Keita Nakane

Emergence of clinical strains of Mycoplasma genitalium harbouring alterations in ParC associated with fluoroquinolone resistance

Surveillance for antimicrobial resistance in Mycoplasma genitalium clinical strains is extremely limited as culturing of strains from clinical specimens is still difficult. We therefore conducted a non-cultural assessment of fluoroquinolone resistance of M. genitalium clinical strains by analysing the quinolone-resistance determining regions (QRDRs) of the gyrA and parC genes. The QRDRs amplified from M. genitalium DNA taken from urine specimens of 28 men with non-gonococcal urethritis positive for M. genitalium by polymerase chain reaction were sequenced. An amino acid change (Phe-108-->Iso) in GyrA was found in one specimen, and the same change was accompanied by an amino acid change (Lys-97-->Arg) in ParC in another specimen. A single amino acid change (Ser-83-->Asn, Asp-87-->Tyr or Asp-87-->Val) in ParC was also found in three other respective specimens without alterations in GyrA. No alterations in GyrA and ParC were found in the remaining 23 specimens. The alterations of Ser-83-->Asn, Asp-87-->Tyr and Asp-87-->Val in ParC found in 3 (10.7%) of 28 specimens were analogous to those commonly observed in fluoroquinolone-resistant mutants of other Mycoplasma and Ureaplasma spp. M. genitalium harbouring mutations associated with fluoroquinolone resistance in the parC gene may have emerged clinically and the prevalence may be ca. 10% in Japan.

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H2O2 were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H2O2 in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H2O2 via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.

Macrolide Resistance–associated 23S rRNA Mutation in Mycoplasma genitalium, Japan

To the Editor: Mycoplasma genitalium is now recognized as a serious pathogen in sexually transmitted infections (1,2). Azithromycin regimens have been commonly used for treatment of M. genitalium infections (3). However, failure of azithromycin treatment has been reported in cases of M. genitalium–positive nongonococcal urethritis (NGU) (4,5), and macrolide-resistant strains of M. genitalium have been isolated from case-patients in Australia, Sweden, and Norway for whom azithromycin treatment has failed (4,5). In these strains, mutations in the 23S rRNA gene were associated with macrolide resistance, and mutations in ribosomal protein genes L4 and L22 were also found (5). Surveillance for antimicrobial resistance of M. genitalium is essential to identify antimicrobial resistant strains and to then determine appropriate treatment. Coculture of patient specimens with Vero cells has improved the primary isolation rate of M. genitalium from clinical specimens and offered some current clinical strains for antimicrobial drug susceptibility testing (6). To determine their antimicrobial susceptibilities, a molecular real-time PCR method has been developed (7,8). However, isolating M. genitalium from clinical specimens and antimicrobial drug susceptibility testing of clinical isolates remain labor-intensive, time-consuming tasks. In addition, no methods are available to directly determine antimicrobial drug susceptibilities of M. genitalium in clinical specimens. To monitor macrolide susceptibilities in clinical strains of M. genitalium in Japan, therefore, we examined M. genitalium DNA found in the urine of men with NGU for the presence of macrolide resistance–associated mutations in the 23S rRNA gene and the ribosomal protein genes L4 and L22. This retrospective study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Gifu, Japan. We collected pretreatment urine specimens from 308 men with NGU who had visited a urologic clinic (iClinic) in Sendai, Japan, during 2006 through 2008 and stored the specimens at –70°C. Each man gave informed consent. Twenty-five of 58 urine specimens confirmed to be positive for M. genitalium by PCR-based assay were randomly chosen for this study and subjected to DNA purification. The 23S rRNA gene and the ribosomal proteins genes L4 and L22 of M. genitalium were amplified from the purified DNA by PCR as reported previously and then sequenced (5). In 1 specimen, we found an A-to-G transition at nucleotide position 2072 in the 23S rRNA gene of M. genitalium, corresponding to position 2059 in Escherichia coli (Table). An A2059 (E. coli numbering) residue in region V of the 23S rRNA gene is critical for the binding of macrolides (9). Mutations of A2058, A2059, and other 23S rRNA residues within the macrolide-binding site can confer a high-level resistance to macrolides in several bacterial species, including M. genitalium (5,9). Therefore, M. genitalium strains that harbor the A2059G (E. coli numbering) mutation in the 23S rRNA gene could be highly macrolide resistant. We also found a T-to-G transition at nucleotide position 2199 in the 23S rRNA gene of M. genitalium, corresponding to position 2185 in E. coli, in 3 specimens, but this mutation has not been associated with macrolide resistance in other bacterial species (9). Table Mutations in the 23S rRNA gene and amino acid changes in L4 and L22 ribosomal proteins of 25 Mycoplasma genitalium strains in the pretreatment urine specimens of men with nongonococcal urethritis, Japan We found amino acid changes in L4 and L22 ribosomal proteins in M. genitalium in 9 specimens. L4 and L22 ribosomal proteins each have extended loops, which converge to form a narrowing in the exit tunnel adjacent to the macrolide-binding site (10). Therefore, macrolide resistance–associated missense mutations in L4 and L22 tend to be localized to Gln62–Gly66 in L4 and Arg88-Ala93 in L22 of E. coli, which are closest to the macrolide-binding site (10). All of the amino acid changes in L4 of M. genitalium found in this study corresponded to those at the downstream regions from Gln62-Gly66 in L4 of E. coli. Of the amino acid changes in L22 of M. genitalium, the only Gly93Glu change found in M. genitalium harboring the A2059G (E. coli numbering) mutation in the 23S rRNA gene was located within the region corresponding to Arg88-Ala93 in L22 of E. coli. In this strain, therefore, the Gly93Glu change in L22 might contribute to the increase of macrolide resistance. The patient with NGU, whose specimen exhibited this strain of M. genitalium that harbored both the A2059G (E. coli numbering) mutation in the 23S rRNA gene and in which the Gly93Glu change in L22 was detected, was given a single dose of 1 g azithromycin and was clinically cured of NGU. However, the present study suggests that M. genitalium strains with high-level macrolide resistance might have already emerged in clinical settings in Japan. The emergence and spread of such a clinical mutant could threaten the ability of macrolides to treat M. genitalium infections. We should continue monitoring macrolide resistance of M. genitalium clinical strains. The nonculture approach used in our study will be useful until culturing of mycoplasmas from clinical specimens and antimicrobial drug susceptibility testing can be performed easily in laboratories.

CCR1/CCL5 interaction promotes invasion of taxane-resistant PC3 prostate cancer cells by increasing secretion of MMPs 2/9 and by activating ERK and Rac signaling

Castration-refractory prostate cancer (CRPC) is treated with taxane-based chemotherapy, but eventually becomes drug resistant. It is thus essential to identify novel therapeutic targets for taxane resistance in CRPC patients. We investigated the role of the chemokine (C-C motif) receptor 1 (CCR1) and its ligand, chemokine (C-C motif) ligand 5 (CCL5), in taxane-resistant CRPC using paclitaxel-resistant prostate cancer cells (PC3PR) established from PC3 cells. We found that the expression levels of CCR1 mRNA and protein were up-regulated in PC3PR cells compared to PC3 cells. In order to investigate the role of increased CCR1 in PC3PR cells, we stimulated cells with CCL5, one of the chemokine ligands of CCR1. In CCL5-stimulated PC3PR cells, siRNA-mediated knockdown of CCR1 expression reduced phosphorylation of ERK1/2 and Rac1/cdc42. Furthermore, CCR1 knockdown and MEK1/2 inhibition decreased CCL5-stimulated secretion of MMPs 2 and 9, which play important roles in cancer cell invasion and metastasis. In the Matrigel invasion assay, knockdown of CCR1 and inhibition of the ERK and Rac signaling pathways significantly decreased the number of invading cells. Finally, the serum CCL5 protein level as measured by ELISA was not different among the three groups of patients: those with negative prostate biopsy, those at initial diagnosis of prostate cancer, and those with taxane-resistant prostate cancer. These results demonstrated for the first time that the interaction of CCR1 with CCL5 caused by increased expression of CCR1 promotes invasion of PC3PR cells by increasing secretion of MMPs 2 and 9 and by activating ERK and Rac signaling. Our findings suggest that CCR1 could be a novel therapeutic target for taxane-resistant CRPC.

Inhibition of cortactin and SIRT1 expression attenuates migration and invasion of prostate cancer DU145 cells

Objectives:  Cortactin is overexpressed in various types of cancer and enhances cell motility. It has been recently reported that silent mating type information regulation 2 homolog 1 interacts with cortactin and promotes cell migration. Here, we examined the role of cortactin and silent mating type information regulation 2 homolog 1 in migration and invasion of prostate cancer cells.

Identification of organoselenium compounds that possess chemopreventive properties in human prostate cancer LNCaP cells

The process of cancer development consists of three sequential stages termed initiation, promotion, and progression. Oxidative stress damages DNA and introduces mutations into oncogenes or tumor suppressor genes, thus contributing to cancer development. Cancer chemoprevention is defined to prevent or delay the development of cancer by the use of natural or synthetic substances. In the present study, we synthesized a series of organoselenium compounds and evaluated their possible chemopreventive properties in human prostate cancer LNCaP cells. Among 42 organoselenium compounds tested, two compounds, 3-selena-1-dethiacephem 13 and 3-selena-1-dethiacephem 14 strongly activated the Nrf2/ARE (antioxidant response element) signaling and thus markedly increased expression of heme oxygenase-1 (HO-1), a phase II antioxidant enzyme. Translocation of Nrf2 to the nucleus preceded HO-1 protein induction by two compounds. The intracellular ROS level was strongly reduced immediately after treatment with these compounds, showing that they are potent antioxidants. Finally, both compounds inhibited cell growth via cell cycle arrest. Our findings suggest that compounds 13 and 14 could not only attenuate oxidative stress through Nrf2/ARE activation and direct ROS scavenging but also inhibit cell growth. Thus, these compounds possess the potential as pharmacological agents for chemoprevention of human prostate cancer.

Emergence and spread of drug resistant Neisseria gonorrhoeae.

PURPOSE The emergence and spread of Neisseria gonorrhoeae with resistance to oral antibiotics have led to difficulty in treating gonorrhea. We review drug resistance in N. gonorrhoeae with a particular emphasis on resistance to fluoroquinolones, cefixime and azithromycin. MATERIALS AND METHODS Literature selected from peer reviewed journals listed in MEDLINE(R)/PubMed(R) from 1943 to 2009 and from resources cited in those articles was reviewed comprehensively. RESULTS Due to the spread of fluoroquinolone resistant N. gonorrhoeae fluoroquinolones are no longer recommended for the treatment of gonorrhea. The emergence of N. gonorrhoeae with a mosaic penicillin-binding protein 2 associated with oral cephalosporin resistance has threatened cefixime treatment for gonorrhea. Emergence of N. gonorrhoeae with high level resistance to azithromycin has also been documented. However, injectable antibiotics (sepctinomycin and ceftriaxone) retain their activity against N. gonorrhoeae. To monitor drug resistance in N. gonorrhoeae several national and international programs have become functional. CONCLUSIONS Oral regimens for the treatment of gonorrhea are limited. At present to our knowledge ceftriaxone is the most reliable and available agent for the treatment of gonorrhea. To prevent the further emergence and international spread of drug resistance, and allow for the selection of appropriate treatments, a comprehensive global program is needed including surveillance for drug resistance in N. gonorrhoeae and collection of patient epidemiological data. Clinicians should effectively treat patients with gonorrhea, always being conscious of local trends of drug resistance in N. gonorrhoeae, and should perform culture and antimicrobial susceptibility testing in those with persistent gonorrhea after treatment.

Serum exosomal P-glycoprotein is a potential marker to diagnose docetaxel resistance and select a taxoid for patients with prostate cancer

OBJECTIVES Docetaxel is used as the first-line chemotherapy for castration-resistant prostate cancer (CRPC), but docetaxel resistance occurs in part owing to induction of P-glycoprotein (P-gp) encoded by multidrug resistance protein 1 (MDR1) gene. A recently developed taxane-cabazitaxel-has poor affinity for P-gp and is thereby effective in docetaxel-resistant CRPC. It has been recently demonstrated that exosomes in the body fluids could serve as a diagnostic marker because they contain proteins and RNAs specific to the cells from which they are derived. In this study, we aimed to investigate if P-gp in blood exosomes could be a marker to diagnose docetaxel resistance and select a taxoid for patients with CRPC. METHODS AND MATERIALS Exosomes were isolated by differential centrifugation from docetaxel-resistant prostate cancer (PC-3) cells (PC-3R) and their parental PC-3 cells and from the serum of patients. Silencing of P-gp was performed by small interfering RNA transfection. Protein expression was examined by Western blot analysis. Viability of cells treated with docetaxel or cabazitaxel was determined by water soluble tetrazolium salt (WST) assay. RESULTS The level of P-gp was higher in exosomes as well as cell lysates from PC-3R cells than in those from PC-3 cells. Cabazitaxel effectively killed PC-3R cells, and MDR1 knockdown improved the sensitivity of PC-3R cells to docetaxel but not to cabazitaxel. The P-gp level in blood exosomes was relatively higher in clinically docetaxel-resistant patients than in therapy-naïve patients. CONCLUSIONS Our results suggest that detection of P-gp in blood exosomes, which is involved in resistance to docetaxel but not to cabazitaxel, could be useful to diagnose docetaxel resistance and select an appropriate taxoid for patients with CRPC-docetaxel or cabazitaxel.

ETS1 promotes chemoresistance and invasion of paclitaxel-resistant, hormone-refractory PC3 prostate cancer cells by up-regulating MDR1 and MMP9 expression.

ETS1, which belongs to the ETS transcription factor family, plays important roles in diverse aspects of cancer such as drug resistance and metastasis. In the present study, we examined the functional roles of ETS1 in paclitaxel resistance and invasion using human prostate cancer PC3 cells and paclitaxel-resistant PC3PR cells established from PC3 cells. Our results showed that ETS1mRNA and protein expression was markedly up-regulated in paclitaxel-resistant PC3PR cells compared with paclitaxel-sensitive PC3 cells. The mRNA levels of MDR1 as well as MMP1, MMP3, MMP9 and uPA were positively correlated with that of ETS1. In PC3PR cells, silencing of ETS1 expression by siRNAs inhibited the activity of the MDR1 promoter containing ETS binding sites, reduced the mRNA and protein levels of MDR1 and suppressed paclitaxel resistance. Furthermore, ETS1 knockdown decreased secretion of MMP9 as well as its intracellular mRNA level, and dramatically inhibited invasion of PC3PR cells. Our results suggest that ETS1 promotes paclitaxel resistance and invasion in part by up-regulating MDR1 and MMP9 expression. Taken together, a novel therapeutic strategy targeting the ETS1 gene could be designed to overcome chemoresistance and metastasis of taxane-resistant, hormone-refractory prostate cancer.

Microbiological outcome of complicated urinary tract infections treated with levofloxacin: a pharmacokinetic/pharmacodynamic analysis.

The pharmacodynamic targets representing 90% probability thresholds for bacterial eradication were determined in patients with complicated urinary tract infections (UTIs) treated with 500mg of levofloxacin every 24h for 7-14 days. Of 241 pre-treatment strains from 156 patients, 21 strains persisted after treatment. In each patient, plasma concentrations of levofloxacin were simulated based on population pharmacokinetic parameters and patient-specific data. Minimum inhibitory concentrations (MICs) of levofloxacin for the pre-treatment strains determined in our previous study were used. The pharmacodynamic targets representing 90% probability thresholds for bacterial eradication were determined by logistic regression analyses. For all the isolates, Gram-negative bacilli, Gram-positive cocci, Escherichia coli and Enterococcus faecalis, the target values of the area under the concentration-time curve (AUC)/MIC were 14.65, 31.46, 4.85, 43.00 and 3.06, respectively, and the targets of the maximum plasma concentration (C(max))/MIC were 1.22, 2.74, 0.39, 3.61 and 0.25, respectively. Such thresholds of AUC/MIC and C(max)/MIC in complicated UTIs would be lower than those in infections of other sites. In particular, the C(max)/MIC thresholds for Gram-positive cocci and E. faecalis were <1. These findings suggested that, in addition to its plasma concentration, the high concentration of levofloxacin in the urine might play a role in eradicating bacteria.