Expression Of ATP-binding Cassette Transporters Research Articles

Exploring the Carbamazepine Interaction with Human Pregnane X Receptor and Effect on ABCC2 Using in Vitro and in Silico Approach.

Over expression of ATP-binding cassette transporters is considered one of the major reasons for non-responsiveness to antiepileptic drugs. Carbamazepine (CBZ), one of first line antiepileptic drug is known to influence ABCC2 expression but its exact molecular mechanism is unknown. We investigated the effect of CBZ on expression of ABCC2 and pregnane X receptor (PXR) in HepG2 cell line and compared with hyperforin (agonist of PXR) and ketoconazole (antagonist of PXR) through realtime PCR and western blot assay. Involvement of PXR was demonstrated through nuclear translocation and RNA interference and related effect of CBZ on ABCC2 through functional activity assay. Molecular docking and dynamic simulation approach was used to understand the interaction of CBZ with PXR. CBZ and hyperforin increased the PXR and ABCC2 expression whereas reversed when present it in combination with ketoconazole. Experiments confirmed CBZ induced ABCC2 expression is PXR dependent. Molecular dynamic (MD) simulation and in vitro experiment indicated possibility of CBZ to be PXR agonist and PXR residue Gln285 to be important for CBZ-PXR interaction. CBZ alters the functional activity of ABCC2 through PXR, which in turn can interfere with therapy. Mutational analysis of residues revealed the importance of Gln285 in ligand interaction.

Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression.

Despite advances in therapy, multiple myeloma remains incurable, with a high frequency of relapse. This suggests the need to identify additional factors that contribute to drug resistance. Our previous studies revealed that bone marrow adipocytes promote resistance to chemotherapy in myeloma through adipocyte-secreted adipokines, but the mechanism underlying this effect and the specific adipokines involved are not well understood. We proposed to determine the role of resistin, an adipokine that is secreted by adipocytes, in chemotherapy resistance in myeloma. We found that resistin abrogated chemotherapy-induced apoptosis in established myeloma cell lines and primary myeloma samples. Resistin inhibited chemotherapy-induced caspase cleavage through the NF-κB and PI3K/Akt pathways. Resistin also increased the expression and drug efflux function of ATP-binding cassette (ABC) transporters in myeloma cells through decreasing the expression of both DNA methyltransferases DNMT1 and DNMT3a and the methylation levels of ABC gene promoters. In vivo studies further demonstrated the protective effect of resistin in chemotherapy-induced apoptosis. Our study thus reveals a new biological function of resistin in the pathogenesis of myeloma, with the implication that targeting resistin could be a potential strategy to prevent or overcome multidrug resistance in myeloma.

The sensitivity of glioma cells to pyropheophorbide-αmethyl ester-mediated photodynamic therapy is enhanced by inhibiting ABCG2.

To study the mechanisms of human glioblastoma cell resistance to methyl ester pyropheophorbide-a-mediated photodynamic therapy (MPPa-PDT) and the relationship between the cells and adenosine triphosphate-binding cassette superfamily G member 2 (ABCG2). The sensitivity of four human glioma cell lines (U87, A172, SHG-44, and U251) to MPPa-PDT was detected with a CCK-8 assay. Cell apoptosis, intracellular MPPa, and singlet oxygen were tested with flow cytometry. The mRNA and protein expression of ATP-binding cassette transporters (ABCG2, MRP1, and MDR1) were detected by PCR and Western blot, respectively. Both the sensitivity to MPPa-PDT and intracellular MPPa in A172 were the lowest among the four cell lines, while expression of ABCG2 mRNA and protein in A172 were the highest. The intracellular MPPa and ROS in A172 receiving MPPa-PDT significantly increased after using the ABCG2 inhibitor fumitremorgin C (FTC). Both cell viability and apoptosis in A172 cells undergoing MPPa-PDT were significantly improved with FTC. ABCG2 plays a significant role in the resistance of A172 to MPPa-PDT. Lasers Surg. Med. 49:719-726, 2017. © 2017 Wiley Periodicals, Inc.

ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling

BackgroundLung cancer (LC) is still the most common cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all LC cases but is not a single entity. It is now accepted that, apart from the characteristic driver mutations, the unique molecular signatures of adeno- (AC) and squamous cell carcinomas (SCC), the two most common NSCLC subtypes should be taken into consideration for their management. Therapeutic interventions, however, frequently lead to chemotherapy resistance highlighting the need for in-depth analysis of regulatory mechanisms of multidrug resistance to increase therapeutic efficiency.MethodsNon-canonical Wnt5a and canonical Wnt7b and ABC transporter expressions were tested in primary human LC (n = 90) resections of AC and SCC. To investigate drug transporter activity, a three dimensional (3D) human lung aggregate tissue model was set up using differentiated primary human lung cell types. Following modification of the canonical, beta-catenin dependent Wnt pathway or treatment with cisplatin, drug transporter analysis was performed at mRNA, protein and functional level using qRT-PCR, immunohistochemistry, immune-fluorescent staining and transport function analysis.ResultsNon-canonical Wnt5a is significantly up-regulated in SCC samples making the microenvironment different from AC, where the beta-catenin dependent Wnt7b is more prominent. In primary cancer tissues ABCB1 and ABCG2 expression levels were different in the two NSCLC subtypes. Non-canonical rhWnt5a induced down-regulation of both ABCB1 and ABCG2 transporters in the primary human lung aggregate tissue model recreating the SCC-like transporter pattern. Inhibition of the beta-catenin or canonical Wnt pathway resulted in similar down-regulation of both ABC transporter expression and function. In contrast, cisplatin, the frequently used adjuvant chemotherapeutic agent, activated beta-catenin dependent signaling that lead to up-regulation of both ABCB1 and ABCG2 transporter expression and activity.ConclusionsThe difference in the Wnt microenvironment in AC and SCC leads to variations in ABC transporter expression. Cisplatin via induction of canonical Wnt signaling up-regulates ABCB1 and ABCG2 drug transporters that are not transporters for cisplatin itself but are transporters for drugs that are frequently used in combination therapy with cisplatin modulating drug response.

Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps.

Melanoma remains one of the most therapy-resistant forms of human cancer despite recent introductions of highly efficacious targeted therapies. The intrinsic therapy resistance of human melanoma is largely due to abundant expression of a repertoire of xenobiotic efflux pumps of the ATP-binding cassette (ABC) transporter family. Here, we report that GH action is a key mediator of chemotherapeutic resistance in human melanoma cells. We investigated multiple ABC efflux pumps (ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1, and ABCG2) reportedly associated with melanoma drug resistance in different human melanoma cells and tested the efficacy of five different anti-cancer compounds (cisplatin, doxorubicin, oridonin, paclitaxel, vemurafenib) with decreased GH action. We found that GH treatment of human melanoma cells upregulates expression of multiple ABC transporters and increases the EC50 of melanoma drug vemurafenib. Also, vemurafenib-resistant melanoma cells had upregulated levels of GH receptor (GHR) expression as well as ABC efflux pumps. GHR knockdown (KD) using siRNA in human melanoma cells treated with sub-EC50 doses of anti-tumor compounds resulted in significantly increased drug retention, decreased cell proliferation and increased drug efficacy, compared to mock-transfected controls. Our set of findings identify an unknown mechanism of GH regulation in mediating melanoma drug resistance and validates GHR as a unique therapeutic target for sensitizing highly therapy-resistant human melanoma cells to lower doses of anti-cancer drugs.

Inhibition of Ceramide Decreased the Expression of ATP-Binding Cassette Transporter G5/8 mRNA in an Animal Model of Cholesterol Gallstone

Background: The increased risk of gallstone has been reported in patients with ATP-binding cassette (ABC) transporter polymorphism. The half-transporters ABCG5 and ABCG8 mediate the efflux of cholesterol in hepatocytes and the intestine. We investigated whether ceramide plays a role in cholesterol efflux through the ABC transporters. Methods: Six-week-old C57BL/6J mice were assigned to 3 groups. The normal group (n = 5) was fed a normal chow diet, the cholesterol group (n = 10) was fed a lithogenic diet, and the myriocin group (n = 15) was fed the lithogenic diet and myriocin, a specific inhibitor of serine-palmitoyl transferase. After 6 weeks, the ABCG5 and ABCG8 transporters were analyzed. Results: The rate of cholesterol gallstone formation in cholesterol group was also higher than that in normal and myriocin groups (0, 70, and 40%, respectively). ABCG5 and ABCG8 mRNA levels were significantly increased in cholesterol group and less increased in myriocin group, relative to that in normal group (p < 0.05). Conclusions: The inhibition of ceramide biosynthesis by myriocin suppressed gallstone formation and ABCG5/8 mRNA expression. We expect that ceramide's role as a regulator of the ABCG5/8 transporter might be linked to cholesterol gallstone formation.

&amp;beta;&lt;sub&gt;3&lt;/sub&gt;-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells

ObjectiveThe aim of this study was to explore the effects of β3-adrenoceptor (β3-AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells.Materials and methodsHepG2 cells were cultured and treated with the β3-AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β3-AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining 3H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed.Resultsβ3-AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β3-AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants.ConclusionActivation of β3-AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression.

The FOXM1\u2013ABCC5 axis contributes to paclitaxel resistance in nasopharyngeal carcinoma cells

Paclitaxel is clinically used as a first-line chemotherapeutic regimen for several cancer types, including head and neck cancers. However, acquired drug resistance results in the failure of therapy, metastasis and relapse. The drug efflux mediated by ATP-binding cassette (ABC) transporters and the survival signals activated by forkhead box (FOX) molecules are critical in the development of paclitaxel drug resistance. Whether FOX molecules promote paclitaxel resistance through drug efflux remains unknown. In this study, we developed several types of paclitaxel-resistant (TR) nasopharyngeal carcinoma (NPC) cells. These TR NPC cells acquired cancer stem cell (CSC) phenotypes and underwent epithelial to mesenchymal transition (EMT), and developed multidrug resistance. TR cells exhibited stronger drug efflux than parental NPC cells, leading to the reduction of intracellular drug concentrations and drug insensitivity. After screening the gene expression of ABC transporters and FOX molecules, we found that FOXM1 and ABCC5 were consistently overexpressed in the TR NPC cells and in patient tumor tissues. Further studies demonstrated that FOXM1 regulated abcc5 gene transcription by binding to the FHK consensus motifs at the promoter. The depletion of FOXM1 or ABCC5 with siRNA significantly blocked drug efflux and increased the intracellular concentrations of paclitaxel, thereby promoting paclitaxel-induced cell death. Siomycin A, a FOXM1 inhibitor, significantly enhanced in vitro cell killing by paclitaxel in drug-resistant NPC cells. This study is the first to identify the roles of FOXM1 in drug efflux and paclitaxel resistance by regulating the gene transcription of abcc5, one of the ABC transporters. Small molecular inhibitors of FOXM1 or ABCC5 have the potential to overcome paclitaxel chemoresistance in NPC patients.

Effect of Oxidative Stress on ABC Transporters: Contribution to Epilepsy Pharmacoresistance.

Epilepsy is a neurological disorder affecting around 1%–2% of population worldwide and its treatment includes use of antiepileptic drugs to control seizures. Failure to respond to antiepileptic drug therapy is a major clinical problem and over expression of ATP-binding cassette transporters is considered one of the major reasons for pharmacoresistance. In this review, we have summarized the regulation of ABC transporters in response to oxidative stress due to disease and antiepileptic drugs. Further, ketogenic diet and antioxidants were examined for their role in pharmacoresistance. The understanding of signalling pathways and mechanism involved may help in identifying potential therapeutic targets and improving drug response.

CD24 Modulates Chemosensitivity of MCF-7 Breast Cancer Cells.

The role of cluster of differentiation (CD) 24 in breast cancer remains unclear; previously, we showed that CD24 suppresses malignant phenotypes by inactivating Hedgehog signaling through signal transducer and activator of transcription (STAT) 1 inhibition. In this study, we examined how CD24 affects chemosensitivity in breast cancer cells. The CD44+CD24+ breast cancer cell line MCF-7 was transfected with CD24 with/without STAT1 siRNA, and chemosensitivity to 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum (CDDP) was measured. CD24 inhibition reduced chemosensitivity to 5-FU, while STAT1 inhibition did not affect chemosensitivity to 5-FU in CD24 siRNA-transfected cells. Conversely, CD24 inhibition did not affect chemosensitivity to CDDP, while STAT1 inhibition reduced chemosensitivity to CDDP in CD24 siRNA-transfected cells. STAT1 inhibition, but not CD24 inhibition, reduced expression of the ATP-binding cassette (ABC) transporter genes, ABCB1 and ABCG2. In conclusion, CD24 inhibition may modulate chemosensitivity according to drug type, but ABC transporter expression appears not to contribute to this mechanism. This study contributes to determining the role of CD24 in breast cancer.

Expression and splicing of ABC and SLC transporters in the human blood-brain barrier measured with RNAseq

The blood-brain barrier (BBB) expresses numerous membrane transporters that supply needed nutrients to the central nervous system (CNS), consisting mostly of solute carriers (SLC transporters), or remove unwanted substrates via extrusion pumps through the action of ATP binding cassette (ABC) transporters. Previous work has identified many BBB transporters using hybridization arrays or qRT-PCR, using targeted probes. Here we have performed next-generation sequencing of the transcriptome (RNAseq) extracted from cerebral cortex tissues and brain microvessel endothelial cells (BMEC) obtained from two donors. The same RNA samples had previously been measured for transporter expression using qRT-PCR (Geier et al., 2013), yielding similar expression levels for overlapping mRNAs (R=0.66, p<0.001). RNAseq confirms a number of transporters highly enriched in BMECs (e.g., ABCB1, ABCG2, SLCO2B1, and SLC47A1), but also detects novel BMEC transporters. Multiple splice isoforms detected by RNAseq are either robustly enriched or depleted in BMECs, indicating differential RNA processing in the BBB. The Complete RNAseq data are publically available (GSE94064).

Abstract WMP46: Exosomes Derived From Bone Marrow Mesenchymal Stem Cells of Type Two Diabetes Rats Promotes Neurorestoration After Stroke in Type Two Diabetic Rats

Background and Purpose: Roughly 1 in every 3 stroke patients is diabetic and suffers from an increased risk of stroke recurrence, worse prognosis, and exacerbated vascular and white matter (WM) injury that hinders long term recovery. Hence, there is a critical need to develop therapeutic strategies specifically to improve neurological functional outcome after stroke in the diabetic population. We investigated the neurorestorative effects of treatment of stroke in rats with type 2 diabetes mellitus (T2DM) with bone marrow stem cell (MSC) derived exosomes (T2DM-MSC-Exo) and the mechanisms underlying the therapeutic effect. Methods: Adult male Wistar rats were induced with T2DM using a combination of high fat diet and low dose Streptozotocin and were subjected to transient middle cerebral artery occlusion (MCAo). Three days after MCAo, rats were randomized (n=10/group) and treated via tail-vein injection with: 1) phosphate-buffered-saline (PBS); or 2) T2DM-MSC-Exo (3х10 11 ). A battery of neurological tests was performed and rats were sacrificed at 28 days after stroke for immunohistochemical evaluation. Results: In T2DM rats, stroke treatment using T2DM-MSC-Exo initiated at a delayed time point (3 days after stroke) significantly (p&lt;0.05 vs. PBS treated T2DM-MCAo rats) improves functional outcome, reduces blood brain barrier (BBB) leakage and hemorrhage, but does not affect blood glucose or lesion volume. Delayed stroke treatment with T2DM-MSC-Exo in T2DM rats, significantly (p&lt;0.05) increases axon and myelin density, increases oligodendrocytes and oligodendrocyte progenitor cell numbers in the ischemic border zone compared to PBS treated T2DM-MCAo rats. Treatment of T2DM stroke rats with T2DM-MSC-Exo significantly (p&lt;0.05) increases the expression of ATP-binding cassette transporter 1 (ABCA1) and Insulin-like growth factor 1 receptor (IGFR1) compared to PBS treated T2DM-MCAo rats. Conclusion: Delayed treatment of T2DM stroke using T2DM-MSC-Exo significantly promotes long term neurological functional recovery and neurorestorative effects. These beneficial effects may be mediated by a decrease in BBB leakage and hemorrhage, increase in axon and myelin density, and up regulation of the ABCA1-IGFR1 pathway.

ATP-binding casette transporter expression in acute myeloid leukemia: association with in vitro cytotoxicity and prognostic markers.

Drug resistance and relapse are considered to be the major reasons for treatment failure in acute myeloid leukemia (AML). There is limited data on the role of ABC transporter expression on in vitro sensitivity to cytarabine (Ara-C) and daunorubicin (Dnr) in primary AML cells. RNA expression levels of 12 ABC transporters were analyzed by real-time quantitative PCR in 233 de novo adult acute myeloid leukemia patients. Based on cytarabine or Dnr IC50, the samples were categorized as sensitive, intermediate and resistant. Role of candidate ABC transporter RNA expression on in vitro cytotoxicity, treatment outcome post therapy as well as the influence of various prognostic markers on ABC transporter expression were analyzed. Expression of ABCC3 and ABCB6 were significantly higher in Dnr-resistant samples when compared with Dnr-sensitive samples. Increased ABCC1 expression was associated with poor disease-free survival in this cohort of patients. This comprehensive analysis suggests ABCC1, ABCC3, ABCB6 and ABCA5 as probable targets which can be modulated for improving chemotherapeutic responses.

A novel mechanism governing the transcriptional regulation of ABC transporters in MDR cancer cells.

P-glycoprotein (P-gp/ABCB1) and multidrug resistance-associated protein 1 (MRP1/ABCC1) are the main drug efflux transporters associated with treatment failure in cancer. Much attention has been focused on the molecular mechanisms regulating the expression of these transporters as a viable approach for identifying novel drug targets in circumventing cancer multidrug resistance (MDR) clinically. In this paper, we examine the role of miR-326 in the context of its intercellular transfer between cancer cells by extracellular membrane vesicles called microparticles (MPs). We observe that cellular suppression of ABCC1 by miR-326 is modulated by the presence of ABCB1 transcript. Specifically, we show that siRNA silencing of MP-transferred ABCB1 transcript reverses the knockdown effects of miRNA-326 on target MRP1/ABCC1 transcripts. We also demonstrate a dominance of ABCB1 transcripts when co-localized with ABCC1 transcripts, which is consistent with the facilitation of miR-326 function by ABCB1. This study identifies a novel pathway regulating the expression of ABC transporters and positions ABCB1 mRNA as a transcriptional regulator of other members of this superfamily in multidrug resistant cells through its actions on miRNAs.

Epithelial-mesenchymal transition: potential regulator of ABC transporters in tumor progression.

Epithelial-mesenchymal transition (EMT) can directly contribute to some malignant phenotypes of tumor cells including invasion, metastasis and resistance to chemotherapy. Although EMT is widely demonstrated to play a critical role in chemoresistance and metastasis, the potential signaling network between EMT and drug resistance is still unclear. The distribution of drugs in the internal and external environment of the tumor cells is tightly linked with ATP-binding cassette (ABC) transporters. Recent studies have shown that ABC transporters expression changed continuously during EMT. We believe that EMT is an important regulator of ABC transporters. In this review, we discuss how EMT regulates ABC transporters and their potential linkages. And we hope the knowledge of EMT and ABC transporters will offer more effective targets to experimental research.

Role of ABC transporters in trans-epithelial transport of vitamin K antagonists.

Vitamin K antagonists (VKAs) remain the oral anticoagulant of choice in venous thromboembolic disease. These drugs are characterized by a large inter-individual variability requiring frequent dose tailoring. Genetic polymorphisms for cytochrome CYP2C9 and VKORC1 explain some of the variability, especially in warfarin and acenocoumarol responses. The aim of this study was to assess, in cell models, the role of ABC transporters in the intestinal transfer of the main coumarin derivatives (warfarin, acenocoumarol) and indanedione derivatives (phenindione, fluindione). The results show a basal to apical polarized transport for fluindione, phenindione and acenocoumarol only. Experimental studies using specific inhibitors of transport protein demonstrate the implication of MRPs and BCRP proteins and to a lesser extent P-gp. Warfarin and acenocoumarol seem to be poor inhibitors of MRPs protein, whereas fluindione and phenindione have a slight or no effect. The regulation of the expression of ABC transporters by exposure to VKAs was also investigated in Caco-2 cells. The expression of mRNA P-gp, MRP1, MRP2 and BCRP was weakly or not modified after 24h of VKAs exposure. In conclusion, the intestinal transfer of indanedione derivatives and acenocoumarol could be influenced by transport proteins of the ABC superfamily. Coumarin derivatives are poor inhibitors of these proteins and AVKs have a slight effect on the mRNA ABC transporter expression level. Copyright © 2016 John Wiley & Sons, Ltd.

3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF-24), a synthetic curcumin analogue, induces apoptosis independently of abc transporters expression in human leukemia cells

3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF-24), a synthetic curcumin analogue, induces apoptosis independently of abc transporters expression in human leukemia cells

Effects of tetramethylpyrazine from Chinese black vinegar on antioxidant and hypolipidemia activities in HepG2 cells

Tetramethylpyrazine is a bioactive compound found in Chinese black vinegar. This work is to investigate the potential effects of tetramethylpyrazine on intracellular cholesterol modulation in HepG2 cells. The results demonstrated that tetramethylpyrazine can induce intracellular cholesterol efflux. Tetramethylpyrazine may also improve endothelial function through its antioxidant effects by inhibiting reactive oxygen species levels and increasing the antioxidant enzymes superoxide dismutase and catalase. Tetramethylpyrazine increased liver X receptor and peroxisome proliferator-activated receptor gene expression in HepG2 cells. Protein expression of ATP-binding cassette transporter 1 was up-regulated in a dose-dependent manner (P < 0.05). The biological significance of tetramethylpyrazine may involve hypolipidemic effects via modulation of intracellular cholesterol efflux, ROS inhibition, increases in SOD and CAT activities, and direct regulation of PPAR and LXR gene expression. Tetramethylpyrazine may improve lipid profiles by elevating the PPARγ-LXRα-ABCA1 pathway.

Role of ABC transporters in the pathology of Alzheimer's disease.

The ATP-binding cassette (ABC) transporter superfamily is a large family of proteins that transport specific molecules across membranes. These proteins are associated with both cholesterol metabolism and Alzheimer's disease (AD). Cholesterol homeostasis has a key role in AD, and ABC transporters are important mediators of lipid transportation. Emerging evidence suggests that decreased expression and hypofunction of ABC transporters are crucial to the occurrence and development of AD. In the present article, we review the current knowledge regarding ABC transporters and speculate on their role in the pathogenesis of AD.

Resistin Induces Multidrug Resistance in Myeloma By Inhibiting Cell Death and Upregulating ABC Transporter Expression

Chemoresistance is a major hurdle in multiple myeloma. Most patients are prone to develop resistance to a wide spectrum of anticancer agents, significantly hampers the patients' long term outcome. Many studies point to bone marrow microenvironment as an important player in myeloma chemoresistance, in which marrow stromal cells and stromal-secreted soluble factors are shown to promote myeloma cell growth and survival. Our previous study has demonstrated that marrow-derived adipocytes protect myeloma cells against chemotherapy-induced apoptosis through adipocyte-secreted adipokines, one of such is leptin. However, the level of leptin expression in myeloma patients is not significantly changed, indicating the involvement of additional adipokines in this process. Interestingly, in a clinical study, an elevation of the adipokine resistin in the serum of myeloma patients after thalidomide treatment were observed as compared with that in patients before treatment, suggesting a potential role of this adipokine in response to chemotherapy. As a 12.5-kDa hormone that is mainly secreted by adipocytes and also secreted by other cells, resistin has a function in production of inflammatory cytokines that are important for cancer development. We thus hypothesized that resistin protects myeloma cells against chemotherapy. In our experiments, human myeloma cell lines and primary myeloma cells isolated from patient bone marrow aspirates were cultured in medium with addition of the recombinant human resistin and chemotherapy drugs melphalan or bortezomib for 24 hours. Cells without resistin served as a control. After cultures, an annexin-V binding assay for assessing apoptosis, western blot analysis for assessing cleavage of caspases and phosphorylation of signaling kinases, and the eFluxx-ID Gold uptake assay for examining ABC transporters activity were performed. In the animal study, myeloma-bearing SCID mice were treated with or without resistin and/or melphalan. Our results showed that resistin treatment reduced melphalan- or bortezomib-induced apoptosis both in vitro and in vivo. This protective effect has been further confirmed by the reduced cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase in myeloma cells. Mechanistic studies showed that culturing myeloma cells with resistin upregulated expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL and downregulated expression of the pro-apoptotic protein Bax via the NF-kB and the PI3K/Akt signaling pathways. Addition of resistin also reduced the intracellular accumulation of eFluxx-ID gold fluorescence in myeloma cells ARP-1 and MM.1S, when compared to that in cells without resistin. In addition, resistin significantly increased the mRNA and protein expression of ATP-binding cassette (ABC) transporters in myeloma cells by downregulating the expression of DNA methyltransferase 1 and 3a, and CpG methylation in the promoters of ABC transporters. Thus, our study demonstrates that resistin is a novel factor contributing to myeloma chemoresistance, and also implicates that disruption of its protective effect can be a potential strategy to improve current chemotherapy in patients and prolong survival. DisclosuresNo relevant conflicts of interest to declare.