Multidrug Resistance Properties Research Articles

Prevalence of Extended Spectrum b-Lactamases (ESBLs) Producers Among Gram-Negative Bacilli in Urinary Tract Infections

Background: Antimicrobial resistance is now proclaimed as the most important challenge worldwide being faced by humanity in its fight against infectious diseases. Extended Spectrum b-Lactamases (ESBLs) producing organisms are increasing in number and causing more severe infections because of their continuous mutation and multidrug resistance property which make its treatment difficult.Aims: The present study was undertaken to detect the prevalence of the ESBLs producing bacteria in urinary tract infection.Methods: Isolated gram-negative bacteria initially screened by Minimum Inhibitory Concentration (MIC) ESBLs breakpoints. Then suspected ESBLs producers were confirmed by phenotypic confirmatory test.Results: 71 (59.17%) bacterial strains were isolated from 120 urine samples of patients of suspected urinary tract infection of which 66(92.96%) were gram-negative and 05(07.04%) were gram-positive. Among the isolated gram-negative bacteria 63(95.45%) were found suspected ESBLs producers of which 35(55.56%) were found as confirmed ESBL producers. The prevalence of ESBLs producing organisms in the present study were found to be 53.03% and Klebsiella spp. as most prevalent ESBLs producers.Conclusion: It is essential to report ESBL production along with routine sensitivity reporting, which will help the clinician in prescribing the proper antibiotics.Chatt Maa Shi Hosp Med Coll J; Vol.14 (2); Jul 2015; Page 17-20

Gain-of-function p53 mutant with 21-bp deletion confers susceptibility to multidrug resistance in MCF-7 cells.

The majority of p53 mutations, which are responsible for gain of oncogenic function, are missense mutations in hotspot codons. However, in our previous study, we demonstrated that a deletion spanning codons 127-133 in the p53 gene (designated as del p53) was detected in doxorubicin-resistant MCF-7 cell lines following various induction processes. In the present study, we aimed to investigate the role of del p53 and its association with the proliferation, metastasis and drug resistance of MCF-7 cells. The MCF-7/del p53 cell line is a representative of the del p53 stably expressed clones which were constructed by transfection of the del p53-containing construct into MCF-7/wt cells. Markers of multidrug resistance (MDR), epithelial-mesenchymal transition (EMT) and stem cell-like properties were examined in the MCF-7/del p53 cells. The results revealed that the MCF-7/del p53 cells expressed full-length p53 and del p53 mRNA and protein, as well as P-glycoprotein (P-gp). The MCF-7/del p53 cells acquired resistance to doxorubicin with increased P-gp efflux function. Using a transient expression assay, the mdr1 promoter was found to be significantly activated by external or integrated del p53 (P<0.001). The inhibition of nuclear factor (NF)-κB by cyclosporine sensitized the MCF-7/del p53 cells to doxorubicin toxicity. In addition, the morphological characteristics of the MCF-7/del p53 and MCF-7/adr were similar. EMT was observed in the MCF-7/del p53 cells as demonstrated by the presence of the mesenchymal markers, Slug and vimentin, and the decrease in the epithelial marker, cadherin 1, type 1, E-cadherin (CDH1), as well as an enhanced migration ability (P<0.001). Furthermore, the number of cells expressing the cancer stem cell-like marker, CD44, increased, accompanied by mammosphere formation. Taken together, these findings indicate that the expression of del p53 in MCF-7/del p53 cells enables the cells to partially acquire doxorubicin resistance characteristics of the MCF-7/adr cells. Thus, del p53 may be an important factor in non-invasive MCF-7 cells, activating NF-κB signaling and the mdr1 promoter and partially attributing to EMT; the cells thus acquire stem cell‑like properties, which facilitates drug resistance. Therefore, the 21-bp deletion of p53 may prove to be a therapeutic strategy with which to prevent cancer cells from acquiring resistance to drugs.

A polymeric nanoparticle formulation of curcumin in combination with sorafenib synergistically inhibits tumor growth and metastasis in an orthotopic model of human hepatocellular carcinoma

Curcumin, a yellow polyphenol extracted from the rhizome of turmeric root (Curcuma longa) has potent anti-cancer properties in many types of tumors with ability to reverse multidrug resistance of cancer cells. However, widespread clinical application of this agent in cancer and other diseases has been limited due to its poor aqueous solubility. The recent findings of polymeric nanoparticle formulation of curcumin (NFC) have shown the potential for circumventing the problem of poor solubility, however evidences for NFC's anti-cancer and reverse multidrug resistance properties are lacking. Here we provide models of human hepatocellular carcinoma (HCC), the most common form of primary liver cancer, in vitro and in vivo to evaluate the efficacy of NFC alone and in combination with sorafenib, a kinase inhibitor approved for treatment of HCC. Results showed that NFC not only inhibited the proliferation and invasion of HCC cell lines in vitro, but also drastically suppressed primary tumor growth and lung metastases in vivo. Moreover, in combination with sorafenib, NFC induced HCC cell apoptosis and cell cycle arrest. Mechanistically, NFC and sorafenib synergistically down-regulated the expression of MMP9 via NF-κB/p65 signaling pathway. Furthermore, the combination therapy significantly decreased the population of CD133-positive HCC cells, which have been reported as cancer initiating cells in HCC. Taken together, NanoCurcumin provides an opportunity to expand the clinical repertoire of this agent. Additional studies utilizing a combination of NanoCurcumin and sorafenib in HCC are needed for further clinical development.

Molecular mechanism of cisplatin to enhance the ability of TRAIL in reversing multidrug resistance in gastric cancer cells

To study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells. MTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis. After combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3β signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations. The activation of DNA-PKcs/Akt/GSK-3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

Marine natural products as breast cancer resistance protein inhibitors.

Breast cancer resistance protein (BCRP) is a protein belonging to the ATP-binding cassette (ABC) transporter superfamily that has clinical relevance due to its multi-drug resistance properties in cancer. BCRP can be associated with clinical cancer drug resistance, in particular acute myelogenous or acute lymphocytic leukemias. The overexpression of BCRP contributes to the resistance of several chemotherapeutic drugs, such as topotecan, methotrexate, mitoxantrone, doxorubicin and daunorubicin. The Food and Drugs Administration has already recognized that BCRP is clinically one of the most important drug transporters, mainly because it leads to a reduction of clinical efficacy of various anticancer drugs through its ATP-dependent drug efflux pump function as well as its apparent participation in drug resistance. This review article aims to summarize the different research findings on marine natural products with BCRP inhibiting activity. In this sense, the potential modulation of physiological targets of BCRP by natural or synthetic compounds offers a great possibility for the discovery of new drugs and valuable research tools to recognize the function of the complex ABC-transporters.

Nano-Coupling of Cephalosporin Antibiotics with Fe&amp;lt;SUB&amp;gt;3&amp;lt;/SUB&amp;gt;O&amp;lt;SUB&amp;gt;4&amp;lt;/SUB&amp;gt; Nanoparticles: Trojan Horse Approach in Antimicrobial Chemotherapy of Infections Caused by &amp;lt;i&amp;gt;Klebsiella spp&amp;lt;/i&amp;gt;.

In the present study we had an aim to develop the methods of functionalizing the surface of magnetite nanoparticles with cefotaxime and ceftriaxone antibiotics. The quantitative analysis of the nanostructured cephalosporins was determined by Atom Absorbance Spectroscopy (AAS) and based on the Lambert-Beer law. The engineered nanostructures were tested on gram-negative microorganisms Klebsiella spp., of Enterobacteriaceae, and gram-positive bacteria Staphylococcus aureus, each having multi-drug resistance properties.

Abstract 1924: Discovery and characterization of EMT positive and chemo-resistant novel population of HCC like stem cells

Abstract Increasing evidence suggests that tumor heterogeneity facilitates relapse of liver cancer in patients during the post therapeutic period. This concept of heterogeneity has gained increasing support from the liver cancer stem cells (LCSC), which have the potential of differentiation and genetic and epigenetic alterations to adapt to the tumor microenvironment. With the technological advancement, researchers were able to identify new population of LCSCs besides CD133+ clones by discovering new surface markers-CD90, EpCAM, CD13 and CD24. These studies corroborated that CD133- population is highly heterogeneous and might have some additional markers. Recently, by screening colorectal cancer patient samples we discovered a novel surface marker vimentin (Vim) on metastatic liver nodules. We further initiated screening of primary hepatocellular carcinoma murine liver tumor samples and discovered the surface expression of Vim in both species of HCC. Interestingly, we purified Vim+CD133- from primary liver tumor cell suspensions. Using sphere assays, limited dilution assays and staining of the stem cell markers Sox2, Nanog and Oct4A, we showed that Vim+CD133- cells have stem cell like properties similar to Vim-CD133+ population. Then we performed differentiation assays with Vim+CD133- cells under suitable condition and showed hepatocyte like phenotypes by upregulating protein expression of albumin, periodic acid schiff's staining and cholangiocyte type morphologies- tubular and star shaped structures. Several studies correlated that cells undergo epithelial-mesenchymal transition (EMT)phenotypes when vimentin is expressed on their surface. Our investigation revealed that Vim+CD133- population supported EMT like phenotypes by presence of the twist and β-catenin inside the nucleus and absence of E-cadherin on cell surface. Contrary to this Vim-CD133+ population did not show EMT like phenotypes. In next step of our characterization of Vim+CD133- cells, we investigated its chemoresistance properties as LCSCs are well known to cause relapse in patients. We treated these cells with highly toxic doses of 5 Fluorouracil and cisplatin for 4 days and interestingly observed significantly more viability compared to Vim-CD133+ population. We also validated multidrug resistance properties such as expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic markers in Vim+CD133- cells. Finally, we validated their tumorigenic potential using anchorage independent assays and in vivo inoculation in immunocompromised NOD-SCID mice. Collectively our studies characterized a novel population Vim+CD133- in heterogeneous LCSCs, which showed EMT like phenotypes and higher chemo-resistance compared to Vim-CD133+ cells. In the era of personalized cancer therapy, this population should be considered as superior druggable target compared to our canonical Vim-CD133+ cells to obtain greater therapeutic outcome. Citation Format: Abhisek Mitra, Arun Satelli, Xueqing Xia, Shulin Li. Discovery and characterization of EMT positive and chemo-resistant novel population of HCC like stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1924. doi:10.1158/1538-7445.AM2014-1924

In vitro activity of taurine-5-bromosalicylaldehyde Schiff base against planktonic and biofilm cultures of methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, implicated in both community and hospital acquired infections. The therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming more difficult because of multidrug resistance and strong biofilmforming properties. Schiff bases have attracted attention as promising antibacterial agents. In this study, we investigated the in vitro activity of taurine-5-bromosalicylaldehyde Schiff base (TBSSB) against MRSA. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) were determined using a microtiter broth dilution method. TBSSB effectively inhibited planktonic MRSA, with an MIC of 32 μg/ml. The time-kill curve confirmed that TBSSB exhibited bactericidal activity against MRSA. TBSSB was also found to significantly inhibit MRSA biofilm formation at 24 h, especially at 1×MIC and sub-MIC levels. Furthermore, scanning electron microscopy and transmission electron microscopy showed remarkable morphological and ultrastructural changes on the MRSA cell surface, due to exposure to TBSSB. This study indicated that TBSSB may be an effective bactericidal agent against MRSA.

Prevalence of exfoliative toxin A and B genes in Staphylococcus aureus isolated from clinical specimens

The pathogenicity of Staphylococcus aureus is based on the production of various virulence factors. The frequency of these factors can markedly differ according to the geographical region. In this study, we investigated the prevalence of two frequent isoforms of exfoliative toxins and mecA genes using PCR in 197 clinical isolates obtained from clinical samples during the years 2011 and 2012. The samples were obtained from an educational hospital in northern Tehran, Iran. In addition, the antibiotic susceptibility pattern was studied for each isolate using the disc diffusion method. In this study, 186 (94.4%), 15 (7.6%) and 172 (86.3%) of the 197 isolates expressed the eta, etb and mecA genes, respectively. In addition, 164 (88.2%) and 12 (80%) strains, which harbored the eta and etb genes, respectively, were MRSA (methicillin resistant S. aureus). Furthermore, the prevalence of the mecA, eta and etb genes was higher among the wound samples (61.2%, 55.8% and 6.09%, respectively). We observed high rates of MDR (multi drug resistance) among our isolates. A significant correlation was detected between the presence of the mecA gene and the resistance to oxacillin, gentamicin, kanamycin, erythromycin, tetracycline, cotrimoxazole, clindamycin, and cephazolin as well as the multi-drug resistant property (P<0.05). In addition to penicillin, the MDR properties and resistances to the tested antibiotics in the etb-positive strains were significantly lower compared to the etb-negative strains (P<0.05). The prevalence of the eta, etb and mecA genes might be due to the specific geographic region.

Identification, Molecular Detection and Antibiogram Profile of Bacteria Isolated from California Mastitis Test Positive Milk Samples of Crossbred Cows of Satkhira District in Bangladesh

Present study was conducted for the isolation, identification, molecular detection and antibiotic sensitivity patterns of the bacteria present in the California Mastitis Test (CMT) positive milk samples of clinical and sub-clinical mastitic crossbred (Friesian cross and Sahiwal cross) cows of Satkhira district. A total number of 598 quarters milk samples from 150 cows were tested by CMT kit, among them 82 quarters from 52 cows were positive for CMT. The bacteria isolated from 52 CMT positive milk samples were coagulase positive Staphylococcus aureus 27 (49.09%) followed by Escherichia coli 15 (27.27%), coagulase negative Staphylococcus (CNS) spp. 10 (18.18%) and Bacillus spp. 3 (5.45%) respectively. In this present study only 13 (48.15%) positive Staphylococcus aureus were positive against nuc gene by PCR. All the isolates of E.coli were further confirmed by PCR with species specific 16srRNA PCR. None of the isolates of E.coli revealed positivity in PCR by stx1 and stx2 genes. All the coagulase positive Staphylococcus aureus isolates were found sensitive to 6 antibiotics and resistant to 5 antibiotics. The Coagulase negative Staphylococcus spp. was also sensitive to 7 antibiotics and acquired resistant properties against 3 antibiotics. Most E.coli isolates were found resistant against 9 antibiotics already possessed multidrug resistant properties and sensitive to only 3 antibiotics. Bacillus spp. isolated from milk samples was highly sensitive against 5 antibiotics compare to other antibiotics of the panel. Antibiogram profile of this study revealed that, all the isolated bacteria were sensitive to Ciprofloxacin only.

Isolation and Identification of non-plasmid Multidrug Resistant E.coli from Poultry Wastes in Chittagong Region, Bangladesh

In two branches of poultry culture; small local ones and big industrial ones, tetracycline is a common antibiotic, which has been taken as a standard antibiotic in this study. 20 isolates were taken from big poultry farms like Agha Ltd and Denm Poultry. 10 isolates were taken from small local poultry farms like Rahat Poultry and Star Poultry. After collection of samples, total numbers of bacteria with and without tetracycline were counted. In both cases numerous bacterial growths were observed. The normal dose of tetracycline is 30 μg/ml which failed extremely to regulate high bacterial growth. Two dilutions (10-3 and 10-4) of sample 1, 2, 3 and 4 were taken and allowed to grow at different concentrations of tetracycline like 30,60 and 100 μg/ml, where bacterial growth was observed. High concentration of antibiotics for example, above 100 μg/ml may be harmful to humans and animals. After performing sensitivity test against other commonly used antibiotics in poultry, it was found that isolated tetracycline-resistant E. coli were 100% resistant to penicillin and erythromycin,100 sensitive to imipenem, 93.34% resistant to tetracycline, 23.03% resistant to gentamycin and 53.33% resistant to chloramphenicol. These indicated the multidrug resistant property of isolates. Subsequent agarose gel electrophoresis showed no plasmid DNA band in the gel indicating non-existence of any bacterial plasmid and also proved that the observed resistance was chromosomal gene-mediated or at least not plasmid mediated.

English

Cholera is a devastating diarrheal disease caused by V. cholera. Two biotypes of V. cholerae O1, classical and El-Tor, are distinguished. Each biotype is further subdivided into two serotypes, termed Inaba and Ogawa. As large deltaic areas of the Ganges and Brahmaputra rivers are considered to be the homeland of cholera, objective of our study was to detect the circulating strain of Vibrio causing Cholera outbreaks in different pockets of West Bengal. Water samples collected from the water sources of the affected areas and stool samples and or rectal swabs of suspected cases were tested according to standard bacteriological protocol. In July 2008, Cholera outbreak was caused by Vibrio cholerae O1 El-Tor Inaba serotype in Darjeeling district affecting 176 persons. Mean age of the affected people was 15years. Thereafter in the June, 2010, there was an outbreak of Vibrio cholerae El Tor Ogawa affecting 87 people in Maldah. Mean age of the cases was 25years. Similar type of Vibrio cholerae O1 El-Tor Ogawa strain was isolated in the outbreak of June to September 2012 in Maldah with lower mean age of cases i.e.7years. A total of 93 patients suffered from Cholera during this outbreak. Cholera outbreak in West Bengal was caused by classical Vibrio cholerae O1 Ogawa serotype affecting 803 people in North 24 Paragana in October, 2013. Mean age of the patients was 31year. As classical Vibrio causes more severe disease than El-Tor, its reemergence with multidrug resistant properties is no doubt an upcoming threat.

Prevalence and Characterization of Multidrug-Resistant ZoonoticEnterobacterspp. in Poultry of Bangladesh

Poultry and poultry products are major contributors of zoonotic pathogens. Limited data are available on Enterobacter spp. as a potent zoonotic pathogen in poultry. The present study is a first endeavor on the emergence of multidrug-resistant zoonotic Enterobacter spp. and its prevalence arising from poultry in Bangladesh. Cloacal swabs from poultry samples of five different farms at Savar, Dhaka, Bangladesh were collected and from 106 isolates, 18 presumptive Enterobacter spp. were obtained. Antibiogram using 19 used antibiotics belonging to 15 major groups revealed that all of the 18 isolates were completely resistant to penicillin and rifampicin, but differed in their drug resistance pattern against ampicillin (94.4%), clindamycin (94.4%), erythromycin (94.4%), vancomycin (88.9%), sulfonamides (72.2%), imipenem (66.6%), streptomycin (55.6%), nitrofurantoin (33.3%), doxycycline (33.3%), tetracyclines (33.3%), cefepime (11.1%), and gentamicin (5.6%). All Enterobacter spp. were found to be plasmid free, implying that multidrug-resistant properties are chromosomal borne. The vanA and sulI were detected by polymerase chain reaction assay in 17 and 13 isolates, respectively. Amplified ribosomal DNA restriction analysis and randomly amplified polymorphic DNA distributed the 18 multidrug-resistant Enterobacter spp. into three genotypes. Phylogenetic analysis of the representatives of the three genotypes using partial 16S rRNA gene sequence (approximately 900 bp) showed that the genotypically diverse groups belonged to Enterobacter hormaechei, E. cloacae, and E. cancerogenus, respectively. The clinical significance of the close relative Enterobacter spp. is indicative of their zoonotic potential. Therefore, urgent intervention is required to limit the emergence and spread of these bacteria in poultry feed as well as prudent use of antibiotics among poultry farmers in Bangladesh.

Antimicrobial Activity of CaO Nanoparticles

The high degree of microbial diseases and their multidrug resistant properties make the researchers to develop new class of antimicrobial agents. A modern and innovative approach of drug development is the use of metallic nanoparticles as new formulations of antimicrobial agents. In this study, microwave irradiated CaO nanoparticles (CaO-NPs) were used to determine their antimicrobial efficacy against gram negative and gram positive bacteria, as well as pathogenic yeast. The physiochemical properties of CaO-NPs were characterized by means of X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). The nanoparticles consist of well dispersed agglomerates of grains with a narrow size distribution of 14-24 nm. The prepared CaO-NPs showed much higher antimicrobial activity against Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus epidermidis (MTCC 435) in comparision to Candida tropicalis (NCIM 3110). The minimum inhibitory concentration (MIC) value of CaO-NPs was found within the range of 2-8 mM for all the above tested strains. This bioactive nanoparticle also inhibits the biofilm formation and may have future applications cheap and non toxic as antimicrobial drug for skin care product development.

JS5-3 - Biological Properties and Targeting of Liver Cancer Stem Cells

ABSTRACT Cancer stem cells (CSCs) are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. In liver cancer, we have identified CD13 (aminopeptidase N) as a candidate liver cancer stem cell marker by the screening of cell surface markers based on microarray database and cell cycle analysis. CD13+ liver CSCs localized in side population (SP) fraction, known as multi-drug resistant cell fraction by ATP-binding cassette (ABC) transporters expression, and also localized in G0 cell fraction. In vivo study clarified CD13+ cells as 5-bromo-2′-deoxy-uridine (BrdU)-long retaining cells and this finding was compatible with the data of cell cycle analysis. In vivo chemosensitivity assay showed high multi-drug resistant property of CD13+ cells. Treatment of liver cancer cells with CD13 inhibitor and CD13 neutralizing antibody efficiently induced cellular apoptosis in vitro suggested CD13 as a liver CSCs target. Immunohistochemical analysis of clinical liver cancer specimens revealed that CD13+ cells typically formed cellular clusters in cancer foci, and by the following treatment, CD13+ cells were enriched along the fibrous capsule. To assess whether regulation of CD13 disrupts cancer maintenances, in vivo cancer treatment model was established. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. Additionally, CD13 inhibition suppressed their self-renewing and tumor-initiating abilities. As CD13 plays key roles in glutathione pathway, expression status of reactive oxygen species (ROS) was assessed. Expression of ROS was low in CD13+ cells compared with CD13- cells, and was increased by CD13 inhibition, suggested that CD13+ cells acquire chemo-resistant properties by ROS regulation. Finally, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.

Emergence of multi drug resistant nonfermentative Acinetobacter spp in a tertiary care hospital

Background Acinetobacter are gram negative, catalase positive, oxidase negative, nonmotile, non fermenting cocobacilli. The emergence of Acinetobacter infection is uncommon in organ systems that have a high fluid content. The nosocomial infections including, respiratory tract, CSF, peritoneal fluid, urinary tract, and endotracheal aspirtes (ET) are the place where they have the capabilities to accumulate and cause in-hospital and in-community infections. The rate of emergence of Acinetobacter spp with multi drug resistance property is increasing in different geographical regions. Due to the properties of MDR the recent treatments of such infections has become difficult. In the present study evaluation of emergence of MDR nonfermentative Acinetobacter spp was done from the niche of organ system of patients with high fluid content.

PPARγ agonists regulate the expression of stemness and differentiation genes in brain tumour stem cells

Background:Brain tumour stem cells (BTSCs) are a small population of cancer cells that exhibit self-renewal, multi-drug resistance, and recurrence properties. We have shown earlier that peroxisome proliferator-activated receptor gamma (PPARγ) agonists inhibit the expansion of BTSCs in T98G and U87MG glioma. In this study, we analysed the influence of PPARγ agonists on the expression of stemness and differentiation genes in BTSCs.Methods:The BTSCs were isolated from T98G and DB29 glioma cells, and cultured in neurobasal medium with epidermal growth factor+basic fibroblast growth factor. Proliferation was measured by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2 H-5-tetrazolio]-1,3-benzene disulphonate) and 3H thymidine uptake assays, and gene expression was analysed by quantitative reverse--transcription PCR and Taqman array. The expression of CD133, SRY box 2, and nanog homeobox (Nanog) was also evaluated by western blotting, immunostaining, and flow cytometry.Results:We found that PPARγ agonists, ciglitazone and 15-deoxy-Δ12,14-ProstaglandinJ2, inhibited cell viability and proliferation of T98G- and DB29-BTSCs. The PPARγ agonists reduced the expansion of CD133+ BTSCs and altered the expression of stemness and differentiation genes. They also inhibited Sox2 while enhancing Nanog expression in BTSCs.Conclusion:These findings highlight that PPARγ agonists inhibit BTSC proliferation in association with altered expression of Sox2, Nanog, and other stemness genes. Therefore, targeting stemness genes in BTSCs could be a novel strategy in the treatment of glioblastoma.

Induction of dendritic cells with multidrug resistance from K562/MDR1 cells

To induce the differentiation of K562/MDR1 cells into dendritic cells (DC) with multidrug resistance property. K562/MDR1 cells and K562 cells were cultured in the presence of GM-CSF and IL-4 to generate DC and matured by TNF-α. On d14 K562/MDR1-DC and K562-DC cells were harvested and the expressions of CD1a, CD83, CD80, CD86, HLA-ABC and HLA-DR were assessed by flow cytometry (FCM). The antigen presentation function of K562/MDR1-DC and K562-DC was determined by allogenic mixed lymphocyte reaction (Allo-MLR). The expression of P-glycoprotein and the intracellular accumulation of daunorubicin (DNR) were detected by FCM. The sensitivity of K562/MDR1-DC and K562-DC cell to vincristine, adriamycin was measured using MTT assay. Both K562/MDR1 and K562 cells were differentiated into dendritic cells in the presence of cytokine cocktails, showing the morphologic and immunophenotypic characteristics of DC. K562/MDR1-DC more markedly enhanced proliferation of allogeneic lymphocytes in MLR than K562-DC. High level expression of P-glycoprotein and efflux of DNR were demonstrated in K562/MDR1-DC. K562/MDR1-DC showed multidrug resistance property, with higher IC(50) to VCR and ADM than that of K562-DCs. K562/MDR1 cells can be differentiated into DC with the presence of cytokines, the induced K562/MDR1-DC cells express high level of P-glycoprotein and acquire the multidrug resistance property.

A novel combination of TRAIL and doxorubicin enhances antitumor effect based on passivetumor-targeting of liposomes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel anticanceragent for non-small cell lung cancer (NSCLC). However, approximately half of NSCLC celllines are highly resistant to TRAIL. Doxorubicin (DOX) can sensitize NSCLC cells toTRAIL-induced apoptosis, indicating the possibility of combination therapy. Unfortunately,the therapeutic effect of a DOX and TRAIL combination is limited by multiple factorsincluding the short serum half-life of TRAIL, poor compliance and applicationdifficulty in the clinic, chronic DOX-induced cardiac toxicity, and the multidrugresistance (MDR) property of NSCLC cells. To solve such problems, we developed thecombination of TRAIL liposomes (TRAIL-LP) and DOX liposomes (DOX-LP).An in vitro cytotoxicity study indicated that DOX-LP sensitized the NSCLCcell line A-549 to TRAIL-LP-induced apoptosis. Furthermore, this combinationtherapy of TRAIL-LP and DOX-LP displayed a stronger antitumor effect onNSCLC in xenografted mice when compared with free drugs or liposomal drugsalone. Therefore, the TRAIL-LP and DOX-LP combination therapy has excellentpotential to become a new therapeutic approach for patients with advanced NSCLC.

TRAIL and doxorubicin combination enhances anti-glioblastoma effect based on passive tumor targeting of liposomes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel anticancer agent for glioblastoma multiforme (GBM). Some GBM cell lines, however, are relatively resistant to TRAIL. Doxorubicin (DOX) can sensitize GBM cells to TRAIL-induced apoptosis, indicating that the combination of DOX and TRAIL may be an effective strategy to kill TRAIL-resistant GBM cells. However, the therapeutic effect is limited by the short serum half-life of TRAIL, chronic cardiac toxicity of DOX, multidrug resistance (MDR) property of GBM cells and poor drug delivery across the blood-brain barrier (BBB). To solve such problems, combination treatment of TRAIL liposomes (TRAIL-LP) and DOX liposomes (DOX-LP) were developed for the first time. The in vitro cytotoxicity study indicated that DOX-LP sensitized GBM cell line U87MG but not normal bovine caruncular epithelial cells (BCECs) to TRAIL-LP-induced apoptosis, demonstrating the safety of the combination treatment. This sensitization was accompanied by up-regulation of death receptor 5 (DR5) expression and caspase activation. Furthermore, the combination therapy of TRAIL-LP and DOX-LP displayed stronger anti-GBM effect than free drugs or liposomal drugs alone in vivo. In summary, the combination treatment reported here showed improved therapeutic effect on GBM. Therefore, it has good potential to become a new therapeutic approach for patients with GBM.