Takeshi Hirano

Association of toxicity of sorafenib and sunitinib for human keratinocytes with inhibition of signal transduction and activator of transcription 3 (STAT3).

Hand–foot skin reaction is a most common multi-kinase inhibitor-related adverse event. This study aimed to examine whether the toxicity of sorafenib and sunitinib for human keratinocytes was associated with inhibiting signal transduction and activator of transcription 3 (STAT3). We studied whether STAT3 activity affects sorafenib- and sunitinib-induced cell growth inhibition in HaCaT cells by WST-8 assay. Stattic enhanced the cell-growth inhibitory and apoptotic effects of sorafenib and sunitinib. HaCaT cells transfected with constitutively-active STAT3 (STAT3C) were resistant to the sorafenib- and sunitinib-induced cell growth inhibition. STAT3 activity decreased after short-term treatment with sorafenib and sunitinib in a dose-dependent manner and recovered after long-term treatment with sorafenib and sunitinib at low doses. Moreover, the expression of survivin and bcl-2 decreased after treatment with sorafenib and sunitinib was concomitant with variations in STAT3 activity. Sorafenib-induced STAT3 inhibition was mediated by regulation via MAPK pathways in HaCaT cells, while sunitinib-induced STAT3 inhibition was not. Thus, STAT3 activation mediating apoptosis suppressors may be a key factor in sorafenib and sunitinib-induced keratinocyte cytotoxicity.

Apoptotic Effects of the Extracts of Cordyceps militaris via Erk Phosphorylation in a Renal Cell Carcinoma Cell Line

Cordyceps militaris (CM) is gaining attention as a traditional medicinal food, but its molecular biological mechanisms for anti‐cancer activity are not identified or clarified. We aimed to elucidate the synthesizing apoptotic effects of CM extracts and to determine the biological effects of CM extract against cordycepin alone in a renal cell carcinoma (RCC) cell line. CM extract showed higher effects of growth inhibition, apoptotic effect, and cell cycle arrest than cordycepin alone. Moreover, CM extract activated extracellular signal‐regulated kinase (Erk) highly more than cordycepin alone. We suggest that cordycepin and CM extract induced apoptosis via the activation of Erk dominantly and AMP‐activated protein kinase slightly; CM extract has more potent effects on apoptotic effects associated with Erk activation than cordycepin alone. Copyright

Induction of Epithelial-Mesenchymal Transition via Activation of Epidermal Growth Factor Receptor Contributes to Sunitinib Resistance in Human Renal Cell Carcinoma Cell Lines

Sunitinib is widely used for treating renal cell carcinoma (RCC). However, some patients do not respond to treatment with this drug. We aimed to study the association between sunitinib sensitivity and epithelial-mesenchymal transition (EMT) regulation via epidermal growth factor receptor (EGFR) signaling, which is a mechanism of resistance to anticancer drugs. Three RCC cell lines (786-O, ACHN, and Caki-1) were used, and then we evaluated cell viability, EMT regulatory proteins, and signal transduction with sunitinib treatment. Cell viability of 786-O cells was maintained after treatment with sunitinib. After treatment with sunitinib, EGFR phosphorylation increased in 786-O cells, resulting in an increase in the phosphorylation of extracellular signal-regulated kinase, nuclear translocation of β-catenin, and expression of mesenchymal markers. These results suggest that sunitinib induced EMT via activation of EGFR in 786-O cells, but not in ACHN and Caki-1 cells. Caki-1/SN cells, a resistant cell line generated by continuous exposure to sunitinib, displayed increased phosphorylation of EGFR. Cell viability in the presence of sunitinib was decreased by erlotinib, as the selective inhibitor of EGFR, treatment in 786-O and Caki-1/SN cells. Similarly, erlotinib suppressed sunitinib-induced EGFR activation and upregulated mesenchymal markers. Thus, we postulate that resistance to sunitinib in RCC may be associated with EMT caused by activation of EGFR.

The intestinal transport mechanism of fluoroquinolones : inhibitory effect of ciprofloxacin, an enoxacin derivative, on the membrane potential-dependent uptake of enoxacin

AbstractPurpose. To clarify the absorption-structure relationship for the fluoroquinolones from the point of view of inhibitory behavior.nMethods. The inhibitory effects of ciprofloxacin on the transport process of enoxacin across the rat intestinal brush-border membrane was examined.nResults. Ciprofloxacin, which has a similar structure to enoxacin, exhibited a pH-dependent interference with enoxacin absorption from rat jejunal loops. The uptake experiments using BBM vesicles showed that ciprofloxacin significantly reduced not only the initial binding of enoxacin to the membrane surface, but also the K + - or H+-diffusion potential-dependent transport across the membrane. Furthermore, an H +-diffusion potential (interior negative) also exhibited a stimulative uptake of ciprofloxacin.nConclusions. These results suggest that the inhibition behavior of ciprofloxacin from the jejunal loop was closely related to the ionic diffusion potential-dependent uptake of enoxacin across the brush-border membrane.

Transient elevation of serum cystatin C concentrations during perioperative cisplatin-based chemotherapy in esophageal cancer patients

PurposeIn the present study, the time–concentration profile of platinum (Pt) in plasma was compared to that of serum cystatin C (Cys C) in Japanese esophageal cancer patients receiving perioperative cisplatin-based chemotherapy.MethodsFive male and one female patients receiving 2 successive cycles of cisplatin-based chemotherapy combined with 5-fluorouracil, the treatment for esophageal squamous cell carcinoma, participated in this study. The pharmacokinetic parameters in each patient were calculated from the individual plasma Pt concentration–time curve after intravenous infusion of cisplatin using the one-compartment model.ResultsWithin a week of starting the first cycle of chemotherapy, serum Cys C concentrations increased in all patients from 122.6 to 143.0xa0%, subsequently returning to baseline levels in approximately 10xa0days. A similar increase in serum Cys C levels also occurred during the second treatment cycle. However, no increase in serum creatinine levels was observed during either treatment cycle. In addition, the concentration of plasma Pt 2xa0days after treatment in the first and second cycles did not correlate with those of either serum Cys C or creatinine. Finally, the half-life of Pt in plasma during the first treatment cycle was not significantly different from that in the second cycle.ConclusionsThese findings suggest that concentration fluctuations in serum Cys C are unlikely to correlate with Pt elimination from the plasma and that renal function estimates based on serum Cys C concentration might be underestimated during perioperative cisplatin-based chemotherapy for esophageal cancer.

Everolimus-induced human keratinocytes toxicity is mediated by STAT3 inhibition

BackgroundMammalian target of rapamycin (mTOR) inhibitors are associated with dermatological adverse events. The chief aim of this study was to examine the relation between the signal transducer and activator of transcription 3 (STAT3) protein and the dermatological adverse events associated with the mTOR inhibitor everolimus.MethodsWe evaluated the effects of STAT3 activity and related signal transduction activities on everolimus-induced cell growth inhibition in the human keratinocyte HaCaT cell line via a WST-8 assay, and on signal transduction mechanisms involved in everolimus treatments via a western blot analysis. Apoptosis was evaluated using an imaging cytometric assay.ResultsThe cell growth inhibitory effects of everolimus were enhanced by stattic or STA-21, which are selective inhibitors of STAT3, treatment in HaCaT cells, although such effects were not observed in Caki-1 and HepG2 cells. Phosphorylation at tyrosine 705 of STAT3 was decreased by treatment with everolimus in a dose-dependent manner in HaCaT cells; in contrast, phosphorylation at serine 727 was not decreased by everolimus, but slightly increased. Furthermore, we found that pretreatment of p38 MAPK inhibitor and transfection with constitutively active form of STAT3 in HaCaT cells resisted the cytostatic activity of everolimus.ConclusionsThese findings suggest that STAT3 activity may be a biomarker of everolimus-induced dermatological toxicity.

Association of Single Nucleotide Polymorphisms in STAT3 with Hand-Foot Skin Reactions in Patients with Metastatic Renal Cell Carcinoma Treated with Multiple Tyrosine Kinase Inhibitors: A Retrospective Analysis in Japanese Patients

BackgroundSignal transducer and activator of transcription (STAT)3 is a reported mediator of molecular-targeted drug-induced keratinocyte toxicity.AimOur purpose was to assess the association of single nucleotide polymorphisms (SNPs) in STAT3 with hand-foot skin reactions (HFSR) in patients with metastatic renal cell carcinoma (mRCC) treated with multiple tyrosine kinase inhibitors (mTKIs).Patients and MethodsSixty-five Japanese patients with clear cell renal cell carcinoma who were treated with any mTKI at Kobe University Hospital were retrospectively genotyped to elucidate a potential association between STAT3 polymorphisms and HFSR development.ResultsThe final analysis included 60 patients. HFSR was observed in 46 patients. The GG, GC, and CC genotypes at rs4796793 were found in 9, 27, and 24 patients, respectively. Three other STAT3 polymorphisms exhibited tight linkage disequilibrium with rs4796793. A significant association was found between the rs4796793 allele and HFSR [G vs. C; odds ratio [OR], 4.33; 95xa0% confidence interval [CI], 1.80–10.45; Pu2009=u20090.001]. The GG genotype had the highest OR compared with GC + CC genotypes (OR, 10.75; 95xa0% CI, 2.38–48.07; Pu2009=u20090.001). In a time-to-event Kaplan–Meier analysis, a statistically significant difference was observed between the GC + CC and the GG genotypes (Pu2009=u20090.009).ConclusionsThe rs4796793 genotype appears to be a novel factor for mTKI-induced HFSR in patients with mRCC. Prospective translational trials with larger numbers of patients are required to confirm our results. This research suggests a potential benefit of STAT3 polymorphism screening in patients treated with mTKIs.

Involvement of l-type amino acid transporter 1 in the transport of gabapentin into human placental choriocarcinoma cells

Gabapentin (GBP) is a widely used antiepileptic drug, with potential for use in the treatment of epilepsy in pregnant women. Although studies have examined GBP transport mechanisms across the blood-brain barrier, kidney, and intestine, the mechanism in the placenta has not been fully elucidated. We previously reported that GBP accumulates at high concentrations in human placental choriocarcinoma BeWo cells. The purpose of this study was to examine the transport mechanism of GBP in placental choriocarcinoma cells (BeWo and JEG-3), and to identify the carrier involved. High concentrations of intracellular GBP accumulations were also found in JEG-3 cells. A kinetic analysis showed that a single carrier system was involved in the uptake of GBP. Furthermore, substrates for l-type amino acid transporter (LAT) and siRNAs targeted to LAT1 significantly decreased GBP uptake. Our observations from this study suggest that LAT1 is the main contributor to GBP transport in placental choriocarcinoma cells.

TNF-α -857C>T genotype is predictive of clinical response after treatment with definitive 5-fluorouracil/cisplatin-based chemoradiotherapy in Japanese patients with esophageal squamous cell carcinoma.

Background: Genotypes of tumor necrosis factor alpha (TNF-α) and its surface receptors, TNFRSF1A and TNFRSF1B, have been examined in terms of the progression, metastasis, clinical efficacy, and prognosis of various cancers; however, little is known about their effects on clinical outcome in patients with esophageal squamous cell carcinoma (ESCC). In this study, TNF-α and TNFRSF1A genotypes were retrospectively evaluated in terms of predicting clinical response, long-term survival, and severe acute toxicities in 46 male Japanese ESCC patients treated with definitive 5-fluorouracil (5-FU)/cisplatin (CDDP)-based chemoradiotherapy (CRT). Methods: A course consisted of the continuous infusion of 5-FU at 400 mg/m2/day for days 1-5 and 8-12, the infusion of CDDP at 40 mg/m2/day on days 1 and 8, and radiation at 2 Gy/day on days 1-5, 8-12, and 15-19, with a second course being repeated after a 2-week interval. The TNF-α -1031T>C (rs1799964), -863C>A (rs1800630), -857C>T (rs1799724), -308G>A (rs1800629), -238G>A (rs361525), TNFRSF1A -609G>T (rs4149570), and 36A>G (rs767455) genotypes were evaluated. Results: The TNF-α -857C>T genotype was found to be predictive of clinical response, i.e., complete response or not (P = 0.010, Fishers exact test), but had no effect on long-term survival (CC-857 vs. CT-857 + TT-857, P = 0.072, Fishers exact test, P = 0.070, Log-rank test). Conclusions: The TNF-α -857C>T genotype was found to be predictive of clinical response and was more likely to predict long-term survival in Japanese ESCC patients receiving definitive 5-FU/CDDP-based CRT. Further clinical investigations with a larger number of patients or experiments in vitro should be performed to assess the predictive value of this genotype following CRT.

Valproic acid transport in the choriocarcinoma placenta cell line JEG-3 proceeds independently of the proton-dependent transporters MCT1 and MCT4

Medication therapy is the first line of treatment in the management of epilepsy. Fetal exposure to valproic acid (VPA), an antiepileptic drug, poses an elevated risk of teratogenicity in early pregnancy. Some studies have reported that monocarboxylate transporters (MCTs) may be involved in the placental transport of VPA. However, it has not been determined which MCTs contribute to VPA transport into the placenta. Therefore, the aim of this study was to determine how MCTs contribute to VPA transport into the placenta using the human placenta choriocarcinoma cell line JEG-3. VPA uptake was investigated using JEG-3xa0cells and radiolabeled VPA. MCT expression in JEG-3xa0cells was detected using RT-PCR and western blotting. Knockdown of MCTs was carried out using siRNAs. VPA uptake into JEG-3xa0cells was pH- and concentration-dependent, and described by using the Michaelis-Menten equation (Kmxa0=xa00.95xa0±xa00.17xa0mM; Vmaxxa0=xa019.3xa0±xa01.21xa0nmol/mg protein/15xa0s). MCT1 and MCT4 expression was found in JEG-3xa0cells, and typical MCT inhibitors significantly inhibited VPA uptakexa0into JEG-3xa0cells. However, knockdown of MCT1 and MCT4 did not alter VPA uptake. In conclusion, VPA transport is mediated by a proton-dependent transporter in JEG-3xa0cells, but not by MCT1 and MCT4.