellular carcinoma, based on results of the phase II Study 202. This post hoc analysis compared efficacy and safety in patients with lower and higher bodyweights. METHODS: This comparison included patients from Study 202 (Japanese, n = 43; Korean, n = 3) and Japanese patients from REFLECT (n = 81) who received lenvatinib. In Study 202, all patients received a starting dose of lenvatinib 12 mg/day; in REFLECT, patients received starting doses based on bodyweight (patients <60 kg, 8 mg/day; ≥60 kg, 12 mg/day). Safety and efficacy were assessed in both studies according to bodyweight. RESULTS: In Study 202, treatment-related, treatment-emergent adverse events (TEAEs) led to dose reductions in 80.8% and 55.0% of patients in the lower and higher bodyweight groups, respectively. In REFLECT, treatment-related TEAEs led to dose reductions in 52.5% and 70.7% of patients in the 8 and 12 mg groups, respectively. In Study 202, median overall survival (OS) was 16.2 months (95% confidence interval [CI], 9.8-25.1) and 21.3 months (95% CI, 10.1-not estimable) in the lower and higher bodyweight groups, respectively. In REFLECT, median OS was 15.8 months (95% CI, 10.4-27.6) and 18.2 months (95% CI, 11.3-26.9) in the 8 and 12 mg groups, respectively. CONCLUSIONS: Comparison between patients in Study 202 and REFLECT demonstrates efficacy was maintained with improved safety in patients with lower bodyweights who received lenvatinib 8 mg/day in REFLECT versus patients who received lenvatinib 12 mg/day in Study 202.

Study of transmembrane ion transport under tonicity imbalance using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model.

Transmembrane ion transport under tonicity imbalance has been investigated using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model, by considering the cell diameterd[m] and the initial intracellular ion concentrationcin[mM] as a function of tonicity expressed by sucrose concentrationcs[mM]. The transmembrane ion transport is influenced by extracellular tonicity conditions, leading to a facilitation/inhibition of ion passage through the cell membrane. The transmembrane transport coefficientP[m s-1], which represents the ability of transmembrane ion transport, is calculated by the extracellular ion concentrations obtained by improved ion transport model and LF-EIS measurement.Pis calculated as 4.11 × 10-6and 3.44 × 10-6m s-1atcsof 10 and 30 mM representing hypotonic condition, 2.44 × 10-6m s-1atcsof 50 mM representing isotonic condition, and 3.68 × 10-6, 5.16 × 10-6, 9.51 × 10-6, and 14.89 × 10-6m s-1atcsof 75, 100, 125 and 150 mM representing hypertonic condition. The LF-EIS results indicate that the transmembrane ion transport is promoted under hypertonic and hypotonic conditions compared to isotonic condition. To verify the LF-EIS results, fluorescence intensityF[-] of extracellular potassium ions is observed to obtain the temporal distribution of average potassium ion concentration within the region of 3.6µm from cell membrane interfacecROI[mM]. The slopes of ∆cROI/cROI1to timetare 0.0003, 0.0002, and 0.0006 under hypotonic, isotonic, and hypertonic conditions, wherecROI1denotes initialcROI, which shows the same tendency with LF-EIS result that is verified by the potassium ion fluorescence observation.

Efficacy of botulinum toxin type B (rimabotulinumtoxinB) in patients with cervical dystonia previously treated with botulinum toxin type A: A post-marketing observational study in Japan.

To date, efficacy data on botulinum toxin type B (rimabotulinumtoxinB) in patients with cervical dystonia (CD) previously treated with botulinum toxin type A in a large population are lacking; thus, we aimed to evaluate type B efficacy in this patient population. In a post-marketing observational cohort study, 150 patients previously treated with botulinum toxin type A were enrolled, of whom 138 were followed up for 1 year after the initial type B injection. Final observation data were available for 122 patients. Efficacy was evaluated using the Toronto Western Spasmodic Torticollis Rating Scale. Total score improved from 39.9 at baseline to 34.3 at 4 weeks after the first injection, and pain score improved from 8.9 to 7.9. Improvements were maintained through six further injections in two subpopulations: patients who showed resistance to botulinum toxin type A and patients who were not type A resistant but switched to type B. For a number of patients, even low doses (<5000 units) of botulinum toxin type B demonstrated efficacy. These findings support the efficacy of botulinum toxin type B in clinical settings for the management of CD symptoms, including pain, even at low doses, regardless of the patient's botulinum toxin type A resistance status.

High absolute lymphocyte counts are associated with longer overall survival in patients with metastatic breast cancer treated with eribulin-but not with treatment of physician's choice-in the EMBRACE study.

BACKGROUND: Eribulin, a nontaxane synthetic inhibitor of microtubule dynamics, is widely used to manage locally advanced or metastatic breast cancer (MBC). Eribulin has demonstrated immunomodulatory activity on the tumour microenvironment. Baseline neutrophil-to-lymphocyte ratio (NLR), a marker of immune status, may predict progression-free survival in eribulin treatment. This post hoc analysis assessed predictors for overall survival (OS). METHODS: The phase 3 open-label study (EMBRACE) of eribulin versus treatment of physician's choice (TPC) in patients with MBC provided source data. Baseline absolute lymphocyte counts (ALCs) and NLR were evaluable in 751 and 713 patients, respectively. RESULTS: Eribulin prolonged OS versus TPC in patients with baseline ALC ≥ 1500/µl (hazard ratio [HR] 0.586; 95% confidence interval [CI] 0.437-0.784; P < 0.001). There was no significant difference by treatment for ALC < 1500/µl (HR 1.002; 95% CI 0.800-1.253; P = 0.989). Univariate and multivariate analyses were performed and identified baseline ALC as a potential predictor of OS in eribulin-treated patients. Interaction analysis of OS supported 1500/µl as a potentially differential cutoff value. NLR at a cutoff value of 3 was associated with prolonged OS (eribulin group). However, similar results were also observed in the TPC group, without apparent interaction effect, suggesting that NLR may be a general prognostic marker rather than a specific predictor of OS for eribulin. DISCUSSION: This hypothesis-generating study speculates that baseline ALC may be an independent predictor for longer OS in eribulin-treated MBC patients and could be clinically impactful because it can be evaluated without the need for additional invasive procedures. TRIAL REGISTRATION: www.ClinicalTrials.gov code: NCT00388726.

A Facile Assay of Epithelial-mesenchymal Transition Based on Cooperativity Quantification of Cellular Autonomous Motions.

Epithelial-mesenchymal transition (EMT), a qualitative change in cell migration behavior during cancer invasion and metastasis, is becoming a new target for anticancer drugs. Therefore, it is crucial to develop in vitro assays for the evaluation of the abilities of drug candidates to control EMT progression. We herein report on a method for the quantification of the EMT based on particle image velocimetry and correlation functions. The exponential fitting of the correlation curve gives an index (λ), which represents transforming growth factor (TGF)-ß1-induced EMT progression and its suppression by inhibitors. Moreover, real-time monitoring of the λ value illustrates a time-dependent EMT progressing process, which occurs earlier than the bio-chemical changes in an EMT marker protein expression. The results demonstrate the usefulness of the present method for kinetic studies of EMT progression as well as EMT inhibitor screening.

Numerical Study of Particle-Fluid Flow Under AC Electrokinetics in Electrode-Multilayered Microfluidic Device.

A particle-fluid flow under alternating current (ac) electrokinetics was numerically simulated to investigate the three-dimensional (3-D) particle motion in a complex electric field of a high conductivity medium generated by an electrode-multilayered microfluidic device. The simulation model coupling thermal-fluid-electrical and dispersed particle problems incorporates three ac electrokinetics (ACEK) phenomena, namely, the ac electrothermal effect (ACET), thermal buoyancy (TB), and dielectrophoresis (DEP). The electrode-multilayered microfluidic device was fabricated with 40 electrodes exposed at the flow channel sidewalls in five cross sections. The governing equations of the simulation model are solved by the Eulerian-Lagrangian method with finite volume discretization. Fluid flow simulations in three cases with or without consideration of ACET and TB are performed to clarify the contributions of these phenomena. The fluid flow is found to be composed of short-range vortices due to ACET and long-range circulation due to TB based on the features of the electrode-multilayered microfluidic device. The 3-D particle trajectory influenced by the fluid flow is compared with four values of the real part of the Clausius-Mossotti (CM) factor to evaluate the DEP phenomenon. The simulation model is validated by experiments using a cell suspension. The pattern of cell trajectories in the upper part of the flow channel measured by particle tracking velocimetry agrees with the simulated pattern. By comparison of the simulation and experiment, it is found that the cells moving straight away from the electrode on the focal plane are decelerated within the region of 60 µm from the electrode by positive-DEP with [Formula: see text]. Furthermore, the 3-D DEP-effective region and the ACET and TB dominant regions for the cells are predicted by evaluating the particle-fluid relative velocity due to DEP force with [Formula: see text]. Consequently, the flow mechanism and dominant region of each ACEK phenomenon in the device are clarified from the 3-D simulation validated by the experiments.

T-type Calcium Channels Determine the Vulnerability of Dopaminergic Neurons to Mitochondrial Stress in Familial Parkinson Disease.

Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.

Mib1 contributes to persistent directional cell migration by regulating the Ctnnd1-Rac1 pathway.

Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiquitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1ta52b mutant showed increased random migration and loss of directional F-actin-based protrusion formation. Knockdown of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1ta52b mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1-Ctnnd1-Rac1 pathway.

Distinct Motion of GFP-Tagged Histone Expressing Cells Under AC Electrokinetics in Electrode-Multilayered Microfluidic Device.

The distinct motion of GFP-tagged histone expressing cells (Histone-GFP type cells) has been investigated under ac electrokinetics in an electrode-multilayered microfluidic device as compared with Wild type cells and GFP type cells in terms of different intracellular components. The Histone-GFP type cells were modified by the transfection of green fluorescent protein-fused histone from the human lung fibroblast cell line. The velocity of the Histone-GFP type cells obtained by particle tracking velocimetry technique is faster than Wild type cells by 24.9% and GFP type cells by 57.1%. This phenomenon is caused by the more amount of proteins in the intracellular of single Histone-GFP type cell than that of the Wild type and GFP type cells. The more amount of proteins in the Histone-GFP type cells corresponds to a lower electric permittivity ϵc of the cells, which generates a lower dielectrophoretic force exerting on the cells. The velocity of Histone-GFP type cells is well agreed with Eulerian-Lagrangian two-phase flow simulation by 4.2% mean error, which proves that the fluid motion driven by thermal buoyancy and electrothermal force dominates the direction of cells motion, while the distinct motion of Histone-GFP type cells is caused by dielectrophoretic force. The fluid motion does not generate a distinct drag motion for Histone-GFP type cells because the Histone-GFP type cells have the same size to the Wild type and GFP type cells. These results clarified the mechanism of cells motion in terms of intracellular components, which helps to improve the cell manipulation efficiency with electrokinetics.

Eribulin shows high concentration and long retention in xenograft tumor tissues.

PURPOSE: Eribulin, a synthetic analog of the natural product halichondrin B, is a microtubule dynamics inhibitor. In this study, we report the pharmacokinetic profiles of eribulin in mice, rats, and dogs following intravenous administrations with optimized and validated bio-analytical methods. METHODS: Eribulin was administered at 0.5 and 2 mg/kg in mice, 0.5 and 1 mg/kg in rats, and 0.08 mg/kg in dogs. Tumor and brain penetration of eribulin was also evaluated in LOX human melanoma xenograft models. Concentrations in plasma, tumor, and brain were measured by the LC-MS/MS method. RESULTS: The profiles of eribulin were characterized by extensive distribution, moderate clearance, and slow elimination in the three species. The pharmacokinetics are linear in mice and rats. In xenograft mice, the penetration into the brain was low, as expected, since eribulin is a P-glycoprotein substrate. In contrast to disposition in brain, the exposure of eribulin was approximately 20-30 times higher in tumor than that in plasma and half-lives were 17.8-35.9 h after both single and multiple dose regimens. CONCLUSIONS: Eribulin was distributed rapidly and eliminated slowly in mice, rats, and dogs. The exposure of eribulin was approximately 20-30 times higher in tumor than in plasma in xenograft mice. These results might be caused by eribulin's mechanism of action including increased perfusion in tumor by vascular remodeling effect.

Dynamic control of cell adhesion on a stiffness-tunable substrate for analyzing the mechanobiology of collective cell migration.

A method was developed for photocontrolling cell adhesion on a gel substrate with defined mechanical properties. Precise patterning of geometrically controlled cell clusters and their migration induction became possible by spatiotemporally controlled photo-irradiation of the substrate. The clusters exhibited unique collective motion that depended on substrate stiffness and cluster geometry.

Effect of elasticity on wall shear stress inside cerebral aneurysm at anterior cerebral artery.

BACKGROUND: Many numerical studies have been published with respect to about flow structures around cerebral aneurysm assuming to be rigid. Furthermore, there is little experimental research concerning aneurysm with elastic wall. Wall shear stress in elastic wall comparing with rigid wall should be clarified in experimental approach and verified in CFD. OBJECTIVE: We have experimentally realized elastic aneurysm model accompanying with wall deformation. Wall shear stress was examined for both rigid and elastic aneurysm models in pulsatile flow. METHODS: Effect of elasticity on wall shear stress inside aneurysm induced at the apex of anterior cerebral artery was experimentally examined by particle image velocimetry in vitro. In order to adjust the wall deformation, the pressure adjustment chamber was specially equipped outside the aneurysm wall. RESULTS: Effect of elasticity on wall shear stress was noticed on the comparison with that of rigidity. Wall elasticity reduced the peak magnitude, the spatial and temporal averaged wall shear stress comparing with those of wall rigidity experimentally. These reductions were endorsed by fluid-structure interaction simulation. CONCLUSION: Elastic wall comparing with rigid wall would reduce the peak magnitude, the spatial and temporal averaged wall shear stress acting on vascular wall.

Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning.

To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions.

Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.

Spatial concentration distribution analysis of cells in electrode-multilayered microchannel by dielectric property measurement.

The spatial concentration distribution of cells in a microchannel is measured by combining the dielectric properties of cells with the specific structure of the electrode-multilayered microchannel. The dielectric properties of cells obtained with the impedance spectroscopy method includes the cell permittivity and dielectric relaxation, which corresponds to the cell concentration and structure. The electrode-multilayered microchannel is constructed by 5 cross-sections, and each cross-section contains 5 electrode-layers embedded with 16 micro electrodes. In the experiment, the dielectric properties of cell suspensions with different volume concentrations are measured with different electrode-combinations corresponding to different electric field distributions. The dielectric relaxations of different cell concentrations are compared and discussed with the Maxwell-Wagner dispersion theory, and the relaxation frequencies are analysed by a cell polarization model established based on the Hanai cell model. Moreover, a significant linear relationship with AC frequency dependency between relative permittivity and cell concentration was found, which provides a promising way to on-line estimate cell concentration in microchannel. Finally, cell distribution in 1 cross-section of the microchannel (X and Y directions) was measured with different electrode-combinations using the dielectric properties of cell suspensions, and cell concentration distribution along the microchannel (Z direction) was visualized at flowing state. The present cell spatial sensing study provides a new approach for 3 dimensional non-invasive online cell sensing for biological industry.

Total Cavopulmonary Connection is Superior to Atriopulmonary Connection Fontan in Preventing Thrombus Formation: Computer Simulation of Flow-Related Blood Coagulation.

The classical Fontan route, namely the atriopulmonary connection (APC), continues to be associated with a risk of thrombus formation in the atrium. A conversion to a total cavopulmonary connection (TCPC) from the APC can ameliorate hemodynamics for the failed Fontan; however, the impact of these surgical operations on thrombus formation remains elusive. This study elucidates the underlying mechanism of thrombus formation in the Fontan route by using a two-dimensional computer hemodynamic simulation based on a simple blood coagulation rule. Hemodynamics in the Fontan route was simulated with Navier-Stokes equations. The blood coagulation and the hemodynamics were combined using a particle method. Three models were created: APC with a square atrium, APC with a round atrium, and TCPC. To examine the effects of the venous blood flow velocity, the velocity at rest and during exercise (0.5 and 1.0 W/kg) was measured. The total area of the thrombi increased over time. The APC square model showed the highest incidence for thrombus formation, followed by the APC round, whereas no thrombus was formed in the TCPC model. Slower blood flow at rest was associated with a higher incidence of thrombus formation. The TCPC was superior to the classical APC in terms of preventing thrombus formation, due to significant blood flow stagnation in the atrium of the APC. Thus, local hemodynamic behavior associated with the complex channel geometry plays a major role in thrombus formation in the Fontan route.

Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors.

The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors-namely,N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)-are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region-specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus-derived neural stem/progenitor cells-namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50= 7.5 ± 0.4 µM; n= 4), AMPA (EC50= 2.5 ± 0.1 µM; n= 3), or KA (EC50= 33.5 ± 1.1 µM; n= 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor-namely, MK-801 for NMDA stimulation (IC50= 0.6 ± 0.1 µM; n= 4) and NBQX for AMPA and KA stimulation (IC50= 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n= 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.

Generation of metabolically functioning hepatocytes from human pluripotent stem cells by FOXA2 and HNF1α transduction.

BACKGROUND & AIMS: Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. We have recently reported that hepatic differentiation is promoted by sequential transduction of SOX17, HEX, and HNF4α into hESC- or hiPSC-derived cells, but further maturation of hepatocyte-like cells is required for widespread use of drug screening. METHODS: To screen for hepatic differentiation-promoting factors, we tested the seven candidate genes related to liver development. RESULTS: The combination of two transcription factors, FOXA2 and HNF1α, promoted efficient hepatic differentiation from hESCs and hiPSCs. The expression profile of hepatocyte-related genes (such as genes encoding cytochrome P450 enzymes, conjugating enzymes, hepatic transporters, and hepatic nuclear receptors) achieved with FOXA2 and HNF1α transduction was comparable to that obtained in primary human hepatocytes. The hepatocyte-like cells generated by FOXA2 and HNF1α transduction exerted various hepatocyte functions including albumin and urea secretion, and the uptake of indocyanine green and low density lipoprotein. Moreover, these cells had the capacity to metabolize all nine tested drugs and were successfully employed to evaluate drug-induced cytotoxicity. CONCLUSIONS: Our method employing the transduction of FOXA2 and HNF1α represents a useful tool for the efficient generation of metabolically functional hepatocytes from hESCs and hiPSCs, and the screening of drug-induced cytotoxicity.

Expressions of cytochrome P450, UDP-glucuronosyltranferase, and transporter genes in monolayer carcinoma cells change in subcutaneous tumors grown as xenografts in immunodeficient nude mice.

Human tumors grown as xenografts in immunodeficient nude mice are widely used to investigate the pharmacological activities of anticancer drugs. Drug-metabolizing enzymes and transporters are expressed in tumor cell lines and changes in drug metabolism and pharmacokinetics (DMPK)-related gene expression after inoculation of the tumor cell may affect the pharmacological activity of the drug under consideration. The aims of the current study were to characterize DMPK-related gene expression profiles and responses to typical cytochrome P450 inducers in monolayer carcinoma cells grown in tissue culture versus those inoculated into a xenograft model. We used the human hepatocellular carcinoma cell line PLC/PRF/5 for this study and comprehensively assessed changes in DMPK-related gene expression by reverse transcription-polymerase chain reaction quantitation. CYP3A4 and UDP-glucuronosyltransferase 1A protein amounts were also analyzed by immunoprecipitation followed by immunoblotting. We found that the expression of many DMPK-related genes was elevated in the inoculated tumor compared with the monolayer carcinoma cells, indicating changes in their gene regulation pathways, presumably due to modulation of the nuclear receptor family of transcription factors. In addition, monolayer carcinoma versus inoculated tumor cells showed different responses to rifampicin, but similar responses to dexamethasone or 3-methylcholanthrene. These results suggest that inoculation of tumor cells results in the activation of drug metabolism and transport function, leading to changes in the responses to pregnane X receptor ligands and consequent discrepancies in the pharmacological activities between in vitro monolayer carcinoma cells and in vivo xenograft models.

Inoculation of human tumor cells alters the basal expression but not the inducibility of cytochrome P450 enzymes in tumor-bearing mouse liver.

The athymic nude mouse is often used to grow tumors for in vivo oncology research, including the identification of anticancer drugs, whereas wild-type mice are usually used to assess the pharmacokinetics (PK) of new chemical entities. The relationship between PK and pharmacodynamics (PD) provides useful mechanistic information and helps guide of the clinical regimen. The aim of this study was to assess whether the inoculation of human hepatocellular carcinoma cells (PLC/PRF/5) into athymic nude mice alters the expression of genes encoding the drug-metabolizing enzymes and transporters in host liver. The livers from nontumor- and tumor-bearing mice were initially subjected to drug metabolism gene microarray analysis. Microarray analysis indicated that tumor inoculation had little effect on drug metabolism-related genes, including several cytochrome P450s: Cyp1a, Cyp2b, and Cyp3a. This result was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR). However, immunoreactive proteins of Cyp1a, Cyp2b, and Cyp3a were suppressed by tumor inoculation. RT-PCR and Western immunoblotting analysis showed that the inducibility of Cyp1a, Cyp2b, and Cyp3a by 3-methylcholanthrene, phenobarbital, and dexamethasone, respectively, was similar between nontumor- and tumor-bearing mice. These results suggest that inoculation of human tumor cells into athymic nude mice suppresses the expression of certain drug-metabolizing enzymes, which may alter the PK and PD of antitumor drugs.