Expression Of Multidrug Resistance-associated Protein Research Articles

A co-delivery nanosystem of chemotherapeutics and DNAzyme overcomes cancer drug resistance and metastasis

Multidrug resistance (MDR) constitutes a major problem in the management of cancer and cancer metastasized from primary-source tumor causes cancer-related deaths. Our new approach is the co-delivery of chemotherapy drugs with a transcription-factor-targeting genetic agent to simultaneously inhibit the growth and metastasis of cancer cells. C-Jun is a transcription factor that regulates multidrug resistance-associated protein 1 (MRP1) pump efflux transcription and tumor metastasis. In this work, we reported that mesoporous silica nanoparticles (MSNs) can be functionalized to co-deliver doxorubicin (Dox) and DNAzyme (Dz) to increase cancer cell killing in an additive fashion. The MSNs were sequentially conjugated with Dox into the MSNs’ nanochannels and Dz onto the MSNs’ outermost surface to target c-Jun as the Dox@MSN-Dz co-delivery system. The Dox-resistant PC-3 cells treated with Dox@MSN-Dz efficiently enhanced the intracellular Dox concentration due to the abrogation of Dox-induced MRP1 expression through the downregulation of c-Jun expression by Dz. Additionally, significant reductions in invasion and migration related to metastasis were also observed in cells treated with Dox@MSN-Dz. Therefore, our results contribute new insight to the treatment of MDR combined metastatic cancer cells, worthwhile for studying its potential for development in clinical translation.

Shengjiang Xiexin Decoction Alters Pharmacokinetics of Irinotecan by Regulating Metabolic Enzymes and Transporters: A Multi-Target Therapy for Alleviating the Gastrointestinal Toxicity.

Shengjiang Xiexin decoction (SXD), a classic traditional Chinese medical formula chronicled in Shang Han Lun, is used in modern clinical practice to decrease gastrointestinal toxicity induced by the chemotherapeutic drug irinotecan (CPT-11). In this study, the effect of SXD on the pharmacokinetics of CPT-11 and its active metabolites (SN-38 and SN-38G), and the underlying mechanisms were further examined. An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of CPT-11, SN-38, and SN-38G in the plasma, bile, liver, intestine, and intestinal contents of control and SXD-pre-treated rats after intravenous administration of CPT-11. SXD pretreatment increased the area under the curve (AUC) and the initial plasma concentration (C0) of CPT-11 but decreased the plasma clearance (CL). The AUC and the maximum plasma concentration (Cmax) of SN-38 decreased, whereas the Cmax of SN-38G increased. Compared with that of the control group, the biliary excretion of CPT-11, SN-38, and SN-38G was inhibited. The CPT-11, SN-38, and SN-38G concentrations in the liver, intestine, and intestinal contents were different between the two groups. Furthermore, the hepatic expression of multidrug resistance-associated protein-2 (Mrp-2), P-glycoprotein (P-gp), and carboxylesterase 2 (CES2) was significantly down-regulated by SXD, while the hepatic and jejunal uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) expression was elevated. The hydrolysis of CPT-11 to SN-38 by CES and the glucuronidation of SN-38 to SN-38G by UGT were affected by liver and jejunum S9 fractions from rats pre-treated with SXD. Therefore, this study demonstrated for the first time that SXD could alter the pharmacokinetics of CPT-11 and its metabolites to alleviate CPT-11-induced diarrhea. And the underlying mechanism of drug interaction between CPT-11 and SXD involves decreasing hepatic Mrp-2 and P-gp expression and altering the activities of CES and UGT.

Ginger Phytochemicals Inhibit Cell Growth and Modulate Drug Resistance Factors in Docetaxel Resistant Prostate Cancer Cell.

Ginger has many bioactive compounds with pharmacological activities. However, few studies are known about these bioactive compounds activity in chemoresistant cells. The aim of the present study was to investigate the anticancer properties of ginger phytochemicals in docetaxel-resistant human prostate cancer cells in vitro. In this study, we isolated 6-gingerol, 10-gingerol, 4-shogaol, 6-shogaol, 10-shogaol, and 6-dehydrogingerdione from ginger. Further, the antiproliferation activity of these compounds was examined in docetaxel-resistant (PC3R) and sensitive (PC3) human prostate cancer cell lines. 6-gingerol, 10-gingerol, 6-shogaol, and 10-shogaol at the concentration of 100 μM significantly inhibited the proliferation in PC3R but 6-gingerol, 6-shogaol, and 10-shogaol displayed similar activity in PC3. The protein expression of multidrug resistance associated protein 1 (MRP1) and glutathione-S-transferase (GSTπ) is higher in PC3R than in PC3. In summary, we isolated the bioactive compounds from ginger. Our results showed that 6-gingerol, 10-gingerol, 6-shogaol, and 10-shogaol inhibit the proliferation of PC3R cells through the downregulation of MRP1 and GSTπ protein expression.

(Keynote) Expression / Functional Activity Relationship of Multidrug Resistance-Associated Protein 1 Investigated By Scanning Electrochemical Microscopy

Cancer cells can develop multidrug resistance (MDR) after prolonged exposure to chemotherapeutic drugs, which is a severe impediment to successful treatment. MDR is typically associated with transmembrane proteins mediating efflux of administered drugs, thereby keeping their intracellular concentration below the threshold required to kill cells. Although expression assays based on flow cytometry and immunostaining have shown that multidrug resistance-associated protein 1 (MRP1) is prevalent in many cancer types, the functional activity of this efflux pump is more difficult to elucidate, especially at the single-cell level. Herein, we report the measurement of MRP1 functional activity in individual cancer cells using scanning electrochemical microscopy (SECM). Cells were cultured onto plastic substrates containing selective adhesion sites. Optical microscopy and SECM revealed that cells adapt to the underlying surface, while MRP1 functional activity increases once the dimensions of the adhesive islands become smaller than those of the cell itself. Time-lapse SECM imaging revealed a suitable window of 30 min to complete each measurement before the cell undergoes blebbing, which is associated with considerable increase in functional activity. Distinct cell populations were produced by performing a doxorubicin drug challenge on two parental cell lines (e.g., wild-type HeLa cells and MRP1-overexpressing HeLa-R cells). Expression and functional activity of MRP1 were determined using flow cytometry and SECM, and our findings show that these parameters do not directly correlate. This suggests that functional activity may represent a powerful indicator of a cancer cell’s response to chemotherapeutic treatment and should improve our understanding of efflux mechanisms based on MRP1.

Dual-phase 99mTc-MIBI imaging and the expressions of P-gp, GST-π, and MRP1 in hyperparathyroidism.

ObjectiveThe aim of this study was to further elucidate the mechanisms of dual-phase technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) parathyroid imaging by exploring the association between early uptake results (EUR), delayed uptake results (DUR), and the retention index (RI) in dual-phase 99mTc-MIBI parathyroid imaging and P glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and glutathione S-transferase-π (GST-π) expression in hyperparathyroidism (HPT).Patients and methodsPreoperative dual-phase (early and delayed) 99mTc-MIBI imaging was performed on 74 patients undergoing parathyroidectomy for HPT. EUR, DUR, and RI were calculated. P-gp, MRP1, and GST-π expressions were assessed using immunohistochemistry in resected tissue from HPT and control patients. The association between P-gp, MRP1, and GST-π expressions and EUR, DUR, and RI in HPT was evaluated.ResultsThe positive rate of dual-phase 99mT c-MIBI imaging was 91.89% (68/74) and the false-negative rate was 8.11% (6/74). P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients (47.37 and 81.5%, P<0.05); there was no difference in MRP1. EUR were associated with P-gp and GST-π expressions, and DUR were associated with MRP1 expression. There was a significant difference in MRP1 expression between RI greater than or equal to 0 and RI less than 0. There was no relationship between the sensitivity of dual-phase 99mTc-MIBI imaging and P-gp, MRP1, and GST-π expressions in resected parathyroid tissue. The six false-negative HPT cases consisted of three P-gp (−)/MRP1 (−) tissues, three P-gp (−)/GST-π (−) tissues, and four MRP1 (−)/GST-π (−) tissues.ConclusionAs P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients, 99mTc-MIBI may wash out faster from normal parathyroid tissue surrounding the lesion compared with the lesion itself, facilitating detection.

Determination of the Relationship between Expression and Functional Activity of Multidrug Resistance-Associated Protein 1 using Scanning Electrochemical Microscopy.

Cancer cells can develop multidrug resistance (MDR) after prolonged exposure to chemotherapeutic drugs, which is a severe impediment to successful treatment. MDR is typically associated with transmembrane proteins mediating efflux of administered drugs, thereby keeping their intracellular concentration below the threshold required to kill cells. Although expression assays based on flow cytometry and immunostaining have shown that multidrug resistance-associated protein 1 (MRP1) is prevalent in many cancer types, the functional activity of this efflux pump is more difficult to elucidate, especially at the single-cell level. Herein, we report the measurement of MRP1 functional activity in individual cancer cells using scanning electrochemical microscopy (SECM). Cells were cultured onto plastic substrates containing selective adhesion sites. Optical microscopy and SECM revealed that cells adapt to the underlying surface, while MRP1 functional activity increases once the dimensions of the adhesive islands become smaller than those of the cell itself. Time-lapse SECM imaging revealed a suitable window of 30 min to complete each measurement before the cell undergoes blebbing, which is associated with a considerable increase in functional activity. Distinct cell populations were produced by performing a doxorubicin drug challenge on two parental cell lines (e.g., wild-type HeLa cells and MRP1-overexpressing HeLa-R cells). Expression and functional activity of MRP1 were determined using flow cytometry and SECM, and our findings show that these parameters do not directly correlate. This suggests that functional activity may represent a powerful indicator of a cancer cell's response to chemotherapeutic treatment and should improve our understanding of efflux mechanisms based on MRP1.

CLIC1 Induces Drug Resistance in Human Choriocarcinoma Through Positive Regulation of MRP1.

Chemotherapy is typically used to treat choriocarcinoma. However, a small proportion of this malignancy develops resistance to common chemotherapeutic drugs such as methotrexate (MTX) and floxuridine (FUDR). This study aimed to investigate the role and potential mechanisms of chloride intracellular channel protein 1 (CLIC1) in the development of chemoresistance in choriocarcinoma JeG3 cells. Two chemoresistant sublines were induced from their parental cell line JeG3 through intermittent exposure to MTX (named JeG3/MTX) or FUDR (named JeG3/FUDR). It was found that expression of CLIC1 was significantly higher in the chemoresistant sublines JeG3/MTX and JeG3/FUDR than in their parental cell line JeG3. Knockdown of CLIC1 by specific siRNA significantly increased cell sensitivity to MTX and FUDR in vitro and in vivo. Moreover, the high expression of CLIC1 in chemoresistant sublines was associated with upregulation of multidrug resistance-associated protein 1 (MRP1). Knockdown of CLIC1 decreased the expression of MRP1 accordingly. While reexpression of CLIC1 in the parental cell JeG3 increased its resistance to MTX and FUDR, depletion of MRP1 significantly blunted CLIC1 reexpression-mediated acquirement of chemoresistance in JeG3 cells. In conclusion, our results suggest that CLIC1 may serve as a critical mediator of chemoresistance in human choriocarcinoma JeG3 cells. The CLIC1-mediated chemoresistance is achieved through positive regulation of MRP1. Depletion of either CLIC1 or its downstream MRP1 may be a promising therapeutic strategy concerning reversing the chemoresistance in human choriocarcinoma JeG3 cells.

MEK inhibitor enhances sensitivity to chemotherapeutic drugs in multidrug resistant hepatocellular carcinoma cells.

The aim of the present study was to investigate the association between the mitogen-activated protein kinase (MAPK) signal transduction pathway and multidrug resistance in hepatocellular carcinoma cells. A Cell Counting Kit-8 assay was used to determine the drug sensitivity of HepG2 and HepG2/ADM hepatocellular carcinoma cell lines in combination with the MAPK/extracellular-signal-regulated kinase kinase (MEK) inhibitor U0126. Flow cytometry was used to analyze the rate of apoptosis. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) mRNA expression following treatment with various concentrations of U0126. P-gp and MRP1 expression levels were measured using Western blot analysis. The half-maximal inhibitory concentration was markedly decreased in combination with U0126. RT-qPCR results demonstrated that the expression of multidrug resistance 1 (MDR1) and MRP1 in HepG2/ADM cells was increased 5.37- and 6-14-fold compared with that in HepG2 cells. Furthermore, the expression levels in HepG2/ADM cells were decreased following U0126 treatment in a dose-dependent manner. The expression of P-gp and MRP1 in HepG2/ADM cells was increased 2.68- and 2.76-fold compared with that in HepG2 cells. Furthermore, the expression levels in HepG/ADM cells were decreased following U0126 treatment in a dose-dependent manner. The results of the present study indicate that the MEK inhibitor U0126 enhances sensitivity to chemotherapeutic drugs by downregulating P-gp and MRP1 expression in resistant hepatocellular carcinoma cells. The combination of MEK inhibitor and conventional chemotherapeutic drugs may provide novel therapeutic prospects for the treatment of drug-resistant hepatocellular carcinoma.

The wound-healing effect of 7,3',4'-trimethoxyflavone through increased levels of prostaglandin E2 by 15-hydroxyprostaglandin dehydrogenase inhibition.

To find an inhibitor of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) that rapidly metabolises Prostaglandin E2 (PGE2) as a mediator of wound healing, we examined seven flavonoids for this role. 7,3',4'-Trimethoxyflavone (TMF) had the lowest IC50 value of 0.34µM for 15-PGDH inhibition but>400µM for cytotoxicity, indicating a high therapeutic index. TMF elevated PGE2 levels in a concentration-dependent manner in both A549 lung cancer and HaCaT cells. It also significantly increased mRNA expression of multidrug resistance-associated protein 4 (MRP4) and of prostaglandin transporter (PGT) slightly in HaCaT cells. In addition, TMF facilitated in vitro wound healing in a HaCaT scratch model, which was completely inhibited by adding both 15-PGDH and NAD+ as cofactor, confirming the involvement of PGE2 in its wound healing effect. TMF with a high therapeutic index can facilitate wound healing through PGE2 elevation by 15-PGDH inhibition.

Kanglaite inhibits the expression of drug resistance genes through suppressing PVT1 in cisplatin-resistant gastric cancer cells.

Kanglaite (KLT) was shown to alleviate the development of multidrug resistance (MDR) clinically. The purpose of this study is to examine the mechanism of KLT for chemotherapy resistance in gastric cancer cells involving the regulation of MDR-related proteins. The cisplatin-resistant BGC823/DPP and SGC7901/DDP cells were treated with 1, 2.5 and 5 µl/ml of KLT for 24, 36 and 48 h. Cell Counting Kit-8 (CCK-8) assay and flow cytometry were performed to detect the cell viability and cell apoptosis, respectively. The expression of MDR1 and multidrug resistance associated protein 1 (MRP1) were examined by quantitative PCR and western blotting in BGC823/DPP cells and SGC7901/DDP cells treated with KLT. The effect of KLT on the expression of PVT1 was investigated. PVT1-overexpression vector was constructed and transfected into BGC823/DPP cells and SGC7901/DDP cells which were treated with KLT. KLT inhibited the cell viability and promoted the cell apoptosis of BGC823/DPP cells and SGC7901/DDP cells in a concentration-dependent manner. KLT suppressed the expression of MDR1 and MRP1 and the level of PVT1. PVT1 overexpression reversed the increased percentage of apoptotic cells induced by KLT. Finally, we found that PVT1 overexpression also abrogated the effect of KLT on the mRNA level and protein level of MDR1 and MRP1 in BGC823/DPP and SGC7901/DDP cells. KLT inhibited the expression of MDR1 and MRP1 via suppressing the expression of PVT1, which suggested the potential mechanism of KLT involving in MDR in gastric cancer.

Effect of Histone Deacetylase Inhibition on the Expression of Multidrug Resistance-associated Protein 2 in a Human Placental Trophoblast Cell Line.

Background:Placental multidrug resistance-associated protein 2 (MRP2), encoded by ABCC2 gene in human, plays a significant role in regulating drugs’ transplacental transfer rates. Studies on placental MRP2 regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the roles of epigenetic mechanisms in regulating placental drug transporters are still unclear. This study aimed to investigate the effect of histone deacetylases (HDACs) inhibition on MRP2 expression in the placental trophoblast cell line and to explore whether HDAC1/2/3 are preliminarily involved in this process.Methods:The human choriocarcinoma-derived trophoblast cell line (Bewo cells) was treated with the HDAC inhibitors-trichostatin A (TSA) at different concentration gradients of 0.5, 1.0, 3.0, and 5.0 μmol/L. Cells were harvested after 24 and 48 h treatment. Small interfering RNA (siRNA) specific for HDAC1/HDAC2/HDAC3 or control siRNA was transfected into cells. Total HDAC activity was detected by colorimetric assay kits. HDAC1/2/3/ABCC2 messenger RNA (mRNA) and protein expressions were determined by real-time quantitative polymerase chain reaction and Western-blot analysis, respectively. Immunofluorescence for MRP2 protein expression was visualized and assessed using an immunofluorescence microscopy and ImageJ software, respectively.Results:TSA could inhibit total HDAC activity and HDAC1/2/3 expression in company with increase of MRP2 expression in Bewo cells. Reduction of HDAC1 protein level was noted after 24 h of TSA incubation at 1.0, 3.0, and 5.0 μmol/L (vs. vehicle group, all P < 0.001), accompanied with dose-dependent induction of MRP2 expression (P = 0.045 for 1.0 μmol/L, P = 0.001 for 3.0 μmol/L, and P < 0.001 for 5.0 μmol/L), whereas no significant differences in MRP2 expression were noted after HDAC2/3 silencing. Fluorescent micrograph images of MRP2 protein were expressed on the cell membrane. The fluorescent intensities of MRP2 in the control, HDAC2, and HDAC3 siRNA-transfected cells were week, and no significant differences were noticed among these three groups (all P > 0.05). However, MRP2 expression was remarkably elevated in HDAC1 siRNA-transfected cells, which displayed an almost 3.19-fold changes in comparison with the control siRNA-transfected cells (P < 0.001).Conclusions:HDACs inhibition could up-regulate placental MRP2 expression in vitro, and HDAC1 was probably to be involved in this process.

Mechanism of Apoptosis for Resveratrol-mediated Reversing the Drug-resistance of AML HL-60/ADR cells

To explore the mechanism of apoptosis for resvertrol-mediated reversing the drug-resistance of AML HL-60/ADR cells. The HL-60/ADR cells were divided into 4 groups: control group, adriamycin (ADR)-treated group, resveratrol(Res)-treated group and ADR+Res-treated group. The inhibitory rate of cell proliferation was analyzed by CCK-8 method. The auto-fluorescence intensity of intracellular ADR and the apoptotic rate of HL-60/ADR cells were detected by flow cytometry. The mRNA expression levels of multidrug-resistance associated protein-1(MRP1), anti-apoptotic gene BCL-2 and pro-apoptotic gene BAX were analyzed by real-time fluorescence quantitative RT-PCR. The protein expression levels of MRP1, BCL-2 and BAX were detected by Western blot. The maximal inhibition rates of cell proliferation were 44%, 61%, 76% and 81%, respectively in different concentration of Res (25, 50, 100, 200 µmol/L) and with concentration-dependent manner(r=0.876, P<0.05). Compared with ADR group (IC50: 8.534±1.111 µmol/L), the half inhibitory concentration (IC50) of HL-60/ADR cells [(1.591±0.373) µmol/L] decreased significantly in ADR+Res group(P<0.05). The auto-fluorescence intensity of ADR in HL-60/ADR cells of ADR+Res group increased significantly (P<0.05). The apoptotic rate of HL-60/ADR cells in Res or ADR+Res group increased significantly (P<0.05). The mRNA expression levels of MRP1 and Bcl-2 in Res or ADR+Res groups decreased and BAX increased significantly (P<0.05). The protein expression levels of MRP1 and BCL-2 were decreased, BAX increased significantly in Res or ADR+Res group (P<0.05). Resveratrol shows the effect of reversing the drug resisitance of HL-60/ADR cells in acute myeloid leukemia, possibly via promoting the apoptosis of HL-60/ADR cells and inhibiting the expression of MRP1, which may be related with the inhibition of BCL-2 expression and the promotion of BAX expression.

MRP-1 and BCRP Promote the Externalization of Phosphatidylserine in Oxalate-treated Renal Epithelial Cells: Implications for Calcium Oxalate Urolithiasis

To investigate the possible involvement of multidrug resistance-associated protein 1 (MRP-1) and breast cancer resistance protein (BCRP) in the oxalate-induced redistribution of phosphatidylserine (PS) in renal epithelial cell membranes. A western blot analysis was used to examine the MRP-1 and BCRP expression levels. Surface-expressed PS was detected by the annexin V-binding assay. The cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate was used to measure the intracellular reactive oxygen species (ROS) level. A rat model of hyperoxaluria was obtained using 0.5% ethylene glycol and 1.0% ammonium chloride. In addition, certain animals received verapamil (50 mg/kg body weight), which is a common inhibitor of MRP-1 and BCRP. The degree of nephrolithiasis was assessed histomorphometrically using sections stained by Pizzolato method and by measuring the calcium oxalate crystal content in the renal tissue. Oxalate produced a concentration-dependent increase in the synthesis of MRP-1 and BCRP. Treatment with MK571 and Ko143 (MRP-1- and BCRP-specific inhibitors, respectively) significantly attenuated the oxalate-induced PS externalization. Adding the antioxidant N-acetyl-l-cysteine significantly reduced MRP-1 and BCRP expression. In vivo, markedly decreased nephrocalcinosis was observed compared with that in the rat model of hyperoxaluria without verapamil treatment. Oxalate induces the upregulation of MRP-1 and BCRP, which act as phospholipid floppases causing PS externalization in the renal epithelial cell membrane. The process is mediated by intracellular ROS production. The ROS-mediated increase in the synthesis of MRP-1 and BCRP can play an important role in hyperoxaluria-promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.

Modulated function of multidrug resistance-associated proteins in cisplatin-induced acute renal failure rats.

The effect of cisplatin-induced acute renal failure (ARF) on the function and expression of multidrug resistanceassociated proteins (MRPs) was evaluated in rats. Rats received an intraperitoneal injection of cisplatin (9 mg/kg), and the induction of ARF state with high plasma concentrations of indoxyl sulfate and creatinine was observed 72 h after cisplatin treatment. The function of MRPs in the liver, kidney and brain was evaluated by measuring the tissue accumulation and biliary excretion of 2,4-dintrophenyl-S-glutathione (DNP-SG), a substrate for MRPs, after administration of 1-chloro-2,4-dintrobenzene (CDNB), a precursor of DNP-SG, in rats. The levels of MRP1-4 expression were evaluated by Western blot analysis. Effect of ARF plasma components on MRP function was also examined by using calcein acetoxymethyl ester (calcein-AM) in HepG2 cells. In ARF rats (72 h after cisplatin treatment), the accumulation of DNP-SG in the liver, kidney and brain was significantly higher than those in control and cisplatin-treated rats (1 h after treatment). In ARF rats, intrinsic biliary excretion clearance of DNP-SG, estimated by dividing the biliary excretion rate of DNP-SG with the liver concentration, was also significantly reduced, though the expression levels of MRP1-4 in the liver remained unchanged. ARF rat plasma (5%) significantly increased the accumulation of calcein, a MRP substrate, in HepG2 cells after application of calcein-AM. In conclusion, MRP function was found to be suppressed not only in the kidney but also in the liver and brain in cisplatin-induced ARF rats, possibly due to the accumulation of some MRP substrates/inhibitors in plasma.

Pantoprazole Induces Apoptosis of Leukemic Cells by Inhibiting Expression of P-Glycoprotein/Multidrug Resistance-Associated Protein-1 Through PI3K/AKT/mTOR Signaling.

This study aims to investigate the effects and mechanism of pantoprazole on multidrug resistant leukemia K562/A02 and K562/ADM cell lines. K562/A02 and K562/ADM cells at logarithmic growth phase were pre-treated with different concentration of pantoprazole (0, 50, 100, 200μg/mL) for 24h. Flow cytometry was used to measure the cell growth cycle and apoptosis. RT-PCR and Western blot were used to measure the expression of p-PI3K, p-AKT, p-mTOR, P-glycoprotein (P-gp) and multidrug resistance-associated protein-1 (MRP1). Pantoprazole pretreatment significantly increased the ratio of G0/G1 phase but decreased the S phase of K562/A02 and K562/ADM cells in dose-dependent manner (p<0.05). Flow cytometry analysis indicated that pretreatment of leukemic cells with pantoprazole induced apoptosis in a dose-dependent manner. RT-PCR and Western blot analysis indicated that pantoprazole pretreatment inhibited the mRNA and protein expression of p-PI3K, p-Akt, p-mTOR, P-gp and MRP1 in K562/A02 and K562/ADM cells in a dose-dependent manner (p<0.05). Pantoprazole arrested cell cycle and induced apoptosis of multidrug resistant leukemic cells by inhibiting the expression of P-gp and MRP1 through PI3K/Akt/mTOR signaling pathway.

Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells.

Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP) activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP), organic anion-transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1), and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP). Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2), OCT1 and bile salt export pump) or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3) in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR)- and nuclear factor erythroid 2-related factor 2 (Nrf2)-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation.

Transfer of Multidrug Resistance Among Acute Myeloid Leukemia Cells Via Extracellular Vesicles and Their Micrornas Cargo

Resistance of cancer cells to chemotherapy is a major issue in acute myeloid leukemia (AML) and is usually due to a clonal selection of resistant leukemic cells. Recently, a horizontal transfer of chemoresistance among tumor cells has been reported via extracellular vesicles (EVs). EVs are vesicles ranging from 0.03 to 1µm generated by almost all cell types. They carry cellular components such as nucleic acids, membrane and cytosolic proteins. By interacting with cells and by transferring their content, these vesicles could modify target cells’ phenotype. Because of their presence in biological fluids, a lot of studies have tried to use EVs as biomarkers.The aim of this research is to investigate the role of EVs in chemoresistance in AML. For this purpose, the sensitive strain of the promyelocytic leukemia HL60 cell line was compared to its multiresistant strain: HL60/AR. The latter overexpresses multidrug resistance-associated protein 1 (MRP1), an efflux pump conferring resistance to anthracyclines. The transfer of this resistance between the two strains is obtained by treating HL60 with EVs generated by HL60/AR.The production of EVs by HL60/AR was confirmed by transmission electron microscopy. EVs from around 100 to 800 nm were highlighted. EVs from HL60/AR were then isolated by ultracentrifugation and their interaction with HL60 was investigated by labeling EVs with a fluorescent lipid membrane linker. The transmission of this fluorescence to HL60 was monitored by flow cytometry and demonstrated a binding or an incorporation of EVs.In order to check if this interaction has an impact on HL60 resistance to daunorubicin, HL60 were treated with EVs, once for 20 hours or four consecutive times for 48 hours each. The resistance to daunorubicin of EVs-treated HL60 was then assessed by a MTT cytotoxicity assay and revealed that EVs increase survival of cells to daunorubicin. The resistant profile between the cells treated once or four times with EVs were somewhat different and highlighted a potential implication of nucleic acids cargo in this resistance transfer. Indeed, nucleic acids transferred by EVs would need more than 20 hours to impact target cells.Afterwards, we investigated the changes in phenotype that could be responsible for this increased resistance of EVs-treated cells. First, the expression of MRP1 was evaluated by a flow cytometry drug retention assay based on the fluorescence exhibited by daunorubicin and consisting in analysis by flow cytometry of the daunorubicin retention of cells with or without the presence of a specific MRP1 inhibitor. This experiment emphasized an expression of MRP1 after EVs treatment. Oxidative stress also plays an important role in daunorubicin cytotoxicity. Thus reactive oxygen species (ROS) generation assays were performed on EVs-treated cells. These cells showed a decreased production of daunorubicin-induced ROS.We finally analyzed the microRNAs content of isolated EVs. This was performed by TaqMan® Human MicroRNA Array on EVs from HL60/AR but also on EVs generated by HL60. This experiment aimed at highlighting the microRNAs cargo potentially involved in this chemoresistance transfer but also at identifying microRNAs differentially expressed between the two strains to serve as biomarker of chemoresistance in vivo. Out of the 380 microRNAs tested, 32 presented a differential expression between HL60 and HL60/AR. Among them, 5 microRNAs presented a more than 8-fold change between the two strains: miR-19b, miR-20a, miR-488, miR-519a and miR-523. miR-519a was the only microRNA more expressed in EVs produced by HL60, compared to EVs from HL60/AR.In conclusion, this study demonstrates the ability of resistant leukemic cells to transfer, at least partially, their chemoresistance via EVs. This could be due to a direct transfer of MRP1 transported by EVs but also to a nucleic acid transfer. In this context, some microRNAs have been highlighted for their high differential expression in EVs between sensitive and chemoresistant cells. These results bring out a potential clinical use of these circulating microRNAs as chemoresistance biomarkers in AML. DisclosuresNo relevant conflicts of interest to declare.

Increased chemosensitivity and radiosensitivity of human breast cancer cell lines treated with novel functionalized single-walled carbon nanotubes.

Hypoxia is a major cause of treatment resistance in breast cancer. Single-walled carbon nanotubes (SWCNTs) exhibit unique properties that make them promising candidates for breast cancer treatment. In the present study, a new functionalized single-walled carbon nanotube carrying oxygen was synthesized; it was determined whether this material could increase chemosensitivity and radiosensitivity of human breast cancer cell lines, and the underlying mechanisms were investigated. MDA-MB-231 cells growing in folic acid (FA) free medium, MDA-MB-231 cells growing in medium containing FA and ZR-75-1 cells were treated with chemotherapy drugs or radiotherapy with or without tombarthite-modified-FA-chitosan (R-O2-FA-CHI)-SWCNTs under hypoxic conditions, and the cell viability was determined by water-soluble tetrazolium salts-1 assay. The cell surviving fractions were determined by colony forming assay. Cell apoptosis induction was monitored by flow cytometry. Expression of B-cell lymphoma 2 (Bcl-2), survivin, hypoxia-inducible factor 1-α (HIF-1α), multidrug resistance-associated protein 1 (MRP-1), P-glycoprotein (P-gp), RAD51 and Ku80 was monitored by western blotting. The novel synthesized R-O2-FA-CHI-SWCNTs were able to significantly enhance the chemosensitivity and radiosensitivity of human breast cancer cell lines and the material exhibited its expected function by downregulating the expression of Bcl-2, survivin, HIF-1α, P-gp, MRP-1, RAD51 and Ku80.

HDAC2 overexpression is a poor prognostic factor of breast cancer patients with increased multidrug resistance-associated protein expression who received anthracyclines therapy.

Previous studies have revealed the association of multidrug resistancewith histone deacetylasesinhibitors treatment in cancer cells. But little data were available for the correlation of histone deacetylases and drug-resistant-related proteins in breast cancer tissue. This study aimed to exploring the association of histone deacetylases expression with clinicopathological features, drug-resistant-related proteins, prognosis and therapeutic responses in breast cancer patients. We performed immunohistochemistry to study the expression of HDAC1 and HDAC2 in 226 breast cancer and 34 breast fibroadenoma patients, and the expression of breast cancer resistance protein, P-glycoprotein, lung resistance protein and multidrug resistance proteinin 226 breast cancer. In breast cancer, HDAC2 expression was significantly increased than in fibroadenoma (P=0.015), and correlated with lymph node metastasis (P=0.002), advanced clinical stages (P=0.016) and high histological grade (P=0.001). Significant positive correlations were found between HDAC2 and Ki67, HDAC1 and multidrug resistance protein, HDAC2 and breast cancer resistance protein, HDAC2 and multidrug resistance protein. HDAC2 positive expression was associated with shorter overall survival (P=0.035) of breast cancer patients. In addition, HDAC2-positive expression was significantly associated with shorter overall survival in multidrug resistance protein-positive patients (P=0.034), but not in multidrug resistance protein-negative patients (P=0.530). HDAC2-positive expression was associated with shorter survival in patients who received chemotherapy containing anthracyclines (overall survival, P=0.041; disease-free survival, P=0.084), but not in patients who received chemotherapy without anthracyclines (overall survival, P=0.679; disease-free survival, P=0.708). HDAC2 overexpression correlated with the metastasis, progression and the increased Ki67, multidrug resistance protein expression in breast cancer, and HDAC2 could be a prognostic factor of breast cancer patients, especially the patients who received anthracyclines therapy.

Expression and relationship between hepaCAM protein and multidrug resistance protein in renal carcinoma

Objective To investigate the expression and relationship between hepatocyte cell adhesion molecule (hepaCAM) protein and some multidrug resistance proteins in renal carcinoma tissue. Methods Expressions of hepaCAM, multidrug resistance associated protein (MRP), P-glycoprotein (P-gp), lung resistance protein (LRP), and topoisomerase Ⅱ (TOPO Ⅱ) protein were detected by immunohistochemistry in different areas of human renal cell carcinoma tissues and their relationships were analyzed. Results In the peripheral zone of renal tumor, hepaCAM, MRP, P-gp and LRP protein were showed positive expression. In the central region of the renal tumor, the expressions of hepaCAM, P-gp and LRP were negative or weakly positive, while the expressions of MRP and TOPO Ⅱ protein were positive. The expressions of MRP and TOPO Ⅱ protein in the central region of tumor were stronger than those in the peripheral zone of tumor (31.23±5.67 vs. 23.89±4.56; 45.66±2.34 vs. 5.23±0.66), with statistically significant differences (t=-6.20, P=0.00; t=-100.16, P=0.00). While the expressions of other proteins (hepaCAM, P-gp and LRP) in the central region of tumor were weaker than those in the peripheral zone of tumor (3.21±1.12 vs. 27.25±2.23; 2.34±0.33 vs. 51.23±3.45; 4.22±1.78 vs. 44.23±1.45), with statistically significant differences (t=60.87, P=0.00; t=90.35, P=0.00; t=107.18, P=0.00). Correlation analysis showed that the expression of hepaCAM protein in the central region of renal carcinoma was related with the expression of MRP protein (r=0.94, P=0.01), but it was not related with the expressions of P-gp, LRP and TOPO Ⅱ protein (r=0.22, P=0.44; r=0.14, P=0.80; r=0.34, P=0.07). Conclusion The expression of hepaCAM protein in renal carcinoma may be related to tumor drug-resistance. Key words: Kidney neoplasms; Cell adhesion molecules; Multidrug resistance-associated proteins