Resistance-related Protein Research Articles

Long noncoding RNA OIP5-AS1 causes cisplatin resistance in osteosarcoma through inducing the LPAATβ/PI3K/AKT/mTOR signaling pathway by sponging the miR-340-5p.

The abnormal expression of long noncoding RNAs (lncRNAs) plays an important role in the regulation of human cancer progression and drug resistance. The lncRNA OPI5-AS1 is a crucial regulator in some cancers; however, its role in cisplatin resistance of osteosarcoma remains unclear. We found that OIP5-AS1 was significantly upregulated in cisplatin-resistant (CR) osteosarcoma cells MG63-CR and SaOS2-CR compared with the corresponding parental cells. OIP5-AS1 silencing suppressed cell growth in vitro and in vivo, and promoted apoptosis of MG63-CR and SaOS2-CR cells, indicating that knockdown of OIP5-AS1 significantly decreased cisplatin resistance in MG63-CR and SaOS2-CR cells. This conclusion was supported by the decreased expression of the drug resistance-related factors multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp) upon OIP5-AS1 silencing. In addition, OIP5-AS1 downregulation suppressed the PI3K/AKT/mTOR signaling pathway. Importantly, we demonstrated that OIP5-AS1 functions as a competing endogenous RNA of miR-340-5p and regulates the expression of lysophosphatidic acid acyltransferase (LPAATβ), which is a target of miR-340-5p. Moreover, downregulation of miR-340-5p partly reversed the inhibitory effect of OIP5-AS1 knockdown on the PI3K/AKT/mTOR pathway and therefore counteracted cisplatin resistance in MG63-CR and SaOS2-CR cells. In conclusion, OIP5-AS1 causes cisplatin resistance in osteosarcoma through inducing the LPAATβ/PI3K/AKT/mTOR signaling pathway by sponging the miR-340-5p. Our results contribute to a better understanding of the function and mechanism of OIP5-AS1 in osteosarcoma cisplatin resistance.

Proteomics of Bulked Rachides Combined with Documented QTL Uncovers Genotype Nonspecific Players of the Fusarium Head Blight Responses in Wheat.

Fusarium head blight (FHB) is a destructive disease of wheat that reduces yield and grain quality. High-throughput proteomic techniques have been used to identify a wide range of candidate proteins involved in host resistance. The majority of the published works on the proteomics of the wheat response to Fusarium graminearum infection are case specific. In the current study, a high-throughput quantitative label-free strategy was employed on bulked rachides of F. graminearum-infected wheat collected from multiple genotypes. Differentially accumulated proteins among the following four pools were identified: mock-inoculated FHB-resistant accessions (RM), mock-inoculated FHB-susceptible accessions (SM), F. graminearum-inoculated FHB-resistant accessions (RFg), and F. graminearum-inoculated FHB-susceptible accessions (SFg). Four pairs of comparisons were made: RFg versus RM, SFg versus SM, RM versus SM, and RFg versus SFg. Proteins were projected onto the consensus intervals of previously reported quantitative trait loci in the FHB-resistant pool by blasting against the Chinese Spring reference sequences. In addition to proteins previously reported in the host response to Fusarium spp., new candidates have emerged in association with resistance or susceptibility, including a group 3 late embryogenesis abundant as a resistance-related protein and a purple acid phosphatase as a susceptibility protein. The protein atlas presented here provides new perspectives on the interaction between F. graminearum and wheat.

Combining Next Generation Proteomics Platforms with Drug Sensitivity Resistance Testing Allows Identification of Physiologically Distinct Sub-Clones That Can Inform Therapeutic and Drug Development Strategies

Introduction: A hallmark of Multiple Myeloma (MM) is the sequel development of drug resistant phenotypes, which may be present initially or emerge during the course of treatment. These drug resistant phenotypes reflect the intra-tumor and inter-patient heterogeneity of this cancer. Most MM cells are sensitive to proteasome inhibitors (PIs), which have become the standard of care in the treatment of newly diagnosed and relapsed MM. However, resistance develops (intrinsic/acquired). Although several novel drugs have recently been approved or are in development for MM, there are few molecular indicators to guide treatment selection. To address this limitation we have combined mass spectrometry-based proteomics analysis together with ex vivo drug response profiles and clinical outcome to elucidate a best possible accurate phenotype of the resistant sub-clones, thus yielding a theranostic profile that will inform therapeutic and drug development strategies.Methods: We performed mass spectrometry-based proteomics analysis on plasma cells isolated from 38 adult MM patient bone marrow aspirates (CD138+). Samples were obtained at diagnosis or prior to commencing therapy. The participating subjects gave written informed consent in accordance with the Declaration of Helsinki that was approved by local ethics committees. For the proteomics analysis, peptides were purified using the filtered aided sample preparation (FASP) method. Subsequently, samples were prepared for label-free liquid chromatography mass spectrometry (LC-MS/MS) using a Thermo Scientific Q-Exactive MS mass spectrometer. Proteins were analysed using the MaxQuant and Perseus software for mass-spectrometry (MS)-based proteomics data analysis, UniProtKB-Swiss Prot database and KEGG Pathway database. In parallel, we undertook a comprehensive functional strategy to directly determine the drug dependency of myeloma plasma cells based on ex vivo drug sensitivity and resistance testing (DSRT)as previously described (1).Results: Our initial proteomic analysis was generated by examining MM patient plasma cells, grouped based on DSRT to 142 anticancer drugs including standard of care and investigational drugs. Each of the 142 drugs was tested over a 10,000-fold concentration range, allowing for the establishment of accurate dose-response curves for each drug in each patient. MM patients were stratified into four distinct subgroups as follows: highly sensitive (Group I), sensitive (Group II), resistant (Group III) or highly resistant (Group IV) to the panel of drugs tested. We then performed blinded analysis on the 4 groups of CD138+ plasma cells divided based on the ex vivo sensitivity profile, identifying a highly significant differential proteomic signature between the 4-chemosensitivity profiles, with Cell Adhesion Mediated-Drug Resistance (CAM-DR) related proteins (e.g. integrins αIIb and β3) significantly elevated in the highly resistant phenotype (Group IV). In addition our results showed that Group I patients displayed significant upregulation of cell proliferation proteins including: MCM2, FEN1, PCNA and RRM2. Furthermore, Group I patients have shorter Progression Free Survival (PFS) as well as Overall Survival (OS) compared to the other subgroups. Figure 1 shows the Heatmap summarizing the expression of proteins (log2 fold change) in the four distinct MM patient subgroups.Conclusions:Our findings suggest that combining a proteomics based study together with drug sensitivity and resistance testing allows for an iterative adjustment of therapies for patients with MM, one patient at a time, thus providing a theranostic approach. Our results suggest that the disease driving mechanisms in the patient subgroups are distinct, with highly resistant patients exhibiting cell adhesion mediated cytoprotection, while highly sensitive patients show an increased cell proliferation protein profile with shorter PFS and OS. Our study aims to guide treatment decisions for individual cancer patients coupled with monitoring of subsequent responses in patients to measure and understand the efficacy and mechanism of action of the drugs. Future work will include the establishment of flow cytometry-based screening assays to identify the different resistant phenotypes at diagnosis/relapse.

Curcumin Reverses 5-Fluorouracil Resistance by Promoting Human Colon Cancer HCT-8/5-FU Cell Apoptosis and Down-regulating Heat Shock Protein 27 and P-Glycoprotein.

To investigate the potential mechanisms that curcumin reverses 5-fluorouracil (5-FU) multidrug resistance (MDR). Cell growth and the inhibitory rate of curcumin (2-25 μg/mL) and/or 5-FU (0.05-1000 μg/mL) on human colon cancer HCT-8 and HCT-8/5-FU (5-FU-resistant cell line) were determined using cell counting kit-8 (CCK-8) assay. Apoptosis and cell cycle after 5-FU and/or curcumin treatment were detected by flow cytometry (FCM) and transmission electron microscopy (TEM). The expression of the multidrug resistance related factors p-glycoprotein (P-gp) and heat shock protein 27 (HSP-27) genes and proteins were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The inhibitory rate of curcumin or 5-FU on HCT-8 and HCT-8/5-FU cells proliferation at exponential phase were in a dosedependent manner, HCT-8 cell line was more sensitive to curcumin or 5-FU when compared the inhibitory rate of HCT-8/5-FU. The 50% inhibitory concentration (IC50) of combination 5-FU and curcumin (4.0 μg/mL) in HCT-8/5-FU was calculated as 179.26 μg/mL, with reversal fold of 1.85. Another IC50 of combination 5-FU and curcumin (5.5 μg/mL) in HCT-8/5-FU was calculated as 89.25 μg/mL, with reversal fold of 3.71. Synergistic effect of 5-FU and curcumin on HCT-8 and HCT-8/5-FU cells were found. The cell cycle analysis performed by FCM showed that HCT-8 and HCT-8/5-FU cells mostly accumulated at G0/G1 phase, which suggested a synergistic effect of curcumin and 5-FU to induce apoptosis. FCM analysis found that the percentage of apoptosis of cells treated with curcumin, 5-FU and their combination were significantly increased compared to the control group (P<0.05), and the percentage of apoptosis of the combination groups were slightly higher than other groups (P<0.05). The mRNA levels of P-gp (0.28±0.02) and HSP-27 (0.28±0.09) in HCT-8/5-FU cells treated with combination drugs were lower than cells treated with 5-FU alone (P-gp, 0.48±0.07, P=0.009; HSP-27, 0.57±0.10, P=0.007). The protein levels of P-gp (0.25±0.06) and HSP-27 (0.09±0.02) in HCT-8/5-FU cells treated with combination drugs were decreased when compared to 5-FU alone (P-gp, 0.46±0.02, P=0.005; HSP-27, 0.43±0.01, P=0.000). Curcumin can inhibit the proliferation of human colon cancer cells. Curcumin has the ability of reversal effects on the multidrug resistance of human colon cancer cells lines HCT-8/5-FU. Down-regulation of P-gp and HSP-27 may be the mechanism of curcumin reversing the drug resistance of HCT-8/5-FU to 5-FU.

Impact of Zn2+ on ABC Transporter Function in Intact Isolated Rat Brain Microvessels, Human Brain Capillary Endothelial Cells, and in Rat in Vivo.

ABCtransporters act as efflux pumps, thereby influencing the pharmacokinetics and efficacy of many drugs. Zinc (Zn) is an essential trace element contributing to cellular growth and differentiation. It is increasingly recognized as an intracellular messenger. The present study aims at investigating the impact of Zn2+ on the function and regulation of ABC transporters at the blood-brain barrier (BBB). ABC transporter function was first studied in isolated rat brain capillaries. Zn2+ rapidly stimulated the activity of the multidrug resistance-related protein 2 (Mrp2), p-glycoprotein (P-gp), and breast cancer resistance protein (Bcrp). These short-term effects were independent of transporter de novo synthesis but based on Zn2+ triggering intracellular signaling to stimulate basal transport activity. Studies focused on Mrp2 and P-gp showed that Zn2+ induced signaling through an endothelin receptor type B (ETB)/nitric oxide synthase (NOS)/protein kinase C (PKC) pathway and caused, specifically, an activation of the isoform PKCα. Studies revealed signaling through the phosphatidylinositol 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway, as well as induction of the downstream target serum- and glucocorticoid-inducible kinase 1 (SGK1). Short-term effects of Zn2+ were also demonstrated in human hCMEC/D3 cells. An initial in vivo study in rats suggested enhanced P-gp transport activity at the BBB due to elevated Zn2+ plasma levels. This work provides the first evidence for Zn2+ being a regulator of basal ABC transporter activity at the BBB, driving a rapid and nongenomic stimulation of transport function.

Expression of LRP Gene in Breast Cancer Patients Correlated with MRP1 as Two Independent Predictive Biomarkers in Breast Cancer

Background:Breast cancer is the most common malignancy in women. Multidrug resistance (MDR) is still a great obstacle of breast cancer chemotherapy. We have previously shown that multidrug resistance-associated protein 1 (MRP1) is associated with response to neoadjuvant chemotherapy. The lung resistance-related protein (LRP) is identified as a prognostic marker and response to treatment factor which has been studied mainly in hematological malignancy and leukemia. In this study, we aimed to analyze LRP expression and possible correlation between the expression level of this gene with MRP1 as a candidate marker for chemotherapy resistance.Materials and Methods:We collected 54 breast tumors and adjacent normal tissues from Iranian breast cancer patients and Real time RT-PCR was employed to measure the gene expression level in our samples.Results:MRP1 and LRP expression level were significantly lower in tumor tissues of the patients responding to chemotherapy compared to non-responding patients. No relation between the expression level of either of these genes and clinicopathology markers was found.Conclusion:Our results suggest that LRP gene expression is correlated to MRP1 in human breast cancer cells and may affect the clinical response to treatment.

Comparative transcriptome analyses of magainin I-susceptible and -resistant Escherichia coli strains

Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, the molecular mechanisms involved in Escherichia coli resistance to magainin I were investigated through comparative transcriptomics. Sub-inhibitory concentrations of magainin I were used to generate four experimental groups, including magainin I-susceptible E. coli, in the absence (C) and presence of magainin I (CM); and magainin I-resistant E. coli in the absence (R) and presence of magainin I (RM). The total RNA from each sample was extracted; cDNA libraries were constructed and further submitted for Illumina MiSeq sequencing. After RNA-seq data pre-processing and functional annotation, a total of 103 differentially expressed genes (DEGs) were identified, mainly related to bacterial metabolism. Moreover, down-regulation of cell motility and chaperone-related genes was observed in CM and RM, whereas cell communication, acid tolerance and multidrug efflux pump genes (ABC transporter, major facilitator and resistance-nodulation cell division superfamilies) were up-regulated in these same groups. DEGs from the C and R groups are related to basal levels of expression of homeostasis-related genes compared to CM and RM, suggesting that the presence of magainin I is required to change the transcriptomics panel in both C and R E. coli strains. These findings show the complexity of E. coli resistance to magainin I through the rearrangement of several metabolic pathways involved in bacterial physiology and drug response, also providing information on the development of novel antimicrobial strategies targeting resistance-related transcripts and proteins herein described.

Reversal of docetaxel resistance in prostate cancer by Notch signaling inhibition.

Acquired docetaxel (Doc) resistance in hormone-refractory prostate cancer (HRPC) remains an ongoing clinical challenge, resulting in failed chemotherapy and tumor recurrence. However, the mechanism of Doc-resistance development in prostate cancer cells is still unclear. Here, we observed a subpopulation of prostate cancer cells, in both Doc-resistant cell lines and the tumors of patients with HRPC, which show stem cell markers and greater tumorigenic potential. Those stem-like prostate cancer cells show high expression of ABCB1, which encodes multidrug resistance-related protein P-glycoprotein, leading to the Doc-resistance in prostate cancer. Moreover, we found that Notch signaling pathway activation in Doc-resistant cell lines and tumor tissues of patients with HRPC correlated with tumorigenicity and the development of Doc resistance. Here, we revealed that a combination of Doc and a Notch signaling inhibitor overcomes Doc resistance and increases the survival of mice with Doc-resistant xenografts. Therefore, targeting the Notch signaling pathway may be a promising strategy to overcome the Doc-resistant cancer in the clinic.

S100A9 regulates cisplatin chemosensitivity of squamous cervical cancer cells and related mechanism.

ObjectiveOur previous research has shown that the expression of S100 calcium-binding protein A9 (S100A9) in tumor cells was associated with neoadjuvant chemotherapy sensitivity in cervical squamous cell carcinoma. In the present study, we altered the expression of S100A9 through infecting lentivirus, investigated its effect on the chemosensitivity to cisplatin of cervical cancer cells and then made a primary exploration of the involved mechanism.Materials and methodsLentivirus was employed to upregulate and downregulate S100A9 expression in SiHa cells. The protein expression level of apoptotic-related proteins Bcl-2 and Bax, drug resistance-related proteins multiple drug resistance protein 1 (MRP1), P glycoprotein (P-gp), glutathione-S-transferase-π (GST-π), lung resistance-related protein (LRP), and FOXO1 signaling pathway related proteins was detected by Western blot. The CCK-8 assay was used to examine chemosensitivity to cisplatin, and the proportion of apoptosis cells was analyzed by the flow cytometry.ResultsS100A9 overexpression could obviously increase the IC50 value of SiHa cells to cisplatin and decrease the apoptosis rate induced by cisplatin. Downregulation of S100A9 led to the opposite results. In S100A9 overexpression SiHa cells, the expression level of Bcl-2, LRP, GST-π, p-AKT, p-ERK, p-FOXO1, and Nanog was significantly increased, while FOXO1 expression was decreased. The opposite results were observed in S100A9 knockdown SiHa cells.ConclusionDownregulation of S100A9 could significantly increase apoptosis rate, resulting in enhancing sensitivity of SiHa cells to cisplatin, which may be related to Bcl-2, GST-π, and LRP protein and by altering the AKT/ERK-FOXO1-Nanog signaling pathway.

Combination of dihydromyricetin and ondansetron strengthens antiproliferative efficiency of adriamycin in K562/ADR through downregulation of SORCIN: A new strategy of inhibiting P-glycoprotein.

Though the advancement of chemotherapy drugs alleviates the progress of cancer, long-term therapy with anticancer agents gradually leads to acquired multidrug resistance (MDR), which limits the survival outcomes in patients. It was shown that dihydromyricetin (DMY) could partly reverse MDR by suppressing P-glycoprotein (P-gp) and soluble resistance-related calcium-binding protein (SORCIN) independently. To reverse MDR more effectively, a new strategy was raised, that is, circumventing MDR by the coadministration of DMY and ondansetron (OND), a common antiemetic drug, during cancer chemotherapy. Meanwhile, the interior relation between P-gp and SORCIN was also revealed. The combination of DMY and OND strongly enhanced antiproliferative efficiency of adriamycin (ADR) because of the increasing accumulation of ADR in K562/ADR-resistant cell line. DMY could downregulate the expression of SORCIN and P-gp via the ERK/Akt pathways, whereas OND could not. In addition, it was proved that SORCIN suppressed ERK and Akt to inhibit P-gp by the silence of SORCIN, however, not vice versa. Finally, the combination of DMY, OND, and ADR led to G2/M cell cycle arrest and apoptosis via resuming P53 function and restraining relevant proteins expression. These fundamental findings provided a promising approach for further treatment of MDR.

Tumor Oxygenation and Hypoxia Inducible Factor-1 Functional Inhibition via a Reactive Oxygen Species Responsive Nanoplatform for Enhancing Radiation Therapy and Abscopal Effects.

Hypoxia, and hypoxia inducible factor-1 (HIF-1), can induce tumor resistance to radiation therapy. To overcome hypoxia-induced radiation resistance, recent studies have described nanosystems to improve tumor oxygenation for immobilizing DNA damage and simultaneously initiate oxygen-dependent HIF-1α degradation. However, HIF-1α degradation is incomplete during tumor oxygenation treatment alone. Therefore, tumor oxygenation combined with residual HIF-1 functional inhibition is crucial to optimizing therapeutic outcomes of radiotherapy. Here, a reactive oxygen species (ROS) responsive nanoplatform is reported to successfully add up tumor oxygenation and HIF-1 functional inhibition. This ROS responsive nanoplatform, based on manganese dioxide (MnO2) nanoparticles, delivers the HIF-1 inhibitor acriflavine and other hydrophilic cationic drugs to tumor tissues. After reacting with overexpressed hydrogen peroxide (H2O2) within tumor tissues, Mn2+ and oxygen molecules are released for magnetic resonance imaging and tumor oxygenation, respectively. Cooperating with the HIF-1 functional inhibition, the expression of tumor invasion-related signaling molecules (VEGF, MMP-9) is obviously decreased to reduce the risk of metastasis. Furthermore, the nanoplatform could relieve T-cell exhaustion via downregulation of PD-L1, whose effects are similar to the checkpoint inhibitor PD-L1 antibody, and subsequently activates tumor-specific immune responses against abscopal tumors. These therapeutic benefits including increased X-ray-induced damage, downregulated resistance, and T-cell exhaustion related proteins expression achieved synergistically the optimal inhibition of tumor growth. Overall, this designed ROS responsive nanoplatform is of great potential in the sensitization of radiation for combating primary and metastatic tumors.

Brassica oleracea resistance-related proteins identified at an early stage of black rot disease

Previously, we analyzed the protein profile of pooled samples (5, 10 and 15 days after inoculation) of Brassica oleracea inoculated with Xanthomonas campestris pv. campestris (Xcc). To search for proteins that may trigger resistance/defense response, here we analyzed B. oleracea plants at an early stage of infection (24 h after Xcc inoculation - hai). Photosynthesis-related proteins showed pronounced reduction in the susceptible plants. Epithiospecifier protein was detected only in resistant inoculated plants, and may influence Xcc colonization. We show that regulation of photosynthesis is crucial for a resistance response against Xcc, even at an early stage of infection.

Proteomic and transcriptomic approaches to identify resistance and susceptibility related proteins in contrasting rice genotypes infected with fungal pathogen Rhizoctonia solani

The devastating sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) causes major yield loss in most rice growing regions of the world. In this study, two moderately tolerant and four susceptible genotypes of rice were selected for R. solani induced proteome analysis using two-dimensional polyacrylamide gel electrophoresis. Forty five differentially expressed proteins (DEPs) were identified and analyzed by Mass Spectrometry. Based on their functions, these proteins were classified into different groups, viz., photosynthesis, resistance and pathogenesis, stress, cell wall metabolism and cytoskeleton development associated proteins, and hypothetical or uncharacterized proteins. Expression of 14 genes encoding DEPs was analyzed by quantitative PCR which showed consistency in transcripts and genes expression pattern. Furthermore, the expression of 16 other genes involved in diverse biological functions was analyzed. Up-regulation of these genes in the tolerant genotype Pankaj during sheath blight disease suggested efficient genetic regulation of this cultivar under stress. Also, expression analysis of conserved microRNAs (miRNAs) and their target genes revealed important role of miRNAs in post-transcriptional gene regulation during development of rice sheath blight disease. Genome-wide discovery of miRNAs and further characterization of DEPs and genes will help in better understanding of the molecular events during sheath blight disease development in rice.

Association Between First Episode Schizophrenia, Metabolic Syndrome and Insulin Resistance-Related Proteins in Female Balb/C Mice

Background: Metabolic syndrome is a group of different disorders mainly includes, insulin resistance, obesity, cerebrovascular disorders, dyslipidemia, which leads to increase mortality. Patients suffering from related psychotic disorders such as schizophrenia are at the higher risk of developing metabolic syndrome. The aim of this study was to evaluate the association between the first episode of schizophrenia, metabolic syndrome and insulin resistance-related proteins in blood and adipose tissue of mice.Materials and Methods: Twelve, female Balb/c mice were randomly divided into two groups; one group was injected intraperitoneal MK-801 (0.6mg/kg/d) to induce schizophrenia, and other group received the 0.9% normal saline for two weeks. Body weight, fasting blood glucose (FBG), oral glucose tolerance (OGT), and Homeostatic model assessment (HOMA), were observed. Blood and adipose tissue were collected and Western blotting was done to evaluate the insulin resistance related proteins (GGPPS, FAT, PTP-1B, GRK2, ATGL, FGF21, and PGC-1α) by using GAPDH as an internal standard. Results: There was a significant increase in mean body weight in schizophrenic group (21.76 vs 22.81, P=004). On day 14, the FBG, insulin concentrations and Homeostatic model assessment and insulin resistance (HOME-IR) were high in schizhphrenic group vs control group, e.g. 5.3±0.6 vs 3.47±0.2 (P=0.0001), 28.9±2.2 vs 23.3±0.6 (P&lt;0.005) and 9.2±1.3 vs 3.9±0.2 (P=0.0001) . Impaired glucose tolerance deranged from 4.8mmol/L to 6.4mmol/L. Western blotting showed a marked increase in the expression of GGPPS, FAT, ATGL, and FGF21 proteins in monocytes and PTP-1B, GRK2, and PGC-1α ratios in adipose tissues.Conclusion: There was a positive relation between schizophrenia and metabolic syndrome e.g. insulin resistance and obesity. Certain proteins in adipocytes and blood were responsible for causing insulin resistance. [GMJ.2018;7:e692]

Association Between First Episode Schizophrenia, Metabolic Syndrome and Insulin Resistance-Related Proteins in Female Balb/C Mice

Background: Metabolic syndrome is a group of different disorders mainly includes, insulin resistance, obesity, cerebrovascular disorders, dyslipidemia, which leads to increase mortality. Patients suffering from related psychotic disorders such as schizophrenia are at the higher risk of developing metabolic syndrome. The aim of this study was to evaluate the association between the first episode of schizophrenia, metabolic syndrome and insulin resistance-related proteins in blood and adipose tissue of mice.&#x0D; Materials and Methods: Twelve, female Balb/c mice were randomly divided into two groups; one group was injected intraperitoneal MK-801 (0.6mg/kg/d) to induce schizophrenia, and other group received the 0.9% normal saline for two weeks. Body weight, fasting blood glucose (FBG), oral glucose tolerance (OGT), and Homeostatic model assessment (HOMA), were observed. Blood and adipose tissue were collected and Western blotting was done to evaluate the insulin resistance related proteins (GGPPS, FAT, PTP-1B, GRK2, ATGL, FGF21, and PGC-1α) by using GAPDH as an internal standard. &#x0D; Results: There was a significant increase in mean body weight in schizophrenic group (21.76 vs 22.81, P=004). On day 14, the FBG, insulin concentrations and Homeostatic model assessment and insulin resistance (HOME-IR) were high in schizhphrenic group vs control group, e.g. 5.3±0.6 vs 3.47±0.2 (P=0.0001), 28.9±2.2 vs 23.3±0.6 (P&lt;0.005) and 9.2±1.3 vs 3.9±0.2 (P=0.0001) . Impaired glucose tolerance deranged from 4.8mmol/L to 6.4mmol/L. Western blotting showed a marked increase in the expression of GGPPS, FAT, ATGL, and FGF21 proteins in monocytes and PTP-1B, GRK2, and PGC-1α ratios in adipose tissues.&#x0D; Conclusion: There was a positive relation between schizophrenia and metabolic syndrome e.g. insulin resistance and obesity. Certain proteins in adipocytes and blood were responsible for causing insulin resistance. [GMJ.2018;7:e692]

Abstract 2669: Identification of a novel small-molecule inhibitor for treatment of human colon cancer patients

Abstract As the representative targeted anticancer drug for colon cancer patients, Erbitux is the EGFR targeted therapeutic antibody and used for treatment of KRAS wild-type colon cancer patients. Even some patients with KRas wt gene did not respond to Erbitux. However, there is no treatment available for Erbitux-resistant patient group with KRAS WT gene, which is almost 50% of KRAS WT gene holders. Recently, our team identified Erbitux primary resistant related proteins named as CRG (Cetuximab-Resistant Gene) by array analysis based Erbitux responder or nonresponder colon cancer patients derived tissues and confirmed by in vitro and in vivo assay system. Based on these results, we synthesized a novel series of CRG targeted inhibitor. CRG inhibitor (CRG i;WM compound) is a lead compound for treating colon cancer patients who do not respond to Erbitux and have KRAS wild-type gene. CRG i has potent in vitro enzyme activity and high anticancer activity against various colon cancer cell lines with good selectivity in in vitro and in vivo system. Furthermore, the compound has good potent ADME/Tox. profiles for optimized lead. CRG i also displays strong anticancer effect in in vivo xenograft models and patient-derived xenograft (PDX) models. In addition, CRG i shows promising signs of overcoming Erbitux resistance in CRG knockout cells-derived xenograft model. We continue to discover improved preclinical candidates with better selectivity and ADME/Tox. profiles and validate predictive biomarker in colon cancer patients. In these efforts, we found some compounds with better profiles than CRG i. We are also trying to develop small molecules having highly potent activity against mutant CRG. In conclusion, active CRG is a promising biomarker and target for Erbitux-resistant KRAS wt colon cancer patients. Our compounds can be promising therapeutic agents for Erbitux-resistant KRAS wt colon cancer patients. Citation Format: Chun-Ho Park, Sun-Chul Hur, Joseph Kim, Dae Hee Lee, Yoon Sun Park, Jae-Sik Shin, Seung-Woo Hong, Jai-Hee Moon, Hyojin Kim, So Hee Lee, Hyebin Park, Joonyee Jung, Mi Jin Kim, Sang Soo Park, Jun Ki Hong, Ji Hee Gong, Jieun Kim, Il-Whea Ku, Hyun Ho Lee, Dong-Hoon Jin. Identification of a novel small-molecule inhibitor for treatment of human colon cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2669.

Effect of AQP-5 silencing by siRNA interference on chemosensitivity of breast cancer cells.

ObjectivesBased on the functionality of AQP-5 characterized in various physiological processes, our study aimed to investigate the effect of AQP-5 silencing by siRNA interference on chemosensitivity of breast cancer cells.Materials and methodsThe expression levels of AQP-5 mRNA in different experimental groups were measured by reverse transcription PCR. The chemosensitivity of the cells to adriamycin (ADR) was detected by a CCK-8 kit. Cell invasion, migration, and apoptosis were assessed. Western blot was used to detect the expression levels of AQP-5, drug resistance-related protein, and apoptosis-related protein.ResultsThe expression level of AQP-5 in MCF-7/ADR cells was significantly reduced by AQP-5 siRNA transfection. The invasion and migration were significantly reduced in MCF-7/ADR after AQP-5 siRNA interference. AQP-5 silencing significantly increased the chemosensitivity of MCF-7/ADR cells to ADR and activated caspase-dependent apoptosis in MCF-7/ADR cells. AQP-5 silencing also decreased the expression levels of drug resistance-related proteins (P-gp and GST-π).ConclusionThe inhibition of AQP-5 expression may reverse the drug resistance and enhance the chemosensitivity of breast cancer cells.

Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins

Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. SignificanceEnterococcal infections, especially bacteremia and endocarditis, are challenging to treat because E. faecium have acquired resistance to multiple classes of antimicrobials, including ampicillin, aminoglycosides, and glycopeptides. Thus, research on different modes of enterococcal pathogenicity is warranted. This study utilized a proteomic approach to identify MV-associated proteins of different nosocomial E. faecium strains representing four clinically relevant sequence types (STs), namely ST17, ST18, ST78, and ST192. The presented data suggest that E. faecium MVs are involved in virulence and antimicrobial resistance.

Comparative Proteomic and Morphological Change Analyses of Staphylococcus aureus During Resuscitation From Prolonged Freezing.

When frozen, Staphylococcus aureus survives in a sublethally injured state. However, S. aureus can recover at a suitable temperature, which poses a threat to food safety. To elucidate the resuscitation mechanism of freezing survived S. aureus, we used cells stored at -18°C for 90 days as controls. After resuscitating the survived cells at 37°C, the viable cell numbers were determined on tryptic soy agar with 0.6% yeast extract (TSAYE), and the non-injured-cell numbers were determined on TSAYE supplemented with 10% NaCl. The results showed that the total viable cell number did not increase within the first 3 h of resuscitation, but the osmotic regulation ability of freezing survived cells gradually recovered to the level of healthy cells, which was evidenced by the lack of difference between the two samples seen by differential cell enumeration. Scanning electron microscopy (SEM) showed that, compared to late exponential stage cells, some frozen survived cells underwent splitting and cell lysis due to deep distortion and membrane rupture. Transmission electron microscopy (TEM) showed that, in most of the frozen survived cells, the nucleoids (low electronic density area) were loose, and the cytoplasmic matrices (high electronic density area) were sparse. Additionally, a gap was seen to form between the cytoplasmic membranes and the cell walls in the frozen survived cells. The morphological changes were restored when the survived cells were resuscitated at 37°C. We also analyzed the differential proteome after resuscitation using non-labeled high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results showed that, compared with freezing survived S. aureus cells, the cells resuscitated for 1 h had 45 upregulated and 73 downregulated proteins. The differentially expressed proteins were functionally categorized by gene ontology enrichment, KEGG pathway, and STRING analyses. Cell membrane synthesis-related proteins, oxidative stress resistance-related proteins, metabolism-related proteins, and virulence factors exhibited distinct expression patterns during resuscitation. These findings have implications in the understanding of the resuscitation mechanism of freezing survived S. aureus, which may facilitate the development of novel technologies for improved detection and control of foodborne pathogens in frozen food.

Association Between First Episode Schizophrenia, Metabolic Syndrome and Insulin Resistance-Related Proteins in Female Balb/C Mice.

Background: Metabolic syndrome is a group of different disorders mainly includes, insulin resistance, obesity, cerebrovascular disorders, dyslipidemia, which leads to increase mortality. Patients suffering from related psychotic disorders such as schizophrenia are at the higher risk of developing metabolic syndrome. The aim of this study was to evaluate the association between the first episode of schizophrenia, metabolic syndrome and insulin resistance-related proteins in blood and adipose tissue of mice. Materials and Methods: Twelve, female Balb/c mice were randomly divided into two groups; one group was injected intraperitoneal MK-801(0.6mg/kg/d) to induce schizophrenia, and other group received the 0.9% normal saline for two weeks. Body weight, fasting blood glucose (FBG), oral glucose tolerance (OGT), and Homeostatic model assessment (HOMA), were observed. Blood and adipose tissue were collected and Western blotting was done to evaluate the insulin resistance related proteins (GGPPS, FAT, PTP-1B, GRK2, ATGL, FGF21, and PGC-1α) by using GAPDH as an internal standard. Results: There was a significant increase in mean body weight in schizophrenic group (21.76 vs 22.81, P=004). On day 14, the FBG, insulin concentrations and Homeostatic model assessment and insulin resistance (HOME-IR) were high in schizhphrenic group vs control group, e.g. 5.3±0.6 vs 3.47±0.2 (P=0.0001), 28.9±2.2 vs 23.3±0.6 (P<0.005) and 9.2±1.3 vs 3.9±0.2 (P=0.0001) . Impaired glucose tolerance deranged from 4.8mmol/L to 6.4mmol/L. Western blotting showed a marked increase in the expression of GGPPS, FAT, ATGL, and FGF21 proteins in monocytes and PTP-1B, GRK2, and PGC-1α ratios in adipose tissues. Conclusion: There was a positive relation between schizophrenia and metabolic syndrome e.g. insulin resistance and obesity. Certain proteins in adipocytes and blood were responsible for causing insulin resistance.