Multidrug Resistance-related Protein Research Articles

Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition

BackgroundThe activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors.MethodsWe studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells.ResultsMDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells.ConclusionsOur work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway

The fucosyltransferase (FUT) family is the key enzymes in cell-surface antigen synthesis during various biological processes such as tumor multidrug resistance (MDR). The aim of this work was to analyze the alteration of FUTs involved in MDR in human hepatocellular carcinoma (HCC) cell lines. Using mass spectrometry (MS) analysis, the composition profiling of fucosylated N-glycans differed between drug-resistant BEL7402/5-FU (BEL/FU) cells and the sensitive line BEL7402. Further analysis of the expressional profiles of the FUT family in three pairs of parental and chemoresistant human HCC cell lines showed that FUT4, FUT6 and FUT8 were predominant expressed in MDR cell lines. The altered levels of FUT4, FUT6 and FUT8 were responsible for changed drug-resistant phenotypes of BEL7402 and BEL/FU cells both in vitro and in vivo. In addition, regulating FUT4, FUT6 or FUT8 expression markedly modulated the activity of the phosphoinositide 3 kinase (PI3K)/Akt signaling pathway and MDR-related protein 1 (MRP1) expression. Inhibition of the PI3K/Akt pathway by its specific inhibitor wortmannin, or by Akt small interfering RNA (siRNA), resulted in decreased MDR of BEL/FU cells, partly through the downregulation of MRP1. Taken together, our results suggest that FUT4-, FUT6- or FUT8-mediated MDR in human HCC is associated with the activation of the PI3K/Akt pathway and the expression of MRP1, but not of P-gp, indicating a possible novel mechanism by which the FUT family regulates MDR in human HCC.

Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

BackgroundMultidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells.MethodsWestern blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter.ResultsMRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues.ConclusionsOur findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors.

Adenovirus-mediated ING4 expression reduces multidrug resistance of human gastric carcinoma cells in vitro and in vivo

Chemotherapy is the primary treatment for both resectable and advanced gastric carcinoma, yet multiple drug resistance (MDR) of gastric carcinoma remains a significant therapeutic obstacle. The development of novel strategies to reduce MDR in gastric carcinoma would yield a better outcome following chemotherapy. ING4, a member of the inhibitor of growth (ING) tumor-suppressor family, possesses antitumor and radiosensitization or chemosensitization effects in a variety of human cancers. The present study investigated the effects and possible mechanisms of action of adenovirus-mediated ING4 (AdVING4) on the reversion of human gastric carcinoma cell MDR invitro and invivo in nude mouse xenografts. The data showed that the expression of ING4 mRNA and protein was dramatically downregulated (or lost) in gastric carcinoma SGC7901/CDDP cells after CDDP-induced MDR phenotype and in the parental SGC7901 cells. AdVING4‑induced ING4 expression reversed MDR and induced apoptosis of SGC7901/CDDP cells invitro and invivo in the SGC7901/CDDP xenograft tumors. Furthermore, AdVING4 substantially downregulated the expression of MDR-related proteins P-gp and MRP1 and apoptosis‑related proteins Bcl-2 and survivin, but upregulated the expression of apoptosis-related protein Bax in the SGC7901/CDDP xenograft tissues. The reversion effects elicited by AdVING4 on gastric cancer cell MDR were closely associated with the downregulation of ATP-binding cassette transporters and activation of apoptotic pathways. Thus, these findings suggest that AdVING4 may be a feasible modulator for the MDR phenotype of gastric carcinoma cells.

Estrogen Receptor α Roles in Breast Cancer Chemoresistance

Resistance to chemotherapy treatment, which may lead to limited efficacy of systemic therapy in breast cancer patients, is multifactorial. Among the mechanisms of resistance to chemotherapy treatment, there are those closely related to estrogen receptor α, P-glycoprotein, multidrug resistance-related protein, glutathione S-transferase pi and topoisomerase-II. ERα is ligand-activated transcription factor that regulates gene expression and plays a critical role in endocrine signaling. In previous preclinical and clinical studies, positive ERα expression in breast cancer cells was correlated with decreased sensitivity to chemotherapy. This article reviews current knowledge on the predictive value of ERα with regard to response to chemotherapy. Better understanding of its role may facilitate patient selection of therapeutic regimens and lead to optimal clinical outcomes.

Multidrug resistance genes and related drug resistance proteins in cancer

Chemotherapy is one of the most important methods of cancer treatment.However,multidrug resistance (MDR) has been the main factors affecting their efficacies.Recent studies show that the amplification of MDR1 gene in tumor cells,the over expression of the related drug resistance protein and the cell cycle and apoptosis pathway in signal transduction are the main causes for the failure of chemotherapy. Key words: Drug resistance, neoplasms ; Multidrug resistance-associated proteins; Cell cycle ; Neoplastic stem cells

A peptide derived from phage display library exhibits anti-tumor activity by targeting GRP78 in gastric cancer multidrug resistance cells

Multidrug resistance (MDR) remains a significant challenge to the clinical treatment of gastric cancer (GC). In the present study, using a phage display approach combined with MTT assays, we screened a specific peptide GMBP1 (Gastric cancer MDR cell-specific binding peptide), ETAPLSTMLSPY, which could bind to the surface of GC MDR cells specifically and reverse their MDR phenotypes. Immunocytochemical staining showed that the potential receptor of GMBP1 was located at the membrane and cytoplasm of MDR cells. In vitro and in vivo drug sensitivity assays, FACS analysis and Western blotting confirmed that GMBP1 was able to re-sensitize MDR cells to chemical drugs. Western blotting and proteomic approaches were used to screen the receptor of GMBP1, and GRP78, a MDR-related protein, was identified as a receptor of GMBP1. This result was further supported by immunofluoresence microscopy and Western blot. Additionally, Western blotting demonstrated that pre-incubation of GMBP1 in MDR cells greatly diminished MDR1, Bcl-2 and GRP78 expression but increased the expression of Bax, whereas downregulation of GRP78, function as a receptor and directly target for GMBP1, only inhibited MDR1 expression. Our findings suggest that GMBP1 could re-sensitize GC MDR cells to a variety of chemotherapeutic agents and this role might be mediated partly through down-regulating GRP78 expression and then inhibiting MDR1 expression. These findings indicate that peptide GMBP1 likely recognizes a novel GRP78 receptor and mediates cellular activities associated with the MDR phenotype, which provides new insight into research on the management of MDR in gastric cancer cells.

Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy

The emergence of resistance to multiple unrelated chemotherapeutic drugs impedes the treatment of several cancers. Although the involvement of ATP-binding cassette transporters has long been known, there is no in situ method capable of tracking this transporter-related resistance at the single-cell level without interfering with the cell's environment or metabolism. Here, we demonstrate that scanning electrochemical microscopy (SECM) can quantitatively and noninvasively track multidrug resistance-related protein 1-dependent multidrug resistance in patterned adenocarcinoma cervical cancer cells. Nonresistant human cancer cells and their multidrug resistant variants are arranged in a side-by-side format using a stencil-based patterning scheme, allowing for precise positioning of target cells underneath the SECM sensor. SECM measurements of the patterned cells, performed with ferrocenemethanol and [Ru(NH3)6](3+) serving as electrochemical indicators, are used to establish a kinetic "map" of constant-height SECM scans, free of topography contributions. The concept underlying the work described herein may help evaluate the effectiveness of treatment administration strategies targeting reduced drug efflux.

Abstract 900: Chemoresistance is induced by butyrate in colon cancer cells.

Abstract Purpose. Butyric acid is a short chain fatty acid produced naturally by fermentation of dietary fiber in the colonic mucosa. It is known to play an important role in preventing the initiation and the progression of colon cancer by suppressing the proliferation of cancer cells. However, some colon cancer still develops and progresses to a more malignant and chemoresistant one despite of the presence of butyric acid. The present study was undertaken to investigate a possibility of butyrate-involved mechanism by which the colon cancer cells acquire resistance to chemotherapeutic drugs. Methods. Human HCT116 colon cancer cells were exposed to progressively increasing concentrations of butyrate for three months to establish a colon cancer cell line with acquired resistance to butyrate. After obtaining butyrate-resistant cells, the sensitivity against multiple chemotherapeutic drugs were compared between parental and butyrate-resistant cells. The expression of proteins related to chemotherapeutic response was also compared by immunoblotting analysis. Results. Butyrate-resistant HCT116 cancer cell lines could be established by culturing cells in the presence of butyrate up to 0.8 mM concentration. The concentrations inhibiting the cell growth by 50% (IC50) of butyrate was 0.62 and 4.56 mM in parental and butyrate-resistant HCT116 cells. Butyrate-resistant HCT116 cells were also resistant to widely used chemotherapeutic drugs: the IC50 after paclitaxel, doxorubicin or 5-FU treatment were 2.10-, 3.98- and 1.6-fold higher in butyrateresistant cells compared with parental cells. The expression of multidrug resistance-related proteins that function as drug efflux pumps such as P-glycoprotein and the multidrug resistance-associated protein 1 were not changed at protein level. Among the apoptosis-involved proteins, the expression of anti-apoptotic BcL-2 protein was increased while that of proapoptotic BAD protein was reduced in butyrate-resistant cells, suggesting that the above chemoresistance was associated with cellular resistance to apoptosis. Conclusion.Our data herein suggests that the colon cancer cell grown in the presence of butyric acid may acquire resistance to apoptotic cell death and thus lead to resistance to chemotherapeutic drugs. Citation Format: Soo-jeong Lim, Hyeon Gyeom Choi, Chae Kyung Jeon, So Hee Kim. Chemoresistance is induced by butyrate in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 900. doi:10.1158/1538-7445.AM2013-900

Adjudin disrupts spermatogenesis by targeting drug transporters: Lesson from the breast cancer resistance protein (BCRP).

For non-hormonal male contraceptives that exert their effects in the testis locally instead of via the hypothalamic-pituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII‒VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be synthesized can improve their efficacy in downregulating BCRP and perhaps other drug efflux transporters in the testis to improve their efficacy to traverse the BTB by modifying their interacting domains.

Multidrug Resistance in Primary Tumors and Metastases in Patients with Esophageal Squamous Cell Carcinoma

Studies have demonstrated that radical esophagectomy can significantly prolong disease-free survival and improve the survival rate of patients with T3 or T4 esophageal cancer and lymph node metastasis. Multidrug resistant cancer cells have active efflux mechanisms that prevent the accumulation of chemotherapeutic drugs in the cells. The purpose of this study was to compare the expression of five MDR related proteins between primary tumors in patients with thoracic esophageal squamous cell carcinoma (ESCC) and metastatic cancer in lymph nodes to explore the clinical significance of heterogeneity in MDR metastatic cancer cells. Fifty-four patients with ESCC and lymph node metastasis were included. All patients underwent subtotal esophagectomy and D2/D3 lymph node resection. The expression of lung resistance-related protein (LRP), P-glycoprotein, topoisomerase-II, thymidylate synthase, and glutathione S-transferase P1-1 (GST-π) were determined in the primary tumors and lymph nodes via immunohistochemistry. The expression of LRP was significantly different between the primary tumors and lymph nodes (P = 0.026). No significant differences were found for the other four proteins, and protein expression was not associated with either degree of differentiation or disease stage. It was also found that GST-π was expressed in all patients in both the primary tumors and lymph nodes, suggesting that the design and application of chemotherapeutic protocols capable of reducing GST-π expression may be beneficial for patients with ESCC. Additional research regarding the clinical utility of MDR protein expression in ESCC is warranted to design effective chemotherapeutic protocols.

Study of the mechanisms underlying the reversal of multidrug resistance of human neuroblastoma multidrug-resistant cell line SK-N-SH/MDR1 by low-intensity pulsed ultrasound

The aim of the present study was to investigate the underlying mechanism(s) involved in reversing multidrug resistance (MDR) of SK-N-SH/MDR1 by low-intensity pulsed ultrasound (LIPUS). Membrane alteration of SK-N-SH/MDR1 cells exposed to LIPUS was analyzed by scanning electron microscopy (SEM). Immunofluorescence and western blotting were used\ to detect changes in the expression of the MDR-related proteins P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and glutathione-s-transferase-π (GST-π) after the optimum ultrasonic. The optimum ultrasonic conditions were 0.3 MHz, 1.0 W/cm², 40 sec and the chemosensitivity of SK-N-SH/MDR1 cells was significantly increased (P<0.05). The optimum ultrasonic-induced perforation of the irradiated cell membranes was observed by SEM. The expression of P-gp was significantly decreased in the group treated by optimum ultrasonic (P<0.05), but not the expressions of MRP1 or GST-π (P>0.05). We demonstrated that LIPUS effectively reverses the MDR of SK-N-SH/MDR1, presumably via augmenting membrane permeability and decreasing the P-gp expression of SK-N-SH/MDR1.

Interactions between human multidrug resistance related protein (MRP2; ABCC2) and excipients commonly used in self-emulsifying drug delivery systems (SEDDS)

The aim of this study was to investigate the multidrug resistance related protein-2 (MRP2) inhibition effect of excipients commonly employed in the formulation of self-emulsifying drug delivery systems (SEDDS). Cytotoxicity and safe dosages of fifteen excipients, including five surfactants, three oils, three co-surfactants and four solid carriers, were compared using MTT assay. Caco-2 cell permeability and MRP2 vesicles transport assays were used in the inhibition analysis. Scutellarin was shown to be a substrate of MRP2 and used as a probe in the inhibition assay. Twelve excipients decreased efflux ratio of scutellarin in Caco-2 model, among them six excipients showed inhibition effect on MRP2 in MRP2 transport model. In Caco-2 model, Cremophor EL, Maisine 35-1, PEG 2000 and β-cyclodextrin reduced efflux ratio of scutellarin the most in the four different excipients groups. The efflux reduction mechanism was further validated by MRP2 transport assay in inhibiting MRP2 activity in Cremophor EL and PEG 2000. It is suggested that scutellarin quantified by LC-MS can be used as a model drug for MRP2 inhibition analysis using Caco-2 permeability and vesicles transport analyses. Together they constitute a sensitive and rapid screening method for assessing interactions between excipients and MRP2 in SEDDS formulation.

Multidrug resistance-related proteins and their relationship with acute leukemia

Multidrug resistance-related proteins (MRP) are identified as ATP-dependent efflux pumps, the expression abnormality of which has close relationship with multidrug resistance (MDR) in a variety kinds of malignancies, leading to the failure of chemotherapy. The relationship between the expression of MRP and acute leukemia remains to be proved, since experiments and clinical researches are still insufficient. In this article, the structure, distribution, function, and relation of MRP to MDR and prognosis of acute leukemia are reviewed.

Expression and implication of Pgp, MRP, LRP, GST-π in bladder urothelial carcinoma

Objective To evaluate the expression of Pgp,MRP,LRP,GST-π,their co-expression,their clinical relativity and significances in bladder urothelial carcinoma.Methods From February 2005 to November 2010,51 patients (40 males and 11 females) with bladder cancer were recruited.The mean age was 60 years (range from 36 to 83 years).All 51 patients were with bladder urothelial carcinomas.Of whom,11 cases were grade Ⅰ tumors,18 cases were grade Ⅱ tumors and 22 cases were grade Ⅲ tumors.34 cases'were multifocal tumors,17 cases were unifocal tumors.In tumor size.There were 30 cases with tumor greater than 3cm in diameter and the other 21 cases with tumor smaller than 3 cm in diameter.Pglycoprotein (Pgp),Multi-drug resistance related protein (MRP),lung resistance protein (LRP),Glutathione s-transteferase-π (GST-r) of all the ladder urothelial carcinomas were detected by immunohistochemistry SP.The relativity and significances of the 51 cases were analyzed at the same time to find out their clinical-pathological features and relationships.Results The positive expression rates of Pgp,MRP,LRP,GST-π were 70.6％ (36/51),21.6％ (11/51),84.3％ (43/51),92.2％ (47/51),respectively.Pgp protein expression rates were 67.7％ (23/34) and 76.5％ (13/17) in multifocal tumors and unifocal tumors; MRP protein expression rates were 17.7％ (6/34) and 29.4％ (5/7) in multifocal tumors and unifocal tumors; LRP protein expression rates were 85.2％ (29/34) and 88.2％ (15/17) in multifocal tumors and unifocal tumors; GST-r protein expression rates were 88.2％ (30/34) and 100.0％ (17/17)in multifocal tumors and unifocal tumors.Pgp protein expression rates were 57.1％ (12/21) and 46.7％(14/30) in tumors larger than 3cm and tumors smaller than 3cm,respectively; MRP protein expression rates were 19.1％ (4/21) and 23.3％ (7/30) in tumors larger than 3cm and tumors smaller than 3cm;LRP protein expression rates were 81.0％ (17/21) and 86.7％ (26/30) in tumors larger than 3cm and tumors smaller than 3 cm; GST-π protein expression rates were 95.2％ (20/21) and 90.0％ (27/30) in tumors larger than 3 cm and tumors smaller than 3 cm.Pgp protein expression rates were 81.8％ (9/11),61.1％ (11/18) and 72.7％ (16/22) in grade Ⅰ,grade Ⅱ and grade Ⅲ tumors; MRP protein expression rates were 36.4％ (4/11),16.7％ (3/18) and 18.2％ (4/22) in grade Ⅰ,grade Ⅱ and grade Ⅲ tumors; LRP protein expression rates were 100.0％ (11/11),88.9％ (16/18) and 72.7％ (16/22) in grade Ⅰ,grade Ⅱ and grade Ⅲ tumors; GST-π protein expression rates were 100.0％ (11/11),94.4％ (17/18) and 86.4％ (19/22) in grade Ⅰ,grade Ⅱ and grade Ⅲ tumors.LRP protein expression rates were 60.0％ (6/10) and 90.2％ (37/41) in patients of different ages (P ＜ 0.05).GST-π protein expression rates were 95.0％ (38/40) in males and 81.8％ (9/11) in females (P ＜ 0.05).The co-expression rates werePgp+MRP 13.7％ (7/51),Pgp+LRP 58.8％ (30/51),Pgp+GST-π 64.7％ (33/51),MRP+LRP 15.7％ (8/51),MRP+GST-π 15.7 ％ (8/51),LRP+GST-π 82.4％ (42/51),Pgp+MRP + LRP + GST-π 13.7％ (7/51),Pgp + LRP + GST-π 56.9％ (29/51).Significant relationships were detected between Pgp and MRP (r =0.077,P ＜ 0.05),Pgp and GST-π (r =0.097,P ＜ 0.05),MRP and LRP (r =0.065,P ＜0.05),MRP and GST-π (r =0.052,P ＜0.05),LRP and GST-π (r =0.093,P ＜ 0.05).But no statistical significance relationship between Pgp and LRP(r =0.097,P ＞ 0.05)was found.Conclusions The multi-drug resistance in bladder urothelial carcinomas is affected by various multi-drug resistance genes.The expression of LRP is related to patient's age.The expression of GST-π is related to patient's gender.No relationship is established between expression of Pgp,MRP,LRP,GST-π and tumor occurrence frequency,tumor numbers,tumor size,histological grade,clinical-pathological features. Key words: Bladder urothelial carcinoma; Multi-drug resistance associated proteins; Immunohistochemistry

Expression of multidrug resistance 1 and multidrug resistance-related protein 1 in C57BL/6 mice treated with benzene

ATP-binding cassette super family (ABC) proteins are considered key to oncology and pharmacology studies. We examined the effect of benzene on ABC pump protein levels in C57BL/6 mouse bone marrow mononuclear cells. After a 2-week gavage (200 mg/kg, 5 days per week), the number of peripheral leukocytes, lymphocytes and basophils dropped significantly; there was also a significant decrease in MDR1 and MRP1 gene expression. A significant reduction in expression of P-gp was found; however, there was no significant decrease in the expression of MRP1 and NF-κB p65. We conclude that regulation of membrane efflux transport protein could be a factor in benzene hematotoxicity.

Mechanisms of verapamil-enhanced chemosensitivity of gallbladder cancer cells to platinum drugs: Glutathione reduction and MRP1 downregulation

Gallbladder cancer(GBC) is highly malignant with a low response rate after chemotherapy and platinum drugs are currently prominent in the treatment of biliary tract cancers. Therefore, the development of novel strategies to enhance the sensitivity of GBC to platinum drugs is required. In the present study, we examined the effects of verapamil, a classic chemosensitizer whose reported mechanisms of action include inhibiting the transport function of P-glycoprotein(MDR1) or stimulating glutathione (GSH) transport by multidrug resistance-related protein1 (MRP1), in combination with cisplatin (CDDP), carboplatin (CBP) or oxaliplatin on the GBC cell lines, SGC996 and GBC-SD. Our results demonstrated that the co-treatment with verapamil markedly enhanced the chemosensitivity of GBC cells in comparison with platinum drug treatment alone. The mechanisms involved included GSH reduction and MRP1 downregulation. Verapamil/CDDP co-treatment inhibited tumor xenograft growth via the downregulation of MRP1 expression. MRP1 was highly expressed in human GBC tissue compared to non-tumorous gallbladder tissue. Our data demonstrate that verapamil may be used as a safe chemosensitizer for platinum drugs in the treatment of GBC. It functions by ROS and ATP-binding cassette transporter-related mechanisms.

Long term myriocin treatment increases MRP1 transport activity

We investigated the effect of myriocin treatment, which extensively depletes sphingolipids from cells, on multidrug resistance-related protein 1 (MRP1) efflux activity in MRP1 expressing cells and isolated plasma membrane vesicles. Our data reveal that both short term (3 days) and long term (7 days) treatment effectively reduce the cellular sphingolipid content to the same level. Intriguingly, a two-fold increase in MRP1-mediated efflux activity was observed following long term treatment, while short term treatment had no impact. Very similar data were obtained with plasma membrane vesicles isolated from myriocin-treated cells. Exploiting the cell-free vesicle system, Michaelis–Menten analysis revealed that the intrinsic MRP1 activity remained unaltered; however, the fraction of active transporter molecules increased. We demonstrate that the latter effect is due to an enhanced recruitment of MRP1 into lipid raft fractions, thereby promoting MRP1 activity.

The association of statins plus LDL receptor‐targeted liposome‐encapsulated doxorubicin increases in vitro drug delivery across blood–brain barrier cells

The passage of drugs across the blood-brain barrier (BBB) limits the efficacy of chemotherapy in brain tumours. For instance, the anticancer drug doxorubicin, which is effective against glioblastoma in vitro, has poor efficacy in vivo, because it is extruded by P-glycoprotein (Pgp/ABCB1), multidrug resistance-related proteins and breast cancer resistance protein (BCRP/ABCG2) in BBB cells. The aim of this study was to convert poorly permeant drugs like doxorubicin into drugs able to cross the BBB. Experiments were performed on primary human cerebral microvascular endothelial hCMEC/D3 cells, alone and co-cultured with human brain and epithelial tumour cells. Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO, which elicits the nitration of critical tyrosine residues on these transporters. Statins also increased the number of low-density lipoprotein (LDL) receptors exposed on the surface of BBB cells, as well as on tumour cells like human glioblastoma. We showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells and to achieve significant anti-tumour cytotoxicity. We suggest that our 'Trojan horse' approach, based on the administration of statins plus a LDL receptor-targeted liposomal drug, might have potential applications in the pharmacological therapy of different brain diseases for which the BBB represents an obstacle.

Impact of ABCC2 polymorphisms on high-dose methotrexate pharmacokinetics in patients with lymphoid malignancy

Human multidrug resistance-related protein 2 (MRP2, encoded by ABCC2) is involved in the transport of anionic drugs such as methotrexate (MTX). We prospectively investigated the influence of four common ABCC2 genetic variants (rs717620, rs2273697, rs8187694 and rs8187710) on MTX pharmacokinetics parameters. MTX concentrations were monitored in 50 patients with lymphoid malignancy (27 males; mean age: 53±17 years) receiving high-dose MTX (5.13±1.88 g m(-)(2) in a 4-h perfusion). The population pharmacokinetics modelling showed that ABCC2 -24T allele (rs717620) had a combined influence on both MTX elimination and distribution. The MTX clearance and distribution volume were significantly higher in carriers of at least one copy of the -24T allele as compared with noncarriers: 8.6±2.2 vs 6.7± 2.5 l h(-1), P<0.01 and 30.7±7.7 vs 22.1±8.8 l, P<0.001, respectively. Consequently, -24T allele carriers were more prone to reach MTX nontoxic levels, 48 h after administration.