Overexpression Of Multidrug Resistance Protein Research Articles

Abstract 7291: Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients

Abstract Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP dependent transmembrane efflux pump that confers to drug resistance. Overexpression of MRP1 has been observed in both pancreatic ductal adenocarcinoma (PDAC) and breast cancer tissues. Numerous proteomics studies have revealed the presence of MRP1 in small extracellular vesicles (sEVs) released from cancer cells. Cancer-derived sEVs are known to be released into the circulation, thus becoming attractive candidates for non-invasive biomarker development. However, whether the MRP1 level in plasma sEVs is indicative of human cancer has not been evaluated. This study examined the MRP1 expression in plasma sEVs derived from patients with PDAC, breast and colon cancer, and age- and gender-matched healthy subjects. The patient plasma samples were obtained primarily from the NCI-sponsored Cooperative Human Tissue Network and Stephenson Cancer Center. Healthy plasma samples were provided by the Oklahoma Blood institute. Human pancreatic cancer cell lines PANC-1, MIA PaCa-2 and BxPC-3, and the pancreatic ductal cell line hTERT-HPNE were tested for MRP1 expression prior to examination of plasma samples. Cellular and plasma sEVs were isolated using the double filtration followed by polymer precipitation method. Western blot was performed to assess the expression level of MRP1 and proteomics was applied to confirm the detection of a truncated form of MRP1 in plasma sEVs. MRP1 expression was detected in the sEVs derived from all three pancreatic cancer cell lines but absent in the sEVs from the HPNE line. Overexpression of a truncated MRP1 protein was detected in plasma sEVs derived from patients with early stage (stage I-IIA) and late stage (III-IV) PDAC, compared with that from matched healthy subjects (n=17 for each group). Similar findings were evident in plasma sEVs from patients with breast cancer (n=7), while the results remained inconclusive in plasma sEVs from patients with colon cancer (n=7). Protein structure analysis indicated that the truncated form of MRP1 likely consists of the regions between the QCRL-1 motif and the C-terminus of the protein. Proteomic analysis detected the peptides derived from the C-terminus region, consistent with the structural analysis. Furthermore, the truncated form of MRP1 can only be detected using antibodies against the epitopes in the C-terminus and QCRL-1 region of MRP1 protein. In summary, we have detected a truncated form of MRP1 protein in plasma sEVs derived from cancer patients, and the level of its expression is significantly elevated in patients with PDAC and breast cancer. Citation Format: Kritisha Bhandari, Jeng Shi Kong, Chao Xu, Ajay Jain, Wei-Qun Ding. Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7291.

A macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation

Adenosine triphosphate-binding cassette (ABC)transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, namedCPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4(LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Emerging role of non-coding RNAs in resistance to platinum-based anti-cancer agents in lung cancer.

Platinum-based drugs are the first line of therapeutics against many cancers, including lung cancer. Lung cancer is one of the leading causes of cancer-related death worldwide. Platinum-based agents target DNA and prevent replication, and transcription, leading to the inhibition of cell proliferation followed by cellular apoptosis. About twenty-three platinum-based drugs are under different stages of clinical trials, among cisplatin, carboplatin, and oxaliplatin are widely used for the treatment of various cancers. Among them, cisplatin is the most commonly used drug for cancer therapy, which binds with RNA, and hinders the cellular RNA process. However, long-term use of platinum-based drugs can cause different side effects and has been shown to develop chemoresistance, leading to poor clinical outcomes. Chemoresistance became an important challenge for cancer treatment. Platinum-based chemoresistance occurs due to the influence of intrinsic factors such as overexpression of multidrug resistance proteins, advancement of DNA repair mechanism, degradation, and deactivation of intracellular thiols. Recently, epigenetic modifications, especially non-coding RNAs (ncRNAs) mediated gene regulation, grasp the attention for reversing the sensitivity of platinum-based drugs due to their reversible nature without altering genome sequence. ncRNAs can also modulate the intrinsic and non-intrinsic mechanisms of resistance in lung cancer cells. Therefore, targeting ncRNAs could be an effective approach for developing novel therapeutics to overcome lung cancer chemoresistance. The current review article has discussed the role of ncRNA in chemoresistance and its underlying molecular mechanisms in human lung cancer.

Host cells with transient overexpression of MDR1 as a novel in vitro model for evaluating on-target effect for activity against the epicellular Cryptosporidium parasite.

To rapidly generate host cells with resistance to multiple compounds for differentiating drug action on parasite target or the host cell target (i.e. on-target or off-target effect) against the zoonotic enteric parasite Cryptosporidium parvum. Transient overexpression of a multidrug resistance protein 1 (MDR1) gene in host cells (HCT-8 cell line) was explored to increase drug tolerance of the host cells to selected anti-cryptosporidial leads. In vitro cytotoxicity and anti-cryptosporidial efficacy of selected compounds were evaluated on the parasite grown in WT parental and transiently transfected HCT-8 cells. The approach was based on the theory that, for an epicellular parasite receiving consistent exposure to compounds in culture medium, overexpressing MDR1 in HCT-8 cells would increase drug tolerance of host cells to selected compounds but would not affect the anti-cryptosporidial efficacy if the compounds acted solely on the parasite target and the drug action on host cell target played no role on the antiparasitic efficacy. Six known anti-cryptosporidial leads were tested. Transient overexpression of MDR1 increased drug tolerance of HCT-8 cells on paclitaxel, doxorubicin HCl and vincristine sulphate (2.11- to 2.27-fold increase), but not on cyclosporin A, daunorubicin HCl and nitazoxanide. Increased drug tolerance in host cells had no effect on antiparasitic efficacy of paclitaxel, but affected that of doxorubicin HCl. Data confirmed that, at efficacious concentrations, paclitaxel acted mainly on the parasite target, while doxorubicin might act on both parasite and host cell targets. This model can be employed for studying the action of additional anti-cryptosporidial leads, and adapted to studying drug action in other epicellular pathogens. The limitation of the model is that the anti-cryptosporidial leads/hits need to be MDR1 substrates.

Extracellular mutation induces an allosteric effect across the membrane and hampers the activity of MRP1 (ABCC1)

Dynamic conformational changes play a major role in the function of proteins, including the ATP-Binding Cassette (ABC) transporters. Multidrug Resistance Protein 1 (MRP1) is an ABC exporter that protects cells from toxic molecules. Overexpression of MRP1 has been shown to confer Multidrug Resistance (MDR), a phenomenon in which cancer cells are capable to defend themselves against a broad variety of drugs. In this study, we used varied computational techniques to explore the unique F583A mutation that is known to essentially lock the transporter in a low-affinity solute binding state. We demonstrate how macro-scale conformational changes affect MRP1’s stability and dynamics, and how these changes correspond to micro-scale structural perturbations in helices 10–11 and the nucleotide-binding domains (NBDs) of the protein in regions known to be crucial for its ATPase activity. We demonstrate how a single substitution of an outward-facing aromatic amino acid causes a long-range allosteric effect that propagates across the membrane, ranging from the extracellular ECL5 loop to the cytoplasmic NBD2 over a distance of nearly 75 Å, leaving the protein in a non-functional state, and provide the putative allosteric pathway. The identified allosteric structural pathway is not only in agreement with experimental data but enhances our mechanical understanding of MRP1, thereby facilitating the rational design of chemosensitizers toward the success of chemotherapy treatments.

Roles of Multidrug Resistance Protein 4 in Microbial Infections and Inflammatory Diseases

Numerous studies have reported the emergence of antimicrobial resistance during the treatment of common infections. Multidrug resistance (MDR) leads to failure of antimicrobial treatment, prolonged illness, and increased morbidity and mortality. Overexpression of multidrug resistance proteins (MRPs) as drug efflux pumps are one of the main contributions of MDR, especially multidrug resistance protein 4 (MRP4/ABCC4) in the development of antimicrobial resistance. The molecular mechanism of antimicrobial resistance is still under investigation. Various intervention strategies have been developed for overcoming MDR, but the effect is limited. Suppression of MRP4 may be an attractive therapeutic approach for addressing drug resistance. However, there are few reports on the involvement of MRP4 in antimicrobial resistance and inflammatory diseases. In this review, we introduced the function and regulation of MRP4, and then summarized the roles of MRP4 in microbial infections and inflammatory diseases as well as polymorphisms in the gene encoding this transporter. Further studies should be conducted on drug therapy targeting MRP4 to improve the efficacy of antimicrobial therapy. This review can provide useful information on MRP4 for overcoming antimicrobial resistance and anti-inflammatory therapy.

The Effect of Nanosystems on ATP-Binding Cassette Transporters: Understanding the Influence of Nanosystems on Multidrug Resistance Protein-1 and P-glycoprotein.

The cancer multidrug resistance is involved in the failure of several treatments during cancer treatment. It is a phenomenon that has been receiving great attention in the last years due to the sheer amount of mechanisms discovered and involved in the process of resistance which hinders the effectiveness of many anti-cancer drugs. Among the mechanisms involved in the multidrug resistance, the participation of ATP-binding cassette (ABC) transporters is the main one. The ABC transporters are a group of plasma membrane and intracellular organelle proteins involved in the process of externalization of substrates from cells, which are expressed in cancer. They are involved in the clearance of intracellular metabolites as ions, hormones, lipids and other small molecules from the cell, affecting directly and indirectly drug absorption, distribution, metabolism and excretion. Other mechanisms responsible for resistance are the signaling pathways and the anti- and pro-apoptotic proteins involved in cell death by apoptosis. In this study we evaluated the influence of three nanosystem (Graphene Quantum Dots (GQDs), mesoporous silica (MSN) and poly-lactic nanoparticles (PLA)) in the main mechanism related to the cancer multidrug resistance such as the Multidrug Resistance Protein-1 and P-glycoprotein. We also evaluated this influence in a group of proteins involved in the apoptosis-related resistance including cIAP-1, XIAP, Bcl-2, BAK and Survivin proteins. Last, colonogenic and MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assays have also been performed. The results showed, regardless of the concentration used, GQDs, MSN and PLA were not cytotoxic to MDA-MB-231 cells and showed no impairment in the colony formation capacity. In addition, it has been observed that P-gp membrane expression was not significantly altered by any of the three nanomaterials. The results suggest that GQDs nanoparticles would be suitable for the delivery of other multidrug resistance protein 1 (MRP1) substrate drugs that bind to the transporter at the same binding pocket, while MSN can strongly inhibit doxorubicin efflux by MRP1. On the other hand, PLA showed moderate inhibition of doxorubicin efflux by MRP1 suggesting that this nanomaterial can also be useful to treat MDR (Multidrug resistance) due to MRP1 overexpression.

Therapeutic Targeting of MDR1 Expression by RORγ Antagonists Resensitizes Cross-Resistant CRPC to Taxane via Coordinated Induction of Cell Death Programs.

Overexpression of ATP-binding cassette subfamily B member 1 (ABCB1)-encoded multidrug resistance protein 1 (MDR1) constitutes a major mechanism of cancer drug resistance including docetaxel (DTX) and cabazitaxel (CTX) resistance in castration-resistant prostate cancer (CRPC). However, no therapeutics that targets MDR1 is available at clinic for taxane sensitization. We report here that retinoic acid receptor-related orphan receptor γ (RORγ), a nuclear receptor family member, unexpectedly mediates MDR1/ABCB1 overexpression. RORγ plays an important role in controlling the functions of subsets of immune cells and has been an attractive target for autoimmune diseases. We found that its small-molecule antagonists are efficacious in resensitizing DTX and CTX cross-resistant CRPC cells and tumors to taxanes in both androgen receptor-positive and -negative models. Our mechanistic analyses revealed that combined treatment with RORγ antagonists and taxane elicited a robust synergy in killing the resistant cells, which involves a coordinated alteration of p53, Myc, and E2F-controlled programs critical for both intrinsic and extrinsic apoptosis, survival, and cell growth. Our results suggest that targeting RORγ with small-molecule inhibitors is a novel strategy for chemotherapy resensitization in tumors with MDR1 overexpression.

ARE THE BCL2 FAMILY INHIBITORS SUBSTRATES FOR THE MULTIDRUG RESISTANCE EFFLUX PUMP MDR1?

The BH3 mimetic compounds, a novel class of targeted anti-cancer agents, are highly efficacious for the treatment of certain haematological malignancies. The BCL2 inhibitor, venetoclax, is the most advanced and has been approved for treating patients with high-risk chronic lymphocytic leukemia (CLL) in many countries. While highly effective, clinical resistance is emerging, and we anticipate that resistance to BH3 mimetic therapy will be a problem in the future. Unlike CLL, the BCL2 relative MCL1 is the key survival factor in multiple myeloma (MM) and clinical trials with inhibitors selective for MCL1 have started in patients with this disease. To anticipate what might drive resistance to MCL1 inhibition, we have undertaken studies using model systems in the laboratory. By culturing a highly sensitive MM cell line, AMO1, in gradually escalating doses of a MCL1 inhibitor, we were able to generate resistant pools of these cells. Interestingly, we found marked amplification of the multidrug resistance protein 1 (MDR1) gene in 3/4 generated resistant pools. We conducted a number of studies which validated MDR1 amplification as a potential mechanism of resistance to this class of MCL1 inhibitors. Firstly, ectopic overexpression of MDR1 in multiple hematopoietic cell lines conferred resistance. Secondly, this resistance is reversed by co-treatment with an advanced MDR1 inhibitor. Finally, we could restore sensitivity to this MCL1 inhibitor when MDR1 function was blocked in the resistant AMO1 pools with MDR1 amplification. Overall, our data provide strong evidence that MDR1 amplification could adversely impact this MCL1 inhibitor activity. We are also testing if other BH3 mimetics are substrates of MDR1. These studies are important because MDR1 amplification is a common mechanism that underpins resistance to multiple anti-cancer agents, both conventional and targeted.

Overexpression of HIF-1α contributes to melphalan resistance in multiple myeloma cells by activation of ERK1/2, Akt, and NF-κB

Multiple myeloma (MM) commonly displays multidrug resistance and is associated with poor prognosis. Therefore, it is important to identify the mechanisms by which MM cells develop multidrug resistance. Our previous study showed that multidrug resistance is correlated with overexpression of multidrug resistance protein 1 (MDR1) and Survivin, and downregulation of Bim expression in melphalan-resistant RPMI8226/L-PAM cells; however, the underlying mechanism of multidrug resistance remains unclear. In the present study, we investigated the mechanism of multidrug resistance in melphalan-resistant cells. We found that RPMI8226/L-PAM and ARH-77/L-PAM cells showed increased phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Akt, and nuclear localization of nuclear factor κB (NF-κB). The combination of ERK1/2, Akt, and NF-κB inhibitors with melphalan reversed melphalan resistance via suppression of Survivin expression and enhanced Bim expression in melphalan-resistant cells. In addition, RPMI8226/L-PAM and ARH-77/L-PAM cells overexpressed hypoxia-inducible factor 1α (HIF-1α) via activation of ERK1/2, Akt, and NF-κB. Moreover, suppression of HIF-1α by echinomycin or HIF-1α siRNA resensitized RPMI8226/L-PAM cells to melphalan through downregulation of Survivin expression and upregulation of Bim expression. These results indicate that enhanced Survivin expression and decreased Bim expression by HIF-1α via activation of ERK1/2, Akt, and NF-κB play a critical role in melphalan resistance. Our findings suggest that HIF-1α, ERK1/2, Akt, and NF-κB inhibitors are potentially useful as anti-MDR agents for the treatment of melphalan-resistant MM.In this study, the authors show that overexpression of hypoxia-inducible factor(HIF)-1α by ERK1/2, Akt and NF-κB activation contributes to melphalan-resistance in multiple myeloma (MM) cells. Additionally, inhibition of HIF-1α resensitizes melphalan-resistant MM cells to melphalan. Therefore, HIF-1α inhibitors may be therapeutically useful as anti-multidrug resistance protein agents in MM.

Multidrug Resistance Protein 1 Deficiency Promotes Doxorubicin-Induced Ovarian Toxicity in Female Mice.

Multidrug resistance protein 1 (MDR1), a phase III drug transporter that exports substrates out of cells, has been discovered in both cancerous and normal tissues. The over expression of MDR1 in cancer cells contributes to multiple drug resistance, whereas the MDR1 in normal tissues protects them from chemical-induced toxicity. Currently, the role of MDR1 in the ovary has not been entirely understood. Our objective is to determine the function of MDR1 in protecting against chemotherapy-induced ovarian toxicity. Using both the in vivo transgenic mouse model and in vitro follicle culture model, we investigated the expression of MDR1 in the ovary, the effect of MDR1 deficiency on doxorubicin (DOX)-induced ovarian toxicity, and the ovarian steroid hormonal regulation of MDR1. Results showed that the MDR1 was expressed in the ovarian epithelial cells, stroma cells, theca cell layers, endothelial cells, and luteal cells. The lack of MDR1 did not affect female ovarian function and fertility; however, its deficiency significantly exacerbated the DOX-induced ovarian toxicity in both in vivo and in vitro models. The MDR1 showed significantly higher expression levels in the ovaries at estrus and metestrus stages than those at proestrus and diestrus stages. However, this dynamic expression pattern was not regulated by the ovarian steroid hormones of estrogen (E2) and progesterone (P4) but correlated to the number and status of corpus luteum. In conclusion, our study demonstrates that the lack of MDR1 promotes DOX-induced ovarian toxicity, suggesting the critical role of MDR1 in protecting female ovarian functions during chemotherapy.

The UDP-glucose ceramide glycosyltransferase (UGCG) and the link to multidrug resistance protein 1 (MDR1)

The UDP-glucose ceramide glycosyltransferase (UGCG) is a key enzyme in the sphingolipid metabolism by generating glucosylceramide (GlcCer), the precursor for all glycosphingolipids (GSL), which are essential for proper cell function. Interestingly, the UGCG is also overexpressed in several cancer types and correlates with multidrug resistance protein 1 (MDR1) gene expression. This membrane protein is responsible for efflux of toxic substances and protects cancer cells from cell damage through chemotherapeutic agents. Studies showed a connection between UGCG and MDR1 overexpression and multidrug resistance development, but the precise underlying mechanisms are unknown. Here, we give an overview about the UGCG and its connection to MDR1 in multidrug resistant cells. Furthermore, we focus on UGCG transcriptional regulation, the impact of UGCG on cellular signaling pathways and the effect of UGCG and MDR1 on the lipid composition of membranes and how this could influence multidrug resistance development. To our knowledge, this is the first review presenting an overview about UGCG with focus on the relationship to MDR1 in the process of multidrug resistance development.

Abstract 254: Lung cancer cells growing under prolonged period of serum starvation display increased stemness

Abstract Purpose: Resistance to anticancer drugs is a common cause of failure in chemotherapy. We recently found that adherent cells growing under prolonged periods (7-12 days) of serum starvation become highly resistant to conventional anticancer drugs such as Paclitaxel, Hydroxyurea and Colchicine but retain sensitivity to therapeutic levels of the cardiac glycoside Digitoxin. Objectives: The main goal of this study is to characterize the biological properties of cells growing under prolonged periods of serum starvation (PPSS), and to elucidate the underlying mechanism of chemoresistance to Paclitaxel and sensitivity to Digitoxin with particular emphasis on Sox2 and the PI3K/AKT signaling pathway. Experimental setup: Distribution of cell cycle was characterized by flow cytometry. Cell viability was assessed using the MTT assay. Western blotting was employed to evaluate the expression of several key protein markers of cancer stemness (Sox2, Wnt), multidrug resistance (MDR1, ABCG2), epithelial mesenchymal transition (Vimentin), and cell survival (Bcl-2, XIAP, PI3K/AKT). Results: Cells grown under PPSS showed a higher proportion of cells in G1/S and G2 compared to cells growing under routine culture conditions (RCCs). Western blot analysis showed that these cells expressed higher levels of Sox2, MDR1, ABCG2, Bcl-2 compared to cells growing under RCCs. Pretreatment of cells for 3 h with Verapamil or Sorafenib (MDR1 and ABCG2 inhibitors, respectively) followed by co-incubation with Paclitaxel for 72 h did not overcome Paclitaxel resistance. Obatoclax (a Bcl-2 inhibitor), Wortmannin or LY294002 (PI3K inhbitors) also failed to overcome Paclitaxel resistance. It was interesting to observe that cells growing under PPSS were highly resistant to Obatoclax, Wortmannin and LY294002 compared to cells growing under RCCs. However, these cells were highly sensitive to treatment with Digitoxin. Conclusions: It is likely that multiple mechanisms (increased stemness, overexpression of multidrug resistance protein and prosurvival proteins) contributes to the resistance of cells under PPSS to Paclitaxel, Obatoclax and PI3K inhibitors. Preliminary results indicates that Digitoxin exerts its antiproliferative effect in part by downregulating Sox2. However, the lack of effect of Wortmannin and LY294002 on Paclitaxel resistance suggests that this effect is not via the PI3K/AKT pathway. Adherent cells growing under PPSS provide a simple yet powerful system to screen for drugs targeting cancer stemness. Citation Format: Rajkumar Venkatadri, Juan Sebastian Yakisich, Neelam Azad, Anand Krishnan V. Iyer. Lung cancer cells growing under prolonged period of serum starvation display increased stemness. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 254.

NWIW cancels Tacoma methanol project following careful review & evaluation

Chemotherapy is one of the main strategies for cancer treatment, but its effective application is seriously limited by the development of drug resistance. In this study, we designed micellar vectors for doxorubicin based on amphiphilic copolymers sequentially linking β-cyclodextrin (β-CD), polylacticacid (PLA) or polycaprolactone (PCL) block, and polyethylene glycol (PEG) block to overcome drug resistance in human acute myeloid leukemia cells (HL60/ADR) overexpressing multidrug resistance protein 1 (MRP1). The significant enhancement in cytotoxicity and inhibited HL60/ADR tumor growth in mouse was achieved. More importantly, several analyses were performed to understand the interactions between various polymers and MRP1 at the cellular level. The results showed that the polymers did not show remarkable correlation of MRP1 gene and protein expression, but could decrease intracellular ATP, mitochondrial membrane potential and glutathione levels, which was greatly dependent on the molecular structure of polymers. In conclusion, these novel micelles can be considered as a kind of promising drug delivery system for tumor therapy to reverse drug resistance related to MRP1 overexpression.

Mechanism study of PEGylated polyester and β-cyclodextrin integrated micelles on drug resistance reversal in MRP1-overexpressed HL60/ADR cells

Chemotherapy is one of the main strategies for cancer treatment, but its effective application is seriously limited by the development of drug resistance. In this study, we designed micellar vectors for doxorubicin based on amphiphilic copolymers sequentially linking β-cyclodextrin (β-CD), polylacticacid (PLA) or polycaprolactone (PCL) block, and polyethylene glycol (PEG) block to overcome drug resistance in human acute myeloid leukemia cells (HL60/ADR) overexpressing multidrug resistance protein 1 (MRP1). The significant enhancement in cytotoxicity and inhibited HL60/ADR tumor growth in mouse was achieved. More importantly, several analyses were performed to understand the interactions between various polymers and MRP1 at the cellular level. The results showed that the polymers did not show remarkable correlation of MRP1 gene and protein expression, but could decrease intracellular ATP, mitochondrial membrane potential and glutathione levels, which was greatly dependent on the molecular structure of polymers. In conclusion, these novel micelles can be considered as a kind of promising drug delivery system for tumor therapy to reverse drug resistance related to MRP1 overexpression.

Comparison of the treatment effect of plasma exchange and no plasma exchange on hyperlipidemia pancreatitis

Chemotherapy is one of the main strategies for cancer treatment, but its effective application is seriously limited by the development of drug resistance. In this study, we designed micellar vectors for doxorubicin based on amphiphilic copolymers sequentially linking β-cyclodextrin (β-CD), polylacticacid (PLA) or polycaprolactone (PCL) block, and polyethylene glycol (PEG) block to overcome drug resistance in human acute myeloid leukemia cells (HL60/ADR) overexpressing multidrug resistance protein 1 (MRP1). The significant enhancement in cytotoxicity and inhibited HL60/ADR tumor growth in mouse was achieved. More importantly, several analyses were performed to understand the interactions between various polymers and MRP1 at the cellular level. The results showed that the polymers did not show remarkable correlation of MRP1 gene and protein expression, but could decrease intracellular ATP, mitochondrial membrane potential and glutathione levels, which was greatly dependent on the molecular structure of polymers. In conclusion, these novel micelles can be considered as a kind of promising drug delivery system for tumor therapy to reverse drug resistance related to MRP1 overexpression.

The expression of multidrug resistance protein 5 and thymydilate synthase on fluorouracil resistance WiDr colon cancer cell line

Background : Colorectal cancer becomes one of the most common cancer in Indonesia. Fluorouracil is an essential drug in colorectal cancer therapy as it has been supplemented in about 69% of current regiments of chemotherapy. However, drug resistance has reduced its clinical applications. Many factors that contribute to fluorouracil resistance have been investigated including overexpression of Multidrug Resistance Protein 5 (MRP 5) and high level expression of Thymidylate Synthase (TS). Therefore, the expression of those proteins may become key indicators to predict the presence of resistance in the model of fluorouracil-resistance-WiDr colon cancer cell line. Objective: The aim of this study is to characterize a 5-FU acquired resistance WiDr colon cancer cell line, focus on the expression of MRP5 and TS. Methods: This study was a post test controlled group design. The expression of MRP5 and TS were analyzed using immunocytochemistry method. Before protein expression analysis, 4 groups of WiDr colon cancer cell line, group 1, 2, 3, and 4 (control) were treated with 4 different concentration of fluorouracil 12.3 I¼M, 6.15 I¼M, 3.08 I¼M, and 0 I¼M respectively. Previous study showed that a significant increased level of IC50 value is observed in group induced with fluorouracil 3.08 I¼M (group 3). Statistical tests for protein expression performed with SPSS version 19 using Kruskall Wallis with 95% confidence level (p<0.05). Results: The expression of MRP5 and TS in the group developed fluorouracil resistance (group 3) were significantly increased compared to control group (p<0.05). In contrast, the other group exposed with higher dose of fluorouracil did not show both significant increased level of IC50 nor significant different of MRP5 and TS expression compared with the control group. Conclusion: The model of fluorouracil-acquired resistant WiDr cell line expresses Thymidilate Synthase and Multidrug Resistance Protein 5. The expressions of these proteins are in accordance with the profile of fluorouracil resistancy.

Upregulation of COX-2 in the lung cancer promotes overexpression of multidrug resistance protein 4 (MRP4) via PGE2-dependent pathway

It is apparent that lung cancer is associated with inflammation, with accompanying hallmark elevations of cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) levels. However, the effects of these changes on MRP efflux transporters have not been thoroughly investigated before. Here, we report that upregulation of COX-2 can induce overexpression of MRP4 in both A549 non-small-cell lung cancer cell lines and mouse lung cancer models. In A549 cells, phorbol 12-myristate 13-acetate (PMA) treatment induced upregulation of COX-2 and MRP4 together, but not other MRP transporters. Transient overexpression of human COX-2 cDNA also specifically increased COX-2 and MRP4. Moreover, COX inhibitor treatment and COX-2-specific siRNA significantly inhibited the upregulation of MRP4. Additionally, PMA-treatment increased extracellular PGE2 levels, likely due to increased MRP4 function. Likewise, COX-2-specific siRNA reduced extracellular PGE2 levels. Furthermore, COX-2 upregulation resulted in an increase in mPGES-1, an enzyme responsible for PGE2 production. Finally, metastasized lung cancer model mice exhibited increased expression levels of COX-2 and MRP4, as well as mPGES-1. In conclusion, the present study suggests that overexpression of MRP4 in lung cancer may be attributable to COX-2 upregulation via a PGE2-dependent pathway.

Pharmacoresistant epilepsy and nanotechnology.

Epilepsy is one of the most common chronic neurological disorders. Furthermore, it is associated to diminished health-related quality of life and is thus considered a major public health problem. In spite of the large number of available and ongoing development of several new antiepileptic drugs (AEDs), a high percentage of patients with epilepsy (35-40%) are resistant to pharmacotherapy. A hypothesis to explain pharmacoresistance in epilepsy suggests that overexpression of multidrug resistance proteins, such as P-glycoprotein, on the endothelium of the blood brain barrier represents a challenge for effective AED delivery and concentration levels in the brain. Proven therapeutic strategies to control pharmacoresistant epilepsy include epilepsy surgery and neuromodulation. Unfortunately, not all patients are candidates for these therapies. Nanotechnology represents an attractive strategy to overcome the limited brain access of AEDs in patients with pharmacoresistant epilepsy. This manuscript presents a review of evidences supporting this idea.

Vincristine and Prednisolone Combination Reduces MDR1 and Microenvironment-Mediated Treatment Resistance In Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer with excellent treatment outcome where >80% are cured. However, relapse and therapy-related toxicities limit further improvements and greatly increase the cost of therapy. Vincristine (VCR) is cheap, well tolerated, and highly effective. Using VCR optimally will help improve the cost-benefit ratio favorably by allowing us to reduce toxicities like infections from myelosuppression and yet improving cure.The highly successful BFM-ALL treatment backbone starts with a single intrathecal methotrexate on Day 1 followed by 7 days of oral prednisolone (PRED). The persistence of absolute blasts count >1,000/µL at Day 8 (D8), known as PRED poor response, confers a significantly poorer treatment outcome. To avoid seeding the CNS with leukemia from traumatic taps, the new Ma-Spore ALL 2010 treatment protocol, omitted intrathecal methotrexate at Day 1 and replaced with VCR at Day 0. By June 2013, a total of 133 patients have been enrolled. We found that the number of poor PRED responders was halved from the historical 9.5% in the previous Ma-Spore ALL 2003 study (Yeoh et al. J Clin Oncol 2013) to only 4.7% of patients in the ALL 2010 study. In addition, the percentage of MRD standard risk patients (Day 33 blast count ≤1x10-4) increased from 38.9% in the Ma-Spore ALL 2003 to 51.8% in the Ma-Spore ALL 2010 study (P<0.001). The 2-year event-free survival (EFS) for good and poor D8 response patients under the Ma-Spore ALL 2010 trial remained similar to the ALL 2003 study despite only half the number of PRED poor responders (Fig. 1). These data taken together suggests that VCR and PRED combination is highly synergistic and can improve early treatment response. [Display omitted] We investigated VCR and PRED combination in PRED and VCR-resistant (VCR-R) cell lines. Specifically, REH cell line is intrinsically resistant to PRED in vitro because of a mutation in its glucocorticoid receptor. We exposed the REH cell line to increasing concentrations of VCR over 6 months and generated a VCR resistant REH cell line (Fig. 2). This VCR-R REH cell line is resistant to both PRED or VCR when exposed individually in vitro. However when exposed to both PRED and VCR in combination, only 30% of the resistant cells survived (P<0.01). [Display omitted] We found that the drug efflux transporter multi-drug resistance protein 1 (MDR1) was preferentially highly expressed in our VCR-R cell line models. To determine if the highly expressed MDR1 is responsible for treatment resistance, we exposed the VCR-R cell lines to VCR, verapamil (an MDR1 inhibitor) and combination of both VCR and verapamil. The combination of VCR and verapamil increased the G2 cell cycle arrest by 3- folds compared to when VCR was used alone (Fig. 3), supporting the role of MDR1 in treatment resistance. [Display omitted] Interestingly we also found that the combination of VCR and PRED led to a decrease in levels of MDR1 expression by western blot, suggesting that depletion of MDR1 may be a mechanism through which VCR and PRED combination therapy enhances leukemic cell killing.We also investigated microenvironment-mediated resistance to VCR and PRED using mesenchymal stromal cells (MSC) co-culture systems. It was found that after co-culture with MSC or in conditional medium containing soluble factors secreted by MSC, leukemic cells were resistant to VCR and PRED mono-treatment, but the resistance was abrogated after combinatorial therapy.In conclusion, VCR in combination with PRED improves D8 peripheral blood treatment response during early induction in our Ma-Spore 2010 trial. This synergistic combination results from its ability to reverse resistance from intrinsic overexpression of MDR1 in resistant leukemia cells and decrease microenvironment-contributed resistance by mesenchymal cells. Disclosures:No relevant conflicts of interest to declare.