Berberine Induces Apoptotic Cell Death via Activation of Caspase-3 and -8 in HL-60 Human Leukemia Cells: Nuclear Localization and Structure-Activity Relationships.

Berberine (BBR), an isoquinoline alkaloid, is a well-known bioactive compound contained in medicinal plants used in traditional and folk medicines. In this study, we investigated the subcellular localization and the apoptotic mechanisms of BBR were elucidated. First, we confirmed the incorporation of BBR into the cell visually. BBR showed antiproliferative activity and promptly localized to the nucleus from 5[Formula: see text]min to 15[Formula: see text]min after BBR treatment in HL-60 human promyelocytic leukemia cells. Next, we examined the antiproliferative activity of BBR (1) and its biosynthetically related compounds (2-7) in HL-60 cells. BBR exerted strongest antiproliferative activity among 1-7 and the results of structures and activity relation suggested that a methylenedioxyl group in ring A, an [Formula: see text]-alkyl group at C-9 position, and the frame of isoquinoline may be necessary for antiproliferative activity. Moreover, BBR showed the most potent antiproliferative activity in HL-60 cells among human cancer and normal cell lines tested. Next, we examined the effect of BBR on molecular events known as apoptosis induction. In HL-60 cells, BBR induced chromatin condensation and DNA fragmentation, and triggered the activation of PARP, caspase-3 and caspase-8 without the activation of caspase-9. BBR-induced DNA fragmentation was abolished by pretreatment with inhibitors against caspase-3 and caspase-8, but not against caspase-9. ERK and p38 were promptly phosphorylated after 15 min of BBR treatment, and this was correlated with time of localization to the nucleus of BBR. These results demonstrated that BBR translocated into nucleus immediately after treatments and induced apoptotic cell death by activation of caspase-3 and caspase-8.

Ribonucleotide reductase is an effective target to overcome gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells with dual resistant factors.

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.

Caspr4 interaction with LNX2 modulates the proliferation and neuronal differentiation of mouse neural progenitor cells.

Contactin-associated protein 4 (Caspr4), also known as contactin-associated protein-like protein (CNTNAP4), is expressed in various regions of the brain. Recent reports suggest that CNTNAP4 is a susceptibility gene of autism spectrum disorders (ASDs). However, the molecular function of Caspr4 in the brain has yet to be identified. In this study, we show an essential role of Caspr4 in neural progenitor cells (NPCs). Caspr4 is expressed in NPCs in the subventricular zone (SVZ), a neurogenic region in the developing cortex. Knocking down of Caspr4 enhances the proliferation of NPCs derived from the SVZ of embryonic day 14 mouse. Neuronal differentiation is increased by overexpression of Caspr4, but decreased by knocking down of Caspr4 in cultured mouse NPCs. Transfection of the intracellular domain of Caspr4 (C4ICD) rescues the abnormal decreased neuronal differentiation of Caspr4-knocking down NPCs. Ligand of Numb protein X2 (LNX2), a binding partner of Numb, interacts with Caspr4 in a PDZ domain-dependent manner and plays a similar role to Caspr4 in NPCs. Moreover, transfection of LNX2 rescues the decreased neuronal differentiation in Caspr4-knocking down NPCs. In contrast, transfection of C4ICD fails to do so in LNX2-knocking down NPCs. These results indicate that Caspr4 inhibits neuronal differentiation in a LNX-dependent manner. Therefore, this study reveals a novel role of Caspr4 through LNX2 in NPCs, which may link to the pathogenesis of ASDs.

Thymidine phosphorylase activates NFκB and stimulates the expression of angiogenic and metastatic factors in human cancer cells.

Thymidine phosphorylase (TP) promotes angiogenesis and metastasis, and confers resistance to anticancer agents in some cancer cell types. We previously reported that TP stimulates the expression of interleukin (IL)-8 in human KB cancer cells by an unknown mechanism. A mutation in the nuclear factor (NF)κB binding site of the IL-8 promoter suppressed promoter activity in KB/TP cells that overexpress TP. Specifically inhibiting NFκB by using BY11-7082 also suppressed TP-induced IL-8 promoter activity and IL-8 expression. Moreover, TP overexpression led to the activation of NFκB and an upregulation in the expression of its target genes, and increased phosphorylated IKKα/ß protein levels, while promoting IκBα degradation as well as p65 phosphorylation and nuclear localization. The activation of NFκB in KB/TP cells was suppressed by the antioxidants N-acetylcysteine and EUK-8. In addition, in gastric cancer tissue samples, the expression of the NFκB-regulated genes, including IL-8, IL-6, and fibronectin-1 was positively correlated with TP expression. These findings indicate that reactive oxygen species mediated NFκB activation by TP increases the expression of genes that promote angiogenesis and metastasis in gastric cancer.

Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression.

Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 µM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3-100 µM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 µM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.

Functional characterization of neural-restrictive silencer element in mouse pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is predominantly localized in the nervous system, but the underlying mechanism in its neuron-specific expression remains unclear. In addition to two neural-restrictive silencer-like element (NRSLE1 and 2), as reported previously, we have identified the third element in -1,601 to -1,581 bp from the translational initiation site of mouse PACAP gene and termed it as NRSLE3, of which, the sequence and location were highly conserved among mouse, rat, and human PACAP genes. In luciferase reporter assay, the deletion or site-directed mutagenesis of NRSLE3 in the reporter gene construct, driven by heterologous SV40 promoter, cancelled the repression of luciferase activity in non-neuronal Swiss-3T3 cells. Furthermore, its promoter activity was significantly repressed in Swiss-3T3 cells, but not in neuronal-differentiated PC12 cells. The electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells demonstrated a specific complex with NRSLE3 probe that exhibited the same migration with the neural-restrictive silencer element (NRSE) probe of rat type II sodium channel gene. During neuronal differentiation of PC12 cells, the increment of PACAP mRNA exhibited the correlation with that of REST4 mRNA, which is a neuron-specific variant form of neural-restrictive silencer factor (NRSF). In undifferentiated PC12 cells, trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which indirectly inhibits NRSF-mediated gene silencing, increased PACAP mRNA level and attenuated the repression of promoter activity of 5' flanking region of mouse PACAP gene containing NRSLEs. These suggest that the NRSE-NRSF system implicates in the regulatory mechanism of neuron-specific expression of PACAP gene.

Regulation of major vault protein expression by upstream stimulating factor 1 in SW620 human colon cancer cells.

Major vault protein (MVP) is the main constituent of the vault ribonucleoprotein particle and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several types of normal tissues, little is known about its physiological role. In the present study, we identified the crucial MVP promoter elements that regulate MVP expression. An examination of tissue expression profiles revealed that MVP was expressed in the heart, placenta, lung, liver, kidney and pancreas. Elements of the MVP promoter contain binding sites for transcription factors, STAT, p53, Sp1, E-box, GATA, MyoD and Y-box. By deletion analysis, a conserved proximal E-box binding site was demonstrated to be important for human MVP promoter transactivation. Introduction of siRNA against upstream stimulating factor (USF) 1, which is known to bind the E-box binding site, decreased the expression of MVP in SW620 and ACHN cells. Using a chromatin immunoprecipitation (ChIP) assay, USF1 bound the MVP promoter in SW620 cells. These findings suggest that USF1 binding to an E-box element may be critical for basal MVP promoter activation. The results of the present study are useful in understanding the molecular mechanisms regulating MVP gene expression, and may aid in elucidating the physiological functions of MVP.

Molecular basis for the expression of major vault protein induced by hyperosmotic stress in SW620 human colon cancer cells.

Major vault protein (MVP) is identical to lung resistance-related protein (LRP), which is the major component of vaults. Vaults are considered to play a protective role against xenobiotics and other types of stress. In a previous study, we reported that the expression levels of MVP in SW620 human colon cancer cells were increased in hypertonic culture medium with sucrose. However, the molecular mechanism behind the induction of MVP expression by osmotic stress has not yet been elucidated. Therefore, in the present study, we investigated the mechanism behind the induction of MVP expression by osmotic stress. Under hyperosmotic stress conditions, the ubiquitination of specificity protein 1 (Sp1) decreased, Sp1 protein levels increased, its binding to the MVP promoter was enhanced, and small interfering RNA (siRNA) for Sp1 suppressed the induction of MVP expression. The inhibition of c-jun N-terminal kinase (JNK) by SP600125, a specific JNK inhibitor, decreased the expression of MVP and Sp1 under hyperosmotic conditions. Our data indicate that the stabilization and upregulation of Sp1 protein expression by JNK participate in the inhibition of the ubiquitination and degradation of Sp1, and thus in the induction of MVP expression under hyperosmotic conditions.

Molecular basis for the regulation of hypoxia-inducible factor-1α levels by 2-deoxy-D-ribose.

The angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, inhibits the upregulation of hypoxia-inducible factor (HIF) 1α, BNIP3 and caspase-3 induced by hypoxia. In the present study, we investigated the molecular basis for the suppressive effect of 2-deoxy-D-ribose on the upregulation of HIF-1α. 2-Deoxy-D-ribose enhanced the interaction of HIF-1α and the von Hippel-Lindau (VHL) protein under hypoxic conditions. It did not affect the expression of HIF-1α, prolyl hydroxylase (PHD)1/2/3 and VHL mRNA under normoxic or hypoxic conditions, but enhanced the interaction of HIF-1α and PHD2 under hypoxic conditions. 2-Deoxy-D-ribose also increased the amount of hydroxy-HIF-1α in the presence of the proteasome inhibitor MG-132. The expression levels of TP are elevated in many types of malignant solid tumors and, thus, 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.

p53R2 is a prognostic factor of melanoma and regulates proliferation and chemosensitivity of melanoma cells.

BACKGROUND: The treatment of melanoma, an aggressive, chemo-resistant skin cancer characterized by rapid metastasis and a poor prognosis, requires the development of innovative therapies with improved efficacy. The p53R2 gene that encodes the ribonucleotide reductase small subunit 2 homologue is induced by several stress signals including DNA-damaging agents that activate p53. The p53R2 gene product increases the deoxynucleotide triphosphate pool in the nucleus; this facilitates DNA repair and synthesis. OBJECTIVE: We examined the expression of p53R2 in melanoma and evaluated whether p53R2 is involved in the growth and proliferation of melanoma cells. Methods We examined the clinicopathological significance of p53R2 in melanoma. To investigate the role of p53R2 in melanoma we used KHm5 and KHm6 melanoma cells that express p53R2, and p53R2-targeting small interfering (si) RNA. RESULTS: p53R2 expression was detected immunohistochemically in 56 of 78 patients (71.8%). The expression of p53R2 was significantly correlated with the depth of invasion and the tumor stage. p53R2-targeting siRNA successfully knocked down p53R2 and significantly inhibited the growth of KHm5 and 6 cells. Moreover, The degree of KHm5 and 6 cell growth inhibition was greater in the presence of both p53R2-targeting siRNA and nimustine (ACNU) than with ACNU alone, suggesting that p53R2 silencing enhanced the chemosensitivity of KHm5 and 6 cells to ACNU. CONCLUSIONS: We propose p53R2 as a therapeutic target to enhance the effectiveness of chemotherapy in patients with p53R2-positive melanoma.

Thymidine phosphorylase enhances reactive oxygen species generation and interleukin-8 expression in human cancer cells.

Thymidine phosphorylase (TP) is an angiogenic factor that plays a pivotal role in tumor angiogenesis. Various kinds of solid tumors express TP and high TP activity is correlated with microvessel density. We have previously reported that TP enhances interleukin-8 (IL-8) expression in KB human epidermoid carcinoma cells. In this study, TP was shown to be involved in enhanced expression of IL-8 in EJ human bladder cancer cells and Yumoto human cervical cancer cells as well as KB human epidermoid carcinoma cells. The enzymatic activity of TP was required for the enhanced expression of IL-8. A degradation product of thymidine was implicated in the enhanced expression of IL-8. TP augmented reactive oxygen species (ROS) generation in KB and Yumoto cells, and the enzymatic activity of TP was again required for the generation of ROS. An antioxidant, N-acetylcysteine (NAC), attenuated the generation of ROS and IL-8 mRNA expression in KB and Yumoto cells, and H2O2 increased IL-8 mRNA expression in Yumoto cells, suggesting that ROS generated by TP caused the increased expression of IL-8 mRNA. Since TP also reduced cellular glutathione levels and transcription of γ-GCS in KB cells, the TP-induced augmentation of ROS may be partially attributed to the decreased glutathione. Our findings suggest that thymidine-derived sugars enhanced ROS generation and consequently increased IL-8 expression.

Pharmacokinetics of ampicillin-sulbactam and the renal function-based optimization of dosing regimens for prophylaxis in patients undergoing cardiovascular surgery.

UNLABELLED: Surgical site infections are a major cause of postoperative morbidity and mortality in cardiovascular surgery. Proper antibiotic prophylaxis can reduce the rate of such infections, but the concentration of antibiotic must be maintained at an adequate level throughout the operation. This study aimed to use renal function to determine the most appropriate timing for intraoperative repeated dosing of ampicillin-sulbactam, a commonly used prophylactic antibiotic, to maintain adequate concentrations throughout the course of surgery. The mean volume of distribution, elimination rate constant, elimination half-life, and total clearance of ampicillin were 13.2 l, 0.652 h⁻¹, 1.32 h, and 8.45 l/h, respectively. A statistically significant (P < 0.0001) correlation (r = 0.771) was observed between the total clearance of ampicillin and creatinine clearance of the patients. Plasma concentrations of ampicillin were simulated with the pharmacokinetic parameters obtained. We developed a nomogram for adjusting the dosing interval according to renal function and predicted ampicillin trough concentrations. We revealed the best dosage and dosing interval for cardiovascular surgery by analyzing the perioperative pharmacokinetics of ampicillin-sulbactam administered prophylactically. We suggest that the dosage and dosing interval for ampicillin-sulbactam should be adjusted to optimize treatment efficacy and safety, on the basis of the MIC90 of methicillin-sensitive Staphylococcus aureus (MSSA) in each institution. TRIAL REGISTRATION: UMIN000007356.

Reduction in the incidence of MRSA with use of alcohol-based hand rub solutions and gloves.

Methicillin-resistant Staphylococcus aureus (MRSA) is highly contagious. It is spread by direct contact with MRSA-infected people or objects. Healthcare workers' hands are the most common vehicle for the transmission of healthcare-associated pathogens from patient to patient and within the healthcare environment. The present study aimed to investigate the correlation between the incidence of MRSA among Staphylococcus aureus recovered from clinical culture and the use of alcohol-based hand rub solutions or gloves and antimicrobial use density (AUD). All data were examined every 6 months between January 2005 and June 2008. The increasing use of alcohol-based hand rub solutions was correlated with a decreasing incidence of recovery of MRSA from clinical cultures (r(2) = 0.58). A statistically significant (P < 0.05) correlation (r(2) = 0.68) was observed between glove use and the incidence of MRSA. On the other hand, we did not find any correlation between the AUD of each antibiotic group and the incidence of MRSA. Thus, we suggest that it is important to use not only alcohol-based hand rubs, but also gloves, because MRSA is transmitted from patient to patient by the hands of healthcare workers.

Safety analysis of liposomal amphotericin B in adult patients: anaemia, thrombocytopenia, nephrotoxicity, hepatotoxicity and hypokalaemia.

Liposomal amphotericin B (L-AmB), which was developed to reduce side effects, has been shown to have a better safety profile than both the deoxycholate and lipid complex forms of amphotericin B; however, the frequency of major side effects is still unclear. Thus, the aim of the present study was to assess retrospectively the frequency of L-AmB-induced anaemia, thrombocytopenia, nephrotoxicity, hepatotoxicity and hypokalaemia as well as the relationship between daily dose of L-AmB and these side effects. A low red blood cell (RBC) count (post-/pre-treatment) and anaemia were observed in 7 and 10 of 21 adult patients, respectively. Thrombocytopenia was observed in 11 of 19 adult patients. Doses of L-AmB that are estimated to cause side effects of a low RBC count, anaemia and thrombocytopenia with 50% probability are 4.0, 3.3 and 3.0mg/kg/day, respectively. Nephrotoxicity was observed in 6 of 22 patients. Variations of total bilirubin, γ-glutamyl transpeptidase, aspartate aminotransferase and alanine aminotransferase used as indices of hepatotoxicity were observed in 6, 7, 8 and 8 of 22 patients, respectively. Hypokalaemia was observed in 4 of 9 patients; however, nephrotoxicity, hepatotoxicity and hypokalaemia were not caused in a dose-dependent manner. In conclusion, the present analyses showed that L-AmB dose-dependently induced anaemia and thrombocytopenia in adult patients. It is important to pay attention to causing anaemia and thrombocytopenia when patients are receiving L-AmB at doses of >3.3mg/kg/day and >3.0mg/kg/day, respectively.

Effect of 5-fluorouracil treatment on SN-38 absorption from intestine in rats.

5-Fluorouracil (5-FU)-based chemotherapies with irinotecan have been applied for the treatment of cancers, and a common dose-limiting toxicity is neutropenia and diarrhea. In this study, we investigated the effect of 5-FU treatment on expression levels of drug transporters for SN-38 transportation and SN-38 absorption from the intestine following 5-FU treatment. Expression levels of several drug transporters and nuclear receptors in rats after 5-FU treatment were evaluated. SN-38 absorption from the intestine was evaluated by SN-38 concentration levels in serum following SN-38 injection into the intestine of 5-FU treated rats. The levels of renal multidrug resistance protein 2 (Mrp2) on day 4 after treatment (400 mg/kg) showed significant upregulation, 359.2 ± 33.2% (mean ± S.E.) of control. Mrp2 levels in the intestine were downregulated to 26.2 ± 8.4% of control. 5-FU treatment (400 mg/kg) also significantly downregurated expression levels of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) to 41.2 ± 14.7%, 15.7 ± 4.3% of control, respectively. To evaluate SN-38 absorption from the intestine, SN-38 was loaded in to the intestine on day 4 after 5-FU treatment. Pretreatment with 5-FU significantly increased SN-38 concentration in the blood 30, 60 and 90 min after SN-38 administration. The area under the curve for SN-38 in the 5-FU group was significantly higher than in vehicle groups. 5-FU treatment decreased expression levels of P-glycoprotein and Bcrp in intestine. The present study suggests that combination chemotherapy of 5-FU with irinotecan (CPT-11) may elevate SN-38 absorption from intestine.

Multidrug resistance protein 2 implicates anticancer drug-resistance to sorafenib.

Sorafenib and sunitinib is a small molecule inhibitor of certain receptor tyrosine kinases, and have improved outcomes for patients with advanced renal cell carcinoma. Inhibitory concentration of 50% cell growth of sorafenib significantly rose to 6.4-fold in a multidrug resistance protein 2 (MRP2) transfected cell line versus control cell line. The concentration of sorafenib was significantly decreased to 74% of control cells after 3 h treatment. In contrast, a tyrosine kinase inhibitor sunitinib did not show alteration of inhibitory concentration of 50% cell growth and accumulation into the cells of MRP2 transfected cells. The present study suggest that sorafenib is a substrate for MRP2, suggesting that MRP2 may implicate drug resistance to sorafenib.

Wnt-5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP-2.

Wnts are secreted ligands that consist of 19 members in humans, regulate cell proliferation, differentiation, motility and fate in many stages including the embryonic stage and tumorigenesis. Wnts bind to cell surface receptors named Frizzleds and LRPs, and transduce their signals through ß-catenin-dependent and -independent intracellular pathways. Gliomas are one of the most common intracranial tumors. Gliomas exhibit a progression associated with widespread infiltration into surrounding neuronal tissues. However, the molecular mechanisms that stimulate the invasion of glioma cells are not fully understood. We established two cell lines from human glioma cases and analyzed the expression of all Wnt and Frizzled members in these cell lines and other well-known glioma cell lines by real-time PCR study. The mRNA of Wnt-5a and -7b and Frizzled-2, -6 and -7 were overexpressed in glioma cells. The elevation of Wnt-5a expression was most remarkable. Although Wnt-5a is reported to have oncogenic and antioncogenic activity in several cancers, the role of Wnt-5a signaling in human glioma cells remains unclear. Immunohistochemical study also revealed high expression of Wnt-5a in 26 (79%) of 33 human glioma cases. The positivity of Wnt-5a expression was correlated with the clinical grade. Knockdown of Wnt-5a expression suppressed migration, invasion and expression of matrix metalloproteinase-2 of glioma cells. Reciprocally, treatment with purified Wnt-5a ligand resulted in stimulation of cell migration and invasion. MMP-2 inhibitor suppressed the Wnt-5a-dependent invasion of U251 cells. These results suggested that Wnt-5a is not only a prognostic factor but also a therapeutic target molecule in gliomas for preventing tumor cell infiltration.

Isolation and characterization of gemcitabine-resistant human non-small cell lung cancer A549 cells.

Gemcitabine is an effective chemotherapy against non-small cell lung cancer (NSCLC). However, resistance to gemcitabine reduces its efficacy. We have isolated gemcitabine-resistant human non-small cell lung cancer A549 cells, termed A549/GR cells. A549/GR cells were resistant to gemcitabine as well as paclitaxel and docetaxel but not carboplatin and irinotecan. The expression level of multidrug resistance protein 7 (MRP7) in A549/GR cells was higher than that in A549 cells, and the inhibitor of MRP7 by cepharanthine increased the sensitivity to gemcitabine in A549/GR cells. These findings indicate that cepharanthine reversed gemcitabine resistance. To determine predictive molecular markers of gemcitabine resistance for more effective treatment of these tumors, we performed PCR array. We identified that CDKN1A/p21, CYP3A5, microsomal epoxide hyrolase 1 (EPHX1) and ABCC6 (MRP6) were up-regulated >5-fold in A549/GR cells. Gemcitabine also induced the expression of p21 and CYP3A5 in A549 cells. A better understanding of the characterization and mechanism of the resistance to gemcitabine in A549/GR cells may help identify agents that reverse clinical gemcitabine resistance in NSCLC.

An initial dosing method for teicoplanin based on the area under the serum concentration time curve required for MRSA eradication.

Teicoplanin is a glycopeptide antibacterial agent that has a long serum half-life and therefore takes time to achieve steady-state conditions. An appropriate initial dosing is needed for teicoplanin to promptly reach an effective serum trough concentration. However, little information is available on tailoring the initial dosing for patients with various characteristics. The objective of this study was to develop a nomogram for determining teicoplanin initial dose to promptly reach an effective trough concentration (≥ 13 µg/mL). A logistic regression analysis was performed to test whether the area under the concentration time curve (AUC) is a significant predictor of microbiological response (persistence 0; eradication 1). The study included 24 adult patients with methicillin-resistant Staphylococcus aureus infections [minimal inhibitory concentration (MIC) for the isolates was <2 µg/mL). Each AUC was estimated using individual dose, creatinine clearance (CL(cr)), and body weight data. The target value, which gives about a 0.9 microbiological eradication probability, was 750 µg h/mL for AUC from zero to 24 h (AUC(0-24 h)). Using published population pharmacokinetic parameters, the dose required to achieve the AUC(0-24 h) target was calculated as dose (mg) = 750 × (0.00498 × CL(cr) (mL/min) + 0.00426 × body weight (kg). For various combinations of CL(cr) and body weight, we checked the calculated doses using a therapeutic drug monitoring (TDM)-supporting software and developed a nomogram. The nomogram would be useful for initial dose adjustment to promptly reach an effective serum trough concentration and avoid adverse events of teicoplanin.

Isolation and characterization of erlotinib-resistant human non-small cell lung cancer A549 cells.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is an effective therapy for non-small cell lung cancer (NSCLC). However, resistance to erlotinib reduces its efficacy. To investigate the basis of erlotinib resistance, we isolated erlotinib-resistant human NSCLC A549 cells, termed A549/ER cells. The A549/ER cells were found to be resistant to erlotinib, as well as paclitaxel and gemcitabine. We then performed a PCR array to investigate the resistance to erlotinib in A549/ER cells. EGFR expression in A549/ER cells was decreased compared to A549 cells. The expression of fibroblast growth factor 2 (FGF2) and p21 in A549/ER was increased when compared to A549 cells. Our results suggest that the down-regulation of EGFR and up-regulation of FGF2 is related to resistance to erlotinib in A549/ER cells.