Lung Resistance-related Protein Research Articles

A transverse colon lipoma complicated with an adenoma is successfully and safely resected en bloc by endoscopic mucosal resection during water immersion

The B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs.We assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases.BMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues.Our study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

B-cell-specific Moloney murine leukemia virus integration site 1 knockdown impairs adriamycin resistance of gastric cancer cells

Background and study aimsThe B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs. Patients and methodsWe assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases. ResultsBMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues. ConclusionOur study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

Expression of Runt-related Transcription Factor 3 in Human Colon Cancer Cell Line HCT-116 Resistant to 5-Fluorouracil and the Mechanism of Drug Resistance

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC50)of 5-FU on the parent line HCT-116 and drug-resistant line HCT-116/5-FU.The cell growth curve was established for the calculation of population doubling time(TD).The mRNA levels and protein levels of RUNX3,P-glycoprotein(P-gp),multidrug resistance-associated protein 1(MRP1),and lung resistance-related protein(LRP)in HCT-116 and HCT-116/5-FU cells were determined by qRT-PCR and Western blotting,respectively.The RUNX3 expression in HCT-116 cells was knocked down by siRNA technique,and the cells were divided into RUNX3 knockdown groups(si-RUNX3-1 group and si-RUNX3-2 group)and negative control group(si-NC group).The knockdown efficiency was verified by qRT-PCR at the mRNA level and Western blotting at the protein level.The IC50 in si-RUNX3 groups and si-NC group was determined with CCK-8 method,and the expression of P-gp,MRP1,and LRP in the two groups was detected by Western blotting.Results A stable human colon cancer drug-resistant cell line HCT-116/5-FU was successfully constructed.HCT-116/5-FU showed the TD 1.38 times as long as that of HCT-116(P=0.002)and changed morphology.The mRNA level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116 cells(P=0.048),and those of P-gp(P=0.008),MRP1(P=0.001),and LRP(P=0.001)showed the opposite trend.The protein level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116(P<0.001),and those of P-gp,MRP1,and LRP presented the opposite trend(all P<0.001).The HCT-116 cell model with low expression of RUNX3 was successfully established.The mRNA level of RUNX3 had no significant difference between si-RUNX3-1 group and si-NC group(P=0.064),while the level in si-RUNX3-2 group was significantly lower than that in si-NC group(P=0.034).The protein levels of RUNX3 in si-RUNX3-1 group and si-RUNX3-2 group were lower than that in si-NC group(both P<0.001).The results demonstrated higher knocking efficiency in si-RUNX3-2 group,which was thus selected to complete the follow-up test.The IC50 of si-RUNX3 group was significantly higher than that of si-NC group(P<0.001),which indicated that the down-regulated expression of RUNX3 could reduce the sensitivity of HCT-116 cells to 5-FU.The relative protein levels of P-gp,MRP1,and LRP in si-RUNX3 group were significantly higher than those in si-NC group(all P<0.001).Conclusion The down-regulation of RUNX3 expression can reduce the sensitivity of HCT-116 cells to 5-FU,which is considered to be related to the up-regulated expression of P-gp,MRP1,and LRP.

Circular RNAcirc_0076305 Promotes Cisplatin (DDP) Resistance of Non-Small Cell Lung Cancer Cells by Regulating ABCC1 Through miR-186-5p.

Background: Lung cancer is a social problem of increasing concern, and non-small cell lung cancer (NSCLC) accounts for 80%-85% incidence of lung cancer. Cisplatin (DDP) is reported as a first-line chemotherapy drug for NSCLC, but the resistance has became a main obstacle for NSCLC treatment. The high level of circular RNA circ_0076305 was related to the DDP resistance in NSCLC. However, the mechanism of circ_0076305 remains unclear in DDP resistance of NSCLC. Materials and Methods: Exosomes were detected by a transmission electron microscope and nanoparticle tracking analysis. The protein levels of CD63, CD81, P-glycoprotein (P-gp), Lung resistance-related protein, and ATP-binding cassette subfamily C member 1 (ABCC1) were examined by Western blot assay. Circ_0076305, microRNA-186-5p (miR-186-5p), and ABCC1 levels were tested by real-time quantitative polymerase chain reaction. DDP resistance was examined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay. The binding relationship between miR-186-5p and circ_0076305 or ABCC1 was predicted by circRNA interactome or starBase, and then verified by dual-luciferase reporter and RNA immunoprecipitation assays. The effect of circ_0076305 on DDP resistance in NSCLC was examined by xenograft tumor model in vivo. Results: Circ_0076305 was increased in NSCLC cell-derived exosomes, DDP-resistant NSCLC tissues and cells. Circ_0076305 knockdown elevated DDP sensitivity in vitro. Mechanically, circ_0076305 enhanced ABCC1 expression through sponging miR-186-5p, thus regulating DDP resistance of NSCLC. Furthermore, circ_0076305 silencing improved DDP sensitivity of NSCLC in vivo. Conclusion: The results from this study disclosed that circ_0076305 knockdown improved DDP sensitivity by the miR-186-5p/ABCC1 axis in NSCLC, hinting a potential circRNA-targeted therapy for NSCLC.

Grape seed proanthocyanidin extract reverses multidrug resistance in HL-60/ADR cells via inhibition of the PI3K/Akt signaling pathway

Background and purposeMultidrug resistance (MDR) is a great challenge and obstacle in cancer treatment. It is a common problem in the treatment of acute myeloid leukemia (AML). Whether grape seed proanthocyanidin extract (GSPE) could reverse MDR in patients with AML is still unknown. The aim of this study was to investigate the MDR reverse ability of GSPE and its possible mechanism in vitro. Materials and methodsHuman leukemia cell line HL-60 cells and HL-ADR cells were used. MTT assay were employed to identify the cytotoxic effects of different chemotherapeutic drugs and reverse ability of GSPE. Flow cytometry assays were used to verify the cell apoptosis induced by GSPE. MDR-related genes expression was tested by real-time polymerase chain reaction (Q-PCR). MDR-related protein expression was assessed by Western blotting assays. The genes and their related protein expression of multidrug resistance-associated protein 1 (MRP1), multidrug resistance protein 1 (MDR1) and lung resistance-related protein (LRP) were tested in this study. Key resultsWe found that HL-60/ADR cells were resistant to a variety of chemotherapeutic drugs, including cytarabine (Ara-C), adriamycin (ADR), vincristine (VCR), daunorubicin (DNR), mitoxantrone (MTZ), pirarubicin (THP), homoharringtonine (HHT) and etoposide (VP16). Co-treatment with GSPE could significant lower the IC50 of Ara-C and ADR in HL-60/ADR cells (P < 0.01). MDR related mRNA and their protein expression of MRP1 and MDR1 were significant highly expressed in HL-60/ADR cells than HL-60 cells (P < 0.01). But only protein expression of LRP was higher in HL-60/ADR cells than HL-60 cells (P < 0.05). GSPE could induce a higher intracellular level of ADR in HL-60/ADR cells. It could also inhibit Akt phosphorylation resulted in the down regulation of MRP1, MDR1 and LRP and induce cell apoptosis. 25.0 μg/mL GSPE significant inhibited the Akt phosphorylation (P < 0.05). Conclusion and implicationsGSPE-reversed MDR of HL-60/ADR cells might be associated with the inhibition of the PI3K/Akt signaling pathway, which resulted in the down-regulation the expression of MRP1, MDR1 and LRP. These results provide that GSPE may serve as a combination therapy in AML chemotherapy for future study.

The Effects Of Sevoflurane On The Progression And Cisplatinum Sensitivity Of Cervical Cancer Cells.

ObjectiveTo investigate the effect of sevoflurane on the progression of cervical cancer cells, and to explore its effect on the cisplatinum (DDP) sensitivity in cervical cancer cells and underlying mechanism.MethodsSiha and Hela cervical cancer cells were cultured and treated with 3% sevoflurane, 10 μmol/L DDP, or the co-treatment of sevoflurane and DDP, respectively. Cell proliferation was evaluated by the CCK8 assay. Cell apoptosis was assessed by flow cytometry. Cell migration was detected by wound healing assay. The expression of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-2 associated X (BAX), Ezrin, matrix metalloproteinase 2 (MMP2), lung resistance-related protein (LRP), multiple drug resistance protein 1 (MRP1), glutathione-S-transferase-π (GST-π), and P glycoprotein (P-gp) protein was determined by Western blotting.ResultsAfter treated with sevoflurane, cell proliferation and migration rate in Siha and Hela cells were significantly elevated, while cell apoptosis was decreased. In addition, the expression of migration-related protein Ezrin and MMP2 was increased accordingly, apoptotic-related protein BCL-2 expression was also increased while BAX protein expression was decreased after sevoflurane treatment. The proliferation, migration rate, and apoptosis of Siha and Hela cells in sevoflurane plus DDP group were not significantly different with those in DDP group. There was no significant difference in apoptotic-related protein, migration-related protein, and drug resistance-associated proteins expression between DDP treatment group and combined treatment group.ConclusionSevoflurane promotes the progression but has no effect on the cisplatinum sensitivity in cervical cancer cells.

Polydatin enhances the chemosensitivity of osteosarcoma cells to paclitaxel.

Despite improvements in the prognosis of osteosarcoma patients, chemotherapy fails in a considerable number of cases due to drug resistance. The development of novel agents may enhance chemosensitivity. This study explored the anticancer function of polydatin and its ability-in combination with paclitaxel-to overcome drug resistance in human osteosarcoma U-2OS and MG-63 cell lines. A cell proliferation assay (celll counting kit-8), a cell-cycle assay, an apoptosis assay (annexin V-fluorescein isothiocyanate/propidium iodide), and a cell migration assay (Transwell) were used to analyze cell activity. Western blot analysis and quantitative reverse-transcription polymerase chain reaction were performed to examine function-related mRNA and protein levels. Treatment with polydatin suppressed cell growth and migration in both cell lines. Moreover, polydatin induced cell apoptosis in both parental and paclitaxel-resistant cells, and cell-cycle arrest in the S phase. Furthermore, it altered the expression of various proteins associated with cell growth (Ki67, p21, cyclin A, cyclin E, and cyclin-dependent kinase 2), migration (matrix metalloproteinase-2 [MMP-2], MMP-9, and tissue inhibitor of metalloproteinase-1), apoptosis (poly[ADP-ribose] polymerase 1 and caspase 3), and drug resistance (p-glycoprotein 1, lung resistance-related protein 1, growth arrest and DNA damage-45α, glutathione S-transferase π, and heat shock protein 27) in paclitaxel-resistant osteosarcoma cells. Cells transfected with myr-Akt caused obvious upregulation and activation of Akt, which were significantly attenuated via polydatin treatment. In conclusion, polydatin may enhance the chemosensitivity of osteosarcoma cells to paclitaxel.

Nanoporous gold electrode prepared from two-step square wave voltammetry (SWV) and its application for electrochemical DNA biosensing of lung resistance related protein (LRP) gene

In this work, considering with the existing inherent deficiencies in the preparation of current porous gold electrode, uniform and ordered nanoporous gold (NPG) electrode prepared from two-step square wave voltammetry (SWV) was developed. The formation process, morphology and electrochemical properties were carefully characterized. Compared with naked gold electrode, NPG electrode illustrated the characteristics of improved surface area, enhanced signal response and repeatable preparation. Using NPG electrode as active surface, a new electrochemical DNA biosensor was established for the detection of lung resistance related protein (LRP) gene combined with thionine as an electroactive indicator. Likewise, the electrochemical behavior of the interaction between thionine and DNA molecular could be improved on this NPG electrode to enhance the detection performance of LRP gene. In a wide range from 200 f. to 7.5nM, the logarithm concentration of LRP gene was linear with the reduction peak current of thionine, and a detection limit of 60 f. was obtained. As a result, it had provided a good basis of experimental research for LRP gene detection. Meanwhile, this technique could be expected to be applied into the real analysis of LRP gene in clinical peripheral blood samples.

Lung resistance-related protein (LRP) predicts favorable therapeutic outcome in Acute Myeloid Leukemia

There is conflicting evidence that MDR1, MRP2 and LRP expression is responsible for chemotherapy resistance. We conducted this study to explore their role in AML therapy outcomes. Bone marrow and peripheral blood samples of 90 AML patients, receiving chemotherapy, were analyzed by real time PCR. Gene expression was calculated by the 2−ΔΔCt method. The patients who had a persistent remission were labelled ‘Good Responder’ (GRes) whereas, those with relapse or drug resistance were labelled ‘Poor Responders’ (PRes). Higher LRP expression in bone marrow, but not in peripheral blood, was positively associated with persistent remission (p = 0.001), GRes (p = 0.002), 1-year overall as well as disease-free survival (p = 0.02 and p = 0.007, respectively). Marrow and blood MDR1 and MRP2 expression did not differ significantly between the above groups. Logistic regression analysis showed that only a diagnosis of acute promyelocytic leukemia (APL; M3) or high marrow LRP expression significantly predicted a favorable therapeutic outcome. This is the first report showing that high bone marrow LRP expression predicts significant favorable therapeutic outcome. Peripheral blood LRP expression as well as marrow and blood MDR1 and MRP2 expression have no predictive value in AML patients treated with standard dose cytarabine and daunorubicin 3+7 regimen.

Poor expression of microRNA-135b results in the inhibition of cisplatin resistance and proliferation and induces the apoptosis of gastric cancer cells through MST1-mediated MAPK signaling pathway.

Gastric cancer (GC) has been classified as the fourth leading cause of cancer-related deaths worldwide. Due to their ability to suppress the expression of target genes, microRNAs (miRNAs) are listed as one of the key elements involved in the formation and development of tumors. This study was therefore conducted to investigate the effects of microRNA-135b (miR-135b) on cisplatin [ cis-diamminedichloroplatinum (CDDP)] resistance of GC cells through the MAPK signaling pathway by targeting mammalian ste20-like kinase 1 (MST1). A microarray-based gene expression analysis was performed to screen the GC-related differentially expressed genes. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was performed to determine the sensitivity of GC cells to CDDP. The bioinformatics database and dual luciferase reporter gene assay were used to check whether MST1 was a direct target gene of miR-135b. GC cell lines were prepared with high CDDP resistance, after which they were cultured and transfected respectively, followed by the administration of transfected cells into nude mice and subsequent treatment with CDDP in an attempt to identify the underlying mechanisms and functions of miR-135b in relation to MST1 in GC progression. The results were highly indicative of the crucial role played by MST1 in the development of GC and the sensitivity of GC to CDDP. miR-135b was found to regulate MST1, which in turn had an impact on the development of GC. MKN28 was observed to be most sensitive to CDDP, whereas MKN45 presented with the poorest sensitivity to CDDP. Furthermore, the down-regulation of miR-135b resulted in inactivation of the MAPK signaling pathway; increased the expression of MST1 and Bax; and decreased expression of p-p38MAPK, p-ERK1/2, P-glycoprotein, p38MAPK, ERK1/2, multidrug resistance protein 1, multidrug resistance-associated protein 1, lung resistance-related protein, and Bcl-2, thus inhibiting CDDP resistance of GC cells. The down-regulation of miR-135b also restrained cell proliferation and induced the apoptosis rate of GC cells. In summary, the results of this study showed that the down-regulation of miR-135b induced apoptosis, and it inhibited proliferation and CDDP resistance of GC cells by inactivating the MAPK signaling pathway and increasing the expression of MST1.-Zhou, J., Chen, Q. Poor expression of microRNA-135b results in the inhibition of cisplatin resistance and proliferation and induces the apoptosis of gastric cancer cells through MST1-mediated MAPK signaling pathway.

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.

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.

Characterization of Drug Resistance Protein Expression Levels in Human Melanoma Cells and Subsequent Treatment with Antifungal Agents

The multidrug resistance (MDR) phenotype allows cancer cells to display decreased sensitivity to chemotherapy. A well‐characterized drug resistance gene in cancer is MDR1, which encodes for the p‐glycoprotein/ATP‐binding cassette (ABC) transporter. Lesser known are the roles of the multidrug resistance‐associated protein 1 (MRP1) and lung resistance‐related protein (LRP) in melanoma. Because of the previously demonstrated presence of MRP1 and LRP in human melanoma cells and the continued difficulty of overcoming MDR in melanoma treatment, here we characterized the expression levels of drug resistance genes in melanoma cell lines derived from human patients and determined their susceptibility to novel antifungal treatment. Expression levels were determined by real‐time PCR (qPCR) and immunoblot analyses. Susceptibility of cell lines to antifungal agents was analyzed by flow cytometry, cell proliferation assays, and immunoblotting after drug treatments.Several lines of human melanoma and normal skin cells were analyzed. MDR1 expression was observed in multiple melanoma lines, while increased levels of MRP1 were observed in normal skin cells and one melanoma line. Interestingly, LRP expression was highest in normal skin cells and several melanoma lines. Novel treatment with clotrimazole, an agent previously shown to eradicate melanoma cells, caused significant decreases in proliferation and increases in cell death in all MDR melanoma lines. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cells. These results indicated that clotrimazole selectively induces cytotoxicity in drug‐resistant melanoma lines. However, decreased efficacy was observed in melanoma lines that overexpressed LRP, as compared to all other melanoma lines that overexpressed MDR1 and/or MRP1. These results indicated that LRP, mostly known for facilitating drug resistance in lung and breast cancer, may also have a significant role in the ability of melanoma cells to resist the effects of conventional melanoma treatments.In conclusion, this study demonstrated the existence and differential expression levels of MDR1, MRP1, and LRP in melanoma and normal skin cell lines. Although all melanoma lines were susceptible to treatment with clotrimazole, decreased efficacy was observed in melanoma lines that overexpressed LRP. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug‐resistant human melanoma cells. However, these preliminary studies also indicated that LRP is potentially a significant facilitator of drug resistance in human melanoma.Support or Funding InformationThis research was funded by the Ohio Northern University Summer Research Stipend.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression and Functions of Formyl Peptide Receptor 1 in Drug-Resistant Bladder Cancer.

Objective:To explore the correlation of formyl peptide receptor 1 expression with drug resistance and the functions of formyl peptide receptor 1 in drug-resistant bladder cancer.Methods:Expression of formyl peptide receptor 1 in T24 and T24/DDP cisplatin-resistant bladder cancer cell lines was tested by quantitative real-time Polymerase Chain Reaction and Western blotting. After incubation of T24/DDP with N-formyl-Met-Leu-Phe, the phosphor proteins were tested by Western blot analysis. We characterized the functions of formyl peptide receptor 1 in T24/DDP cells by assessing proliferation, migration, and changes of cell cycles.Results:Formyl peptide receptor 1 was expressed in both T24 and T24/DDP, and it was overexpressed in T24/DDP compared with T24. Formyl peptide receptor 1 activation promoted the expression of the messenger RNA of resistance-related proteins, such as multidrug resistance-associated protein 1 (MRP1) and lung resistance-related protein (LRP). The expression of 4 signal pathway proteins were upregulated: signal transducer and activator of transcription 3, Janus kinase 2, extracellular regulated protein kinases, and protein kinase B, while the expression of phosphatidylinositol 3-kinase was observed to be downregulated in drug-resistant bladder cancer cells. Formyl peptide receptor 1 activation also improved the expression of phospho-signal transducer and activator of transcription 3 and phospho-extracellular regulated protein kinases 1/2 and promoted the proliferation and migration of T24/DDP cells. In addition, formyl peptide receptor 1 inhibition led to the change in the cell cycle in T24/DDP.Conclusions:The overexpression of formyl peptide receptor 1 may be related to drug-resistant bladder cancer and promotes the deterioration of drug-resistant bladder cancer.

Niclosamide enhances the cytotoxic effect of cisplatin in cisplatin-resistant human lung cancer cells via suppression of lung resistance-related protein and c-myc.

Lung cancer is a leading cause of cancer-associated mortality worldwide. The cisplatin (DDP)-based chemotherapy remains the foundation of treatment for the majority of patients affected by advanced non-small cell lung cancer (NSCLC). However, DDP-resistance limits the clinical utility of this drug in patients with advanced NSCLC. The aim of the present study was to investigate the inhibitory effect of niclosamide on human lung cancer cell growth and to investigate the possible underlying mechanism. The effects of niclosamide on the proliferation of human lung adenocarcinoma (A549) and DDP-resistant (CR) human lung adenocarcinoma (A549/DDP) cells were examined by Cell Counting kit-8 assay. The impact of niclosamide on the apoptosis of A549/DDP cells was detected by Annexin V-fluorescein isothiocyanate/propidium iodide assay. The expression levels of cisplatin-resistant-associated molecules (lung resistance-related protein and c-myc) following niclosamide treatment in A549/DDP cells were evaluated by western blot analysis. The results indicated that niclosamide in combination with DDP demonstrated a synergistic effect in A549/DDP cells and directly induced apoptosis, which may be associated with caspase-3 activation. Furthermore, niclosamide decreased the expression level of c-myc protein, which may influence DDP sensitivity of A549/DDP cells. Thus, the present study indicates that niclosamide combined with DDP exerts a synergistic effect in cisplatin-resistant lung cancer cells and may present as a promising drug candidate in lung cancer therapy.

Kinesin KIF4A is associated with chemotherapeutic drug resistance by regulating intracellular trafficking of lung resistance-related protein.

Multidrug resistance (MDR) is the major impediment to cancer chemotherapy. The expression of lung resistance-related protein (LRP), a non-ATP-binding cassette (ABC) transporter, is high in tumor cells, resulting in their resistance to a variety of cytotoxic drugs. However, the function of LRP in tumor drug resistance is not yet explicit. Our previous studies had shown that Kinesin KIF4A was overexpressed in cisplatin (DDP)-resistant human lung adenocarcinoma cells (A549/DDP cells) compared with A549 cells. The expression of KIF4A in A549 or A549/DDP cells significantly affects cisplatin resistance but the detailed mechanisms remain unclear. Here, we performed co-immunoprecipitation experiments to show that the tail domain of KIF4A interacted with the N-terminal of LRP. Immunofluorescence images showed that both the ability of binding to LRP and the motility of KIF4A were essential for the dispersed cytoplasm distribution of LRP. Altogether, our results shed light on a potential mechanism in that motor protein KIF4A promotes drug resistance of lung adenocarcinoma cells through transporting LRP-based vaults along microtubules towards the cell membrane. Thus KIF4A might be a cisplatin resistance-associated protein and serves as a potential target for chemotherapeutic drug resistance in lung cancer.

Association between Twist and multidrug resistance gene-associated proteins in Taxol®-resistant MCF-7 cells and a 293 cell model of Twist overexpression.

Multidrug resistance (MDR) severely limits the effectiveness of chemotherapy. Previous studies have identified Twist as a key factor of acquired MDR in breast, gastric and prostate cancer. However, the underlying mechanisms of action of Twist in MDR remain unclear. In the present study, the expression levels of MDR-associated proteins, including lung resistance-related protein (LRP), topoisomerase IIα (TOPO IIα), MDR-associated protein (MRP) and P-glycoprotein (P-gp), and the expression of Twist in cancerous tissues and pericancerous tissues of human breast cancer, were examined. In order to simulate Taxol® resistance in cells, a Taxol®-resistant human mammary adenocarcinoma cell subline (MCF-7/Taxol®) was established by repeatedly exposing MCF-7 cells to high concentrations of Taxol® (up to 15 µg/ml). Twist was also overexpressed in 293 cells by transfecting this cell line with pcDNA5/FRT/TO vector containing full-length hTwist cDNA to explore the dynamic association between Twist and MDR gene-associated proteins. It was identified that the expression levels of Twist, TOPO IIα, MRP and P-gp were upregulated and LRP was downregulated in human breast cancer tissues, which was consistent with the expression of these proteins in the Taxol®-resistant MCF-7 cell model. Notably, the overexpression of Twist in 293 cells increased the resistance to Taxol®, Trichostatin A and 5-fluorouracil, and also upregulated the expression of MRP and P-gp. Taken together, these data demonstrated that Twist may promote drug resistance in cells and cancer tissues through regulating the expression of MDR gene-associated proteins, which may assist in understanding the mechanisms of action of Twist in drug resistance.

Abstract 16: Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP)

Abstract Drug resistance to chemotherapy, mediated in part by overexpression of P-glycoprotein (MDR-1), multidrug resistance-associated protein (MRP), or lung resistance-realted protein (LRP), continues to be a major problem in the treatment of melanoma. Because we previously demonstrated the ability of clotrimazole to enhance cytotoxicity in human metastatic melanoma cells, here we evaluated the ability of clotrimazole to treat drug-resistant human melanoma cells that overexpress MRP and/or LRP. We utilized human melanoma cell lines that overexpress either MDR or LRP, and human melanoma cell lines that overexpress both. Cell growth and cell death were analyzed by flow cytometry, proliferation assays, and immunoblotting after drug treatments and RNA interference. Clotrimazole caused significant decreases in proliferation and increases in cell death in both drug-sensitive melanoma lines and those overexpressing MRP and LRP. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cell lines. These results indicate that clotrimazole selectively induces cytotoxicity in both drug-sensitive and drug-resistant melanoma lines. As clotrimazole is known to inhibit various transient receptor potential (TRP) ion channels, we pretreated these melanoma lines with TRP inhibitors or RNAi. Inhibitors of the TRP melastatin-8 channel (TRPM8) or TRP vanilloid-1 (TRPV1) failed to produce comparable levels of cell death caused by clotrimazole. However, RNAi silencing of the TRPM2 cation channel caused significant levels of cell death in both drug-sensitive and drug-resistant melanoma lines. These results indicated that inhibition of TRPM2 channels may have a primary role in the ability of clotrimazole to treat drug-sensitive and drug-resistant melanoma lines. In conclusion, this study demonstrated that clotrimazole selectively increases cell death in drug-resistant melanoma lines, with minimal deleterious effects in normal skin cells. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug-resistant human melanoma cells. Thus, these preliminary studies indicate that clotrimazole has the potential to successfully treat drug resistant melanoma in the future. Citation Format: Steven D. Blake, Shelby G. McKamey, Christopher M. Tweed, David W. Koh. Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2017-16

Visfatin mediates doxorubicin resistance in human colorectal cancer cells via up regulation of multidrug resistance 1 (MDR1).

Colorectal cancer (CRC) is one of the prevalent and deadly cancers worldwide. Chemotherapy resistance is one of the most challenging problems for CRC and other cancer treatments. Recent studies indicated that increasing levels of visfatin are correlated with worse clinical prognosis of CRC patients, while the effects and mechanisms of visfatin on progression of CRC remain unclear. Our present study established doxorubicin (Dox)-resistant CRC HCT-116 and SW480 cells (named HCT-116 Dox/R and SW480 Dox/R). The expression of visfatin, while not IL-6, IL-8, or TGF-β, in CRC Dox-resistant cells was significantly greater than that in their parental cells, while knockdown of visfatin by its specific siRNAs can elevate Dox sensitivity of CRC-resistant cells. In addition, si-visfatin can significantly down regulate the expression of multidrug resistance 1 (MDR1), while not multidrug resistance-associated protein 1 or lung resistance-related protein, in both HCT-116 Dox/R and SW480 Dox/R cells. Visfatin can regulate the transcription of MDR1 via modulating its promoter activities. Si-visfatin can also decrease the activation and nuclear localization of p65, one of the most important transcription factors for the expression of MDR1. Chromatin immunoprecipitation (ChIP) indicated that si-visfatin can suppress the binding between p65 and MDR1 promoter. Collectively, our present study revealed that visfatin mediates the Dox resistance of CRC cells via up regulation of MDR1. It indicated that targeted inhibition of visfatin might be helpful for overcoming Dox resistance of CRC therapy.

Identification of Sensitivity Predictors of Neoadjuvant Chemotherapy for the Treatment of Adenocarcinoma of Gastroesophageal Junction.

The identification of reliable predictors of chemotherapy sensitivity and early screening of adenocarcinoma of gastroesophageal junction (AGEJ) patients who are resistant to chemotherapy has become an important area of clinical and translational research. We aimed to investigate the predictive value of seven cancer-associated cellular proteins for neoadjuvant chemotherapy in AGEJ patients. Clinical data of 93 patients who received neoadjuvant chemotherapy for locally advanced AGEJ between June 2010 and December 2014 were reviewed. All patients were administered the combination regimen of S-1 and oxaliplatin (SOX). Expression of P-glycoprotein (P-gp), glutathione S-transferase-π (GST-π), topoisomerase II (topo II), multidrug resistance gene-associated protein (MRP), lung resistance-related protein (LRP), Ki-67, and p53 was determined by immunohistochemistry (IHC) in AGEJ tissues before neoadjuvant chemotherapy. Chemotherapeutic efficacy was evaluated according to RECIST 1.0 standards and histopathological results, and the relationship between the expression of the cellular proteins and chemotherapy efficacy was analyzed. The SOX regimen was associated with an overall response rate of 46.2%. The frequency of expression of the seven cancer-associated factors in the AGEJ tissues was as follows: P-gp, 64.5%; GST-π, 39.8%; topo II, 72.0%; MRP, 33.3%; LRP, 68.8%; Ki-67, 62.4%; and p53, 40.9%. Expression of Ki-67 (p = 0.003) and p53 (p = 0.009) was significantly correlated with chemotherapy sensitivity. Elevated Ki-67 expression and decreased p53 expression predict for SOX insensitivity in AGEJ, and the cellular expression of these respective proteins may provide a useful reference for designing individualized chemotherapy regimens for AGEJ patients in the future.