<b>OBJECTIVEb>To predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin (L-OHP) in human colorectal cancer cells.

<b>METHODSb>L-OHP-resistant human colorectal cancer cell line (HCT116/L-OHP) was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control (NC-miRNA) were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPover-expressing miR-145 and HCT116/L-OHP. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98(GPR98) as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPover-expressing GPR98 and HCT116/L-OHP. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHP) were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation (A value) and sensitivity to L-OHP (the lower the IC50, the stronger the sensitivity) in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein (gp), multiple drug-resistance protein 1(MRP1), cancer-inhibition gene PTEN.

<b>RESULTSb>HCT116/L-OHP cell line was successfully established with ICof (42.34±1.05) mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than (9.81±0.95) mg/L (t=39.784, P=0.000) and lower than 1.00±0.09 (t=13.021, P=0.000) in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPcells were established successfully, with relative miR-145 expression of 10.01±1.05, which was higher than 1.06±0.14 in HCT116/L-OHPand 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHPcells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHP(mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHPcells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHPcells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHPcells was lower as compared to HCT116/L-OHPcells (1.41±0.16 vs. 1.98±0.13, P<0.05).

<b>CONCLUSIONb>MiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.

ATP Binding Cassette Transporter, Sub-Family B ; drug effects ; ATP-Binding Cassette, Sub-Family B, Member 1 ; drug effects ; Cell Line, Tumor ; drug effects ; physiology ; Colorectal Neoplasms ; physiopathology ; Down-Regulation ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; physiology ; HCT116 Cells ; drug effects ; physiology ; Humans ; In Vitro Techniques ; MicroRNAs ; genetics ; pharmacology ; Multidrug Resistance-Associated Proteins ; drug effects ; Organoplatinum Compounds ; pharmacology ; PTEN Phosphohydrolase ; drug effects ; RNA, Messenger ; Receptors, G-Protein-Coupled ; drug effects ; genetics

Multidrug resistance (MDR) remains the major obstacle to the success of clinical cancer chemotherapy. P-glycoprotein (P-gp), encoded by the MDR1, is an important part with complex mechanisms associated with the MDR. In order to overcome the MDR of tumors, we in the present experimental design incorporated small interfering RNA (siRNA) targeting MDR1 gene and anticancer drug paclitaxel (PTX) into the solid lipid nanoparticles (SLNs) to achieve the combinational therapeutic effects of genetherapy and chemotherapy. In this study, siRNA-PTX-SLNs were successfully prepared. The cytotoxicity of blank SLNs and siRNA-PTX-SLNs in MCF-7 cells and MCF-7/ADR cells were detected by MTT; and the uptake efficiency of PTX in MCF-7/ADR cells were detected via HPLC method; quantitative real-time PCR and flow cytometry were performed to investigate the silencing effect of siRNA-PTX- SLNs on MDR1 gene in MCF-7/ADR cells. The results showed that PTX loaded SLNs could significantly inhibit the growth of tumor cells, and more importantly, the MDR tumor cells treated with siRNA-PTX-SLNs showed the lowest viability. HPLC study showed that SLNs could enhance the cellular uptake for PTX. Meanwhile, siRNA delivered by SLNs significantly decreased the P-gp expression in MDR tumor cells, thus increased the cellular accumulation of rhodamine123 as a P-gp substrate. In conclusion, the MDR1 gene could be silenced by siRNA-PTX-SLNs, which could promote the growth inhibition efficiency of PTX on tumor cells, leading to synergetic effect on MDR tumor therapy.
ATP Binding Cassette Transporter, Sub-Family B ; genetics ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; pathology ; Drug Delivery Systems ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; Lipids ; chemistry ; MCF-7 Cells ; drug effects ; Nanoparticles ; chemistry ; Paclitaxel ; pharmacology ; RNA, Small Interfering ; pharmacology ; Real-Time Polymerase Chain Reaction

The study aimed to investigate the effects of small ubiquitin-related modifier (SUMO) specific protease 1 (SENP1) on human PXR-mediated MDR1 transcriptional activity and mRNA expression. Empty vector and expression plasmids, including PXR, SENP1 and SENP1 mutant (SENP1m) were transiently transfected into HepG2 and LS174T cells using Lipo2000. Transcriptional activity was detected by dual luciferase reporter gene assay, and mRNA level was measured using real-time polymerase chain reaction. The results showed that SENP1 could remarkably reduce the rifampicin (RIF)-induced MDR1 reporter activity and mRNA level in hPXR over expressed HepG2 and LS174T cells (P < 0.05), whereas adding SENP1m restored the RIF-induced increases (P < 0.05). These results indicated that SENP1 could repress the RIF-induced hPXR-mediated MDR1 transcriptional activity and mRNA expression.
ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Cysteine Endopeptidases ; Endopeptidases ; metabolism ; Gene Expression ; Hep G2 Cells ; Humans ; Peroxisome-Targeting Signal 1 Receptor ; RNA, Messenger ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Transcriptional Activation

To study the effect of Siwu decoction on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells. The Real-time quantitative poly-merase chain reaction (Q-PCR) was used to analyze the mRNA expression of MDR1 gene in Caco-2 cells. Flow cytometer was used to study the effect of Siwu decoction on the uptake of Rhodamine 123 in Caco-2 cells, in order to evaluate the efflux function of P-gp. Western blotting method was used to detect the effect of Siwu decoction on the P-gp protein expression of Caco-2 cells. Compared with the blank control group, after Caco-2 incubation with Siwu decoction at concentrations of 3.3, 5.0, 10.0 g x L(-1) for 24, 48, 72 h, the mRNA expression of MDR1 was up-regulated, suggesting the effect of Siwu decoction in inducing the expression of MDR1. After the administration with Siwu decoction in Caco-2 cells for 48 h, the uptake of Rhodamine 123 in Caco-2 cells decreased by respectively 16.6%, 22.1% (P < 0.05) and 45.4% (P < 0.01), indicating that the long-term administration of Siwu decoction can enhance the P-gp efflux function of Caco-2 cells. After the incubation of Caco-2 cells with Siwu decoction for 48 h, the P-gp protein expression on Caco-2 cell emebranes, demonstrating the effect of Siwu decoction in inducing the protein expression of P-gp.
ATP Binding Cassette Transporter, Sub-Family B ; genetics ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Caco-2 Cells ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Up-Regulation ; drug effects

Pain perception is influenced by multiple factors. The single nucleotide polymorphisms (SNPs) of some genes were found associated with pain perception. This study aimed to examine the association of the genotypes of ABCB1 C3435T, OPRM1 A118G and COMT V108/158M (valine 108/158 methionine) with pain perception in cancer patients. We genotyped 146 cancer pain patients and 139 cancer patients without pain for ABCB1 C3435T (rs1045642), OPRM1 A118G (rs1799971) and COMT V108/158M (rs4680) by the fluorescent dye-terminator cycle sequencing method, and compared the genotype distribution between groups with different pain intensities by chi-square test and pain scores between groups with different genotypes by non-parametric test. The results showed that in these cancer patients, the frequency of variant T allele of ABCB1 C3435T was 40.5%; that of G allele of OPRM1 A118G was 38.5% and that of A allele of COMT V108/158M was 23.3%. No significant difference in the genotype distribution of ABCB1 C3435T (rs1045642) and OPRM1 A118G (rs1799971) was observed between cancer pain group and control group (P=0.364 and 0.578); however, significant difference occurred in the genotype distribution of COMT V108/158M (rs4680) between the two groups (P=0.001). And the difference could not be explained by any other confounding factors. Moreover, we found that the genotypes of COMT V108/158M and ABCB1 C3435T were associated with the intensities of pain in cancer patients. In conclusion, our results indicate that the SNPs of COMT V108/158M and ABCB1 C3435T significantly influence the pain perception in Chinese cancer patients.
ATP Binding Cassette Transporter, Sub-Family B ; genetics ; Adult ; Aged ; Aged, 80 and over ; Alleles ; Breast Neoplasms ; complications ; diagnosis ; genetics ; pathology ; Catechol O-Methyltransferase ; genetics ; Female ; Gastrointestinal Neoplasms ; complications ; diagnosis ; genetics ; pathology ; Gene Expression ; Gene Frequency ; Genital Neoplasms, Female ; complications ; diagnosis ; genetics ; pathology ; Genital Neoplasms, Male ; complications ; diagnosis ; genetics ; pathology ; Genotype ; Humans ; Lung Neoplasms ; complications ; diagnosis ; genetics ; pathology ; Male ; Middle Aged ; Pain ; complications ; diagnosis ; genetics ; pathology ; Pain Measurement ; Pain Perception ; Polymorphism, Single Nucleotide ; Receptors, Opioid, mu ; genetics

<b>OBJECTIVEb>To investigate the effects of miR-21 on paclitaxel-resistance in human breast cancer MCF-7/PR and SKBR-3/PR cells.

<b>METHODSb>Paclitaxel-resistant human breast cancer cell lines MCF-7/PR and SKBR-3/PR were established by stepwise selection in increasing concentration of paclitaxel. Cellular morphology, mRNA and protein level of MDR1, BCRP and MRP1 in MCF-7/PR and SKBR-3/PR cells were determined. The expression of Bax, Bcl-2 and miR-21 in parental and paclitaxel-resistant cells was detected by RT-PCR and Western blotting. The synthetic miR-21 inhibitor or miR-21 mimic were transfected into MCF-7/PR, SKBR-3/PR and MCF-7, SKBR-3 cells with Lipofectamine 2000. The miR-21 levels were determined by RT-PCR, and P-gp, Bcl-2 and Bax protein levels were examined by Western blotting. MTT assay was used to measure the cell viability, and flow cytometry was performed to analyze the cell cycle and apoptosis.

<b>RESULTSb>The levels of MDR1, BCRP, MRP1, Bcl-2/Bax and miR-21 in MCF-7/PR and SKBR-3/PR cells were significantly higher than those in MCF-7 and SKBR-3 cells. The protein levels of P-gp, Bcl-2 were up-regulated, and Bax was down-regulated compared with parental cells. MiR-21 was significantly down-regulated after miR-21 inhibitor was transfected; and the levels of MDR1, BCRP, MRP1 and Bcl-2/Bax (P <0.05) were also down-regulated. MiR-21 inhibitors significantly suppressed G0/G1 transition of the cell cycle, and induced cell apoptosis in MCF-7/PR and SKBR-3/PR cells. MTT results showed that miR-21 inhibitors induced sensitivity of MCF-7/PR and SKBR-3/PR cells to paclitaxel. And miR-21 mimic can increase the expression of MDR1, Bcl-2/Bax and change cell morphology from parental cells to resistant cells.

<b>RESULTSb>The established MCF-7/PR and SKBR-3/PR breast cancer cells show typical multidrug resistance characteristics, which can be used as the model for drug resistance study. Down-regulated miR-21 expression in MCF-7/PR and SKBR-3/PR breast cancer cells can enhance cell sensitivity to paclitaxel.

ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Apoptosis ; Breast Neoplasms ; metabolism ; Cell Line, Tumor ; Down-Regulation ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs ; metabolism ; Paclitaxel ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; Up-Regulation ; bcl-2-Associated X Protein ; metabolism

The aim of this study was to investigate the effects of digoxin on the chemoresistance of human breast cancer cell line MCF-7/adriamycin (ADR) and its underlying mechanism. MCF-7 and MCF-7/ADR cells were designated as control and ADR groups, respectively. MCF-7/ADR cells in ADR + digoxin group received 48 h of digoxin (10 nmol/L) treatment; MCF-7/ADR cells transfected with pLKO.1-shHIF-1α and pLKO.1-shcontrol plasmids were named shHIF-1α and shcontrol groups, respectively. CCK-8 assay was employed to detect the cytotoxic effect of ADR on MCF-7/ADR cells, and IC50 value and resistance index were calculated according to CCK-8. RT-PCR was used to measure the mRNA levels of hypoxia inducible factor-1α (HIF-1α) and multidrug resistance-1 (MDR1). Western blot was used to analyze the protein levels of HIF-1α and MDR1. Flow cytometry was used to determine the apoptosis. The result showed that the resistance index of MCF-7/ADR cells was 115.6, and it was reduced to 47.2 under the action of digoxin (P < 0.05). In comparison with control group, ADR groups showed increased protein and mRNA levels of HIF-1α and MDR1 (P < 0.05). Digoxin reduced the protein levels of HIF-1α and MDR1, as well as the mRNA level of MDR1, but did not affect the mRNA level of HIF-1α. After HIF-1α gene was silenced, the protein levels of HIF-1α and MDR1 were down-regulated (P < 0.05), and the pro-apoptotic effect of ADR on MCF-7/ADR cells was enhanced. Although it was also observed that digoxin promoted cell apoptosis in both shcontrol and shHIF-1α groups, the difference between the two groups was not significant. In conclusion, the results suggest that digoxin may partially reverse the ADR resistance in human breast cancer cell line MCF-7/ADR by means of down-regulating the expression levels of HIF-1α and MDR1 and promoting apoptosis via HIF-1α-independent pathway.
ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Digoxin ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; MCF-7 Cells ; drug effects ; RNA, Messenger ; Transfection

<b>OBJECTIVEb>To investigate the association between ABCB1 polymorphisms and chemosensitivity of paclitaxel in Chinese advanced gastric cancer(AGC) patients.

<b>METHODSb>Clinical data and peripheral blood prior to chemotherapy of 412 AGC patients treated with first-line capecitabine plus paclitaxel(pactitaxel group, n=268) or cisplatin(cisplatin group, n=268) in Peking University Cancer Hospital from December 2008 to April 2013 were retrospectively collected. ABCB1 G2677T/A polymorphisms were determined using PCR amplification and Sanger Sequencing. Clinical response evaluation and survival analysis were performed using RECIST1.1 criteria and Kaplan-Meier curve, respectively. The associations of ABCB1 G2677T/A polymorphisms with clinical response and survival were analyzed statistically.

<b>RESULTSb>The genotypes of ABCB1 were detected in all the patients and the frequency of wild type(G2677G), single allele variants(G2677T+G2677A), and two allele variants (T2677T+T2677A+A2677A) was 22.8%(94/412), 49.8%(205/412), and 27.4%(113/412), respectively. In paclitaxel group, the disease control rate(DCR)[89.9%(116/129)] and median progression-free survival(PFS)(190 days) of patients with single allele variants of G2677T/A were significantly higher than those of wild type patients[76.1%(51/67) and 110 days, all P<0.05], and did not differ statistically from those with two allele variants. In cisplatin group, no significant differences were observed among patients with different genotypes of ABCB1 in terms of the DCR or PFS(all P>0.05).

<b>CONCLUSIONSb>ABCB1 G2677T/A polymorphisms are associated with chemosensitivity of paclitaxel in gastric cancer.

ATP Binding Cassette Transporter, Sub-Family B ; Antineoplastic Agents ; Capecitabine ; Cisplatin ; Deoxycytidine ; analogs & derivatives ; Disease-Free Survival ; Fluorouracil ; analogs & derivatives ; Genotype ; Humans ; Neoplasm Staging ; Paclitaxel ; Polymorphism, Single Nucleotide ; Retrospective Studies ; Stomach Neoplasms ; Survival Analysis

<b>OBJECTIVEb>To compare the biological behaviors of two drug-resistant testicular cancer cell lines established by different methods.

<b>METHODSb>Drug-resistance was induced in testicular cancer cell lines exposure of the cells to increasing concentrations of or a high dose of cisplatin (I-10/DDPi and I-10/DDPh cell lines, respectively). The morphological characteristics of the two cell lines were observed microscopically. The resistance index of the cells was determined with MTT assay, and the cell growth curves were drawn. The cellular expression of resistance-associated proteins MDR1 and P-gp was detected by Western blotting. The cell invasion ability was assessed with Transwell assay.

<b>RESULTSb>Normal testicular cancer cell line I-10 and the two resistant cell lines all showed an adherent growth pattern. Compared with I-10 cells, I-10/DDP cells exhibited slightly heterogenous cell sizes, irregular shapes, the presence of microvilli tentacles on the cell surface, and a scattered arrangement. The cisplatin resistance index of I-10/DDPi and I-10/DDPh cells were 3.924 and 3.099, respectively. Compared with I-10, the drug-resistant cell lines showed extended doubling time with increased expressions of MDR1 and P-gp and increased cell invasiveness, which was especially obvious in I-10/DDPi cells.

<b>CONCLUSIONb>Both increasing dose exposure and high-dose exposure to cisplatin can induce cisplatin resistance in testicular cancer cells, and the resistant cells established by the latter method better mimics clinical drug-resistant tumor cells.

ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Antineoplastic Agents ; pharmacology ; Cell Cycle ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Male ; Neoplasms, Germ Cell and Embryonal ; pathology ; Testicular Neoplasms ; pathology

<b>OBJECTIVEb>To explore the impact of extracellular acidic environment on the expression and activity of P-glycoprotein (P-gp) and on the P-gp-mediated cytotoxicity of daunomycin in cancer cells by using microfluidic chip technology.

<b>METHODSb>The A549 cells cultured on a microfluidic chip were divided into experiment group and control group. The experiment group was exposed to an acidic cell culture medium (pH 6.6), while the control group was treated with a neutral cell culture medium (pH 7.4). The expression of P-gp was detected by cell immunofluorescense analysis and the activity of P-gp was evaluated by Rhodamine 123 efflux experiment. Meanwhile, the cytotoxicity of daunomycin was analyzed by cell live/dead fluorescence staining method.

<b>RESULTSb>Microfluidic chip designed in this study could provide a suitable microenvironment for the growth of A549 cells and the A549 cells reached the confluence of 90% after inoculation for 72 h. Treatment of the acidic cell culture media on A549 cells did not make a significant difference on the expression level of P-gp. However, the activity of P-gp was significantly enhancement and peaked at 6 h after treatment with acidic cell culture media. Meanwhile, the cytotoxicity of daunomycin reduced significantly after treatment with acidic cell culture medium for 6 h,and a reversal effect was obtained when synergy with verapamil.

<b>CONCLUSIONSb>Microfluidic chip technology can shorten the analysis time and reduce the reagent consumption. It can be used as a new technology platform for understanding the mechanisms of multi-drug resistance and for screening highly efficient multi-drug resistance reversal agents.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; Cell Culture Techniques ; Cell Line, Tumor ; Culture Media ; Daunorubicin ; Extracellular Space ; Humans ; Hydrogen-Ion Concentration ; Microfluidics