MDR1-mediated Drug Research Articles

Investigating cross-species differences of MDR1-mediated drug transport

Investigating cross-species differences of MDR1-mediated drug transport

Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a.

Simple SummaryOur study identified the differential expression and potential effects of microRNAs in retinoblastoma vs. pediatric retina and advanced vs. non-advanced tumors. We provide evidence of the epithelial–mesenchymal transition (EMT) and chemoresistance programs in advanced tumors, which were potentially attributed to miR-181a-5p. We analyzed the differential expression of relevant EMT- and chemoresistance-related proteins in advanced vs. non-advanced tumors and chemotherapy-adapted Y79 cells to assess whether EMT and chemoresistance mechanisms were linked. We further examined the possible role of TGFβ as a potential regulator of such differences and highlighted the role of miR-181a-5p in EMT- and chemoresistance-related gene expression and drug sensitivity.Advanced retinoblastoma (Rb) tumors display high metastatic spread to distant tissues, causing a potent threat to vision and life. Through transcriptomic profiling, we discovered key upregulated genes that belonged to the epithelial–mesenchymal transition (EMT) and chemotherapy resistance pathways in advanced Rb tumors. Through in vitro models, we further showed that Rb null tumor cells under prolonged chemo drug exposure, acquires a metastasis-like phenotype through the EMT program mediated by ZEB1 and SNAI2 and these cells further acquires chemotherapeutic resistance through cathepsin-L- and MDR1-mediated drug efflux mechanisms. Using a miRNA microarray, we identified miR-181a-5p as being significantly reduced in advanced Rb tumors, which was associated with an altered EMT and drug-resistance genes. We showed that enhancing miR-181a-5p levels in Rb null chemo-resistant sublines reduced the ZEB1 and SNAI2 levels and halted the mesenchymal transition switch, further reducing the drug resistance. We thus identified miR-181a-5p as a therapeutically exploitable target for EMT-triggered drug-resistant cancers that halted their invasion and migration and sensitized them to low-dose chemotherapy drugs.

Antisense oligonucleotide gapmers containing phosphoryl guanidine groups reverse MDR1-mediated multiple drug resistance of tumor cells

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3′ and 5′ ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2′-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central “window” of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.

Hierarchically Self-Assembled Supramolecular Host-Guest Delivery System for Drug Resistant Cancer Therapy.

In this report, a new star-like copolymer β-CD- g-(PNIPAAm- b-POEGA) x, consisting of a β-CD core, grafted with temperature-responsive poly( N-isopropylacrylamide) (PNIPAAm) and biocompatible poly(oligo(ethylene glycol) acrylate) (POEGA) in a block copolymer of the arms, was used to deliver chemotherapeutics to drug resistant cancer cells and tumors. The first step of the self-assembly process involves the encapsulation of chemotherapeutics through host-guest inclusion complexation between the β-cyclodextrin cavity and the anticancer drug. Next, the chain interaction of the PNIPAAm segment at elevated temperature drives the drug-loaded β-CD- g-(PNIPAAm- b-POEGA) x/PTX inclusion complex to hierarchically self-assemble into nanosized supramolecular assemblies at 37 °C, whereas the presence of poly(ethylene glycol) (PEG) chains in the distal end of the star-like copolymer arms impart enhanced stability to the self-assembled structure. More interestingly, this supramolecular host-guest nanocomplex promoted the enhanced cellular uptake of chemotherapeutics in MDR-1 up-regulated drug resistant cancer cells and exhibited high therapeutic efficacy for suppressing drug resistant tumor growth in an in vivo mouse model, due to the increased stability, improvement in aqueous solubility, enhanced cellular uptake, and partial membrane pump impairment by taking the advantage of PEGylation and supramolecular complex between this star-like copolymer and chemotherapeutics. This work signifies that temperature-sensitive PEGylated supramolecular nanocarriers with good biocompatibility are effective in combating MDR-1 mediated drug resistance in both in vitro and in vivo models, which is of significant importance for the advanced drug delivery platform designed to combat drug resistant cancer.

Targeting multidrug-resistant ovarian cancer through estrogen receptor α dependent ATP depletion caused by hyperactivation of the unfolded protein response.

Ovarian cancers often recur and tumors acquire resistance to chemotherapy due to overexpression of the ATP-dependent efflux pump, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1). Nontoxic small molecule inhibitors targeting MDR1 have remained largely elusive. Instead, in a novel application of our recently described estrogen receptor α (ERα) biomodulator, BHPI, we targeted MDR1’s substrate, ATP. BHPI depletes intracellular ATP and nearly blocks MDR1-mediated drug efflux in ovarian cancer cells by inducing toxic hyperactivation of the endoplasmic reticulum stress sensor, the unfolded protein response (UPR). BHPI increased sensitivity of MDR1 overexpressing multidrug resistant OVCAR-3 ovarian cancer cells to killing by paclitaxel by >1,000 fold. BHPI also restored doxorubicin sensitivity in OVCAR-3 cells and in MDR1 overexpressing breast cancer cells. In an orthotopic OVCAR-3 xenograft model, paclitaxel was ineffective and the paclitaxel-treated group was uniquely prone to form large secondary tumors in adjacent tissue. BHPI alone strongly reduced tumor growth. Notably, tumors were undetectable in mice treated with BHPI plus paclitaxel. Compared to control ovarian tumors, after the combination therapy, levels of the plasma ovarian cancer biomarker CA125 were at least several hundred folds lower; moreover, CA125 levels progressively declined to undetectable. Targeting MDR1 through UPR-dependent ATP depletion represents a promising therapeutic strategy.

MPT0B169, a novel tubulin inhibitor, induces apoptosis in taxol-resistant acute myeloid leukemia cells through mitochondrial dysfunction and Mcl-1 downregulation.

Acute myeloid leukemia (AML) is a hematological malignant disorder. AML cells are not susceptible to chemotherapeutic drugs because of their multidrug resistance (MDR). Antitubulin agents are currently employed in cancer treatments; however, drug resistance results in treatment failures because of MDR1 expressing cancer cells. We previously synthesized a new tubulin inhibitor, 2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-acetamide (MPT0B169), which inhibits AML cell proliferation by arresting cell cycle at the G2/M phase. In this study, we explored the effect of MPT0B169 on apoptosis in AML HL60 and NB4 cells and MDR1-mediated taxol-resistant HL60/TaxR cells and the underlying mechanism. MPT0B169 induced concentration- and time-dependent apoptosis in these cancer cells, as observed through annexin V/propidium iodide double staining and flow cytometry. Furthermore, DNA fragmentation analysis confirmed MPT0B169-induced apoptosis. MPT0B169 induced a loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, cleavage and activation of caspase-9 and caspase-3, and consequently cleavage of poly (ADP ribose) polymerase. Western blot analysis showed that MPT0B169 markedly reduced Mcl-1 (an antiapoptotic protein) levels; however, it caused no changes in Bcl-2 or BAX (a proapoptotic protein). Knockdown of Mcl-1 using small interfering RNA (siRNA) slightly induced growth inhibition and apoptosis in the HL60 and HL60/TaxR cells. Further investigation revealed that Mcl-1 siRNA enhanced the sensitivity of HL60 and HL60/TaxR cells to MPT0B169-induced growth inhibition and apoptosis. Together, these results demonstrated that MPT0B169-induced apoptosis in nonresistant and MDR1-mediated taxol-resistant AML cells through Mcl-1 downregulation and a mitochondria-mediated pathway. MPT0B169 can overcome MDR1-mediated drug resistance in AML cells.

MDR1 Mediated Drug Resistance to a Histone Methyltansferase Inhibitor (KMT)

Aberrant histone methylation is emerging as a major contributor to oncogenesis, and inhibitors of histone lysine methyltransferases (KMTs) are starting to enter early clinical studies. Currently, inhibitors for two KMTs are in phase I/II clinical trials, an inhibitor of the histone 3 lysine 79 (H3K79) methyltransferase DOT1L for MLL-rearrange leukemias (NCT02141828), and inhibitors for the H3K27 methyltransferase EZH2 for lymphomas or solid tumors with activating EZH2 mutations (NCT01897571 and NCT02082977). The next few months and years are likely to see more KMT inhibitors entering clinical development, such as compounds directed against the H3K36 methyltransferase MMSET/NSD2/WHSC1, which is commonly translocated in multiple myeloma and carries activating mutations in a subset of acute lymphoblastic leukemia. Typically, KMT inhibitors are structurally similar and are derived from the methyl donor S-adenosyl-methionine (SAM). Very little is known about possible mechanisms of resistance to these compounds.We exposed a panel of leukemia cell lines to varying concentrations of the DOT1L inhibitor EPZ4777 and assessed the decrease of H3K79 di-methylation by Western Blotting. Our analysis revealed a dose-dependent decrease of H3K79 dimethylation in most cell lines. Interestingly, we identified a pair of cell lines, KG-1 and KG-1a, which were near completely resistant to EPZ4777 mediated DOT1L inhibition, with no effect of 10 μM EPZ4777 (4 day exposure). Even after exposure to EPZ4777 for 15 days, KG-1 showed only a slight decrease in H3K79me2 marking and no decrease in marking was seen for KG-1a up to a concentration of 3 μM. We searched the encode database for expression levels of DOT1L, as well as higher expression of proteins previously described as mediating drug resistance in AML cell lines. We found no significantly higher DOT1L expression level, suggesting that overexpression of the target is not the predominant mechanisms of resistance in KG-1/KG-1a. We confirmed similar baseline H3K79 methylation levels in KG1/KG1a and the other cell lines in our panel by Western Blotting. However, we noted a higher expression of multidrug resistance protein 1 (MDR1/p-glycoprotein/ABCB1) in the KG-1 and KG-1a cell lines compared to the other cell lines in our panel. To explore whether DOT1L inhibition by EPZ4777 could be restored in KG1 and KG1a cells when MDR1 was inhibited, cells were exposed to 0.3 μM EPZ4777 and varying concentrations of Cyclosporine A (CSA), a known inhibitor of MDR1. Increasing concentration of CSA with a fixed dose of 0.3 μM EPZ4777 led to a dose dependent decrease in H3K79me2 in KG-1/KG-1a, suggesting that inhibition of MDR1 increased the intracellularly effective dose of EPZ4777. CSA at a concentration of 0.3 μM and 0.3 μM EPZ4777 near completely abolished H3K79 methylation. In summary, our results suggest that KMT inhibitors can be subject to classic drug resistance mechanisms such as the MDR1/p-glycoprotein/ABCB1 efflux pump. These data provide insight for the use and design of small-molecule inhibitors for KMTs. DisclosuresNo relevant conflicts of interest to declare.

Single-cell pharmacokinetic imaging reveals a therapeutic strategy to overcome drug resistance to the microtubule inhibitor eribulin.

Eribulin mesylate was developed as a potent microtubule-targeting cytotoxic agent to treat taxane-resistant cancers, but recent clinical trials have shown that it eventually fails in many patient subpopulations for unclear reasons. To investigate its resistance mechanisms, we developed a fluorescent analog of eribulin with pharmacokinetic (PK) properties and cytotoxic activity across a human cell line panel that are sufficiently similar to the parent drug to study its cellular PK and tissue distribution. Using intravital imaging and automated tracking of cellular dynamics, we found that resistance to eribulin and the fluorescent analog depended directly on the multidrug resistance protein 1 (MDR1). Intravital imaging allowed for real-time analysis of in vivo PK in tumors that were engineered to be spatially heterogeneous for taxane resistance, whereby an MDR1-mApple fusion protein distinguished resistant cells fluorescently. In vivo, MDR1-mediated drug efflux and the three-dimensional tumor vascular architecture were discovered to be critical determinants of drug accumulation in tumor cells. We furthermore show that standard intravenous administration of a third-generation MDR1 inhibitor, HM30181, failed to rescue drug accumulation; however, the same MDR1 inhibitor encapsulated within a nanoparticle delivery system reversed the multidrug-resistant phenotype and potentiated the eribulin effect in vitro and in vivo in mice. Our work demonstrates that in vivo assessment of cellular PK of an anticancer drug is a powerful strategy for elucidating mechanisms of drug resistance in heterogeneous tumors and evaluating strategies to overcome this resistance.

The novel toluidine sulphonamide EL102 shows pre-clinical in vitro and in vivo activity against prostate cancer and circumvents MDR1 resistance

Background:Taxanes are routinely used for the treatment of prostate cancer, however the majority of patients eventually develop resistance. We investigated the potential efficacy of EL102, a novel toluidine sulphonamide, in pre-clinical models of prostate cancer.Methods:The effect of EL102 and/or docetaxel on PC-3, DU145, 22Rv1 and CWR22 prostate cancer cells was assessed using cell viability, cell cycle analysis and PARP cleavage assays. Tubulin polymerisation and immunofluorescence assays were used to assess tubulin dynamics. CWR22 xenograft murine model was used to assess effects on tumour proliferation. Multidrug-resistant lung cancer DLKPA was used to assess EL102 in a MDR1-mediated drug resistance background.Results:EL102 has in vitro activity against prostate cancer, characterised by accumulation in G2/M, induction of apoptosis, inhibition of Hif1α, and inhibition of tubulin polymerisation and decreased microtubule stability. In vivo, a combination of EL102 and docetaxel exhibits superior tumour inhibition. The DLKP cell line and multidrug-resistant DLKPA variant (which exhibits 205 to 691-fold greater resistance to docetaxel, paclitaxel, vincristine and doxorubicin) are equally sensitive to EL102.Conclusion:EL102 shows potential as both a single agent and within combination regimens for the treatment of prostate cancer, particularly in the chemoresistance setting.

Egr-1 enhances drug resistance of breast cancer by modulating MDR1 expression in a GGPPS-independent manner

The multidrug resistance 1 (MDR1) gene product P-glycoprotein is an ATP-dependent efflux pump associated with chemotherapy failure in breast cancer. In the present study, we show that paclitaxel induces MDR1 expression in the MCF-7 breast cancer cell line in a MAPK/Egr-1-dependent manner. Paclitaxel exposure activated the Erk1/2/MAPK pathway and promoted the accumulation of the early response transcription factor Egr-1 in MCF-7 cells. Egr-1 binds to the GC element on the proximal MDR1 promoter to enhance MDR1 transcription. Loss of Egr-1 function in paclitaxel-resistant MCF-7 cells decreased MDR1 expression, whereas inhibiting Erk1/2 activity reduced both Egr-1 accumulation and MDR1 expression. These findings suggest that Erk1/2-induced Egr-1 accumulation activates MDR1 transcription and thereby induces the drug resistance observed in paclitaxel-resistant MCF-7 cells. Further mechanistic studies indicate that Egr-1 most likely does not induce the constitutive activation of Erk1/2 through its target gene geranylgeranyl diphosphate synthase (GGPPS), which regulates Ras prenylation. Indeed, our results suggest a novel pathway by which paclitaxel induces MDR1 expression, possibly illuminating a potential target pathway for the prevention of MDR1-mediated drug resistance.

Prostate cancer inhibitory activity of a novel dual inhibitor, EL102, in combination with docetaxel, and its effects on MDR1-mediated drug resistance in vitro.

e15126 Background: EL102 is a dual-action drug promoting apoptosis and inhibiting angiogenesis. It exerts its action though the inhibition of Hif1a induced hypoxic signalling and induction of the Caspase 3/7 apoptotic cascade. The drug has equal activity in normoxia and hypoxia indicating it may be equally active in these different tumor compartments. We tested its ability to circumvent chemotherapeutic drug resistance. Methods: We assessed the ability of EL102 to inhibit prostate cancer cell proliferation and motility in vitro, calculating IC50s for CWR22, 22Rv2, PC3 and DU145 prostate cancer cell lines, comparing sensitivity between androgen dependent, androgen independent and metastatic prostate cancer. Additionally we assessed the activity of EL102 in combination with docetaxel in vitro and in murine CWR22 xenografts. The ability to overcome MDR1 and BCRP mediated drug resistance was also tested using DLKP drug resistant variants which exhibit 200 fold resistance to doxorubicin, docetaxel, paclitaxel and vincristine. Results: We found that prostate cancer cell lines are sensitive to EL102 with IC50s in the region of 10-50nM. Of particular interest was the identical sensitivity of the androgen independent 22Rv1 and its androgen dependent parent CWR22, suggesting ability to overcome hormone refractory prostate cancer. Additionally we demonstrate dose response for inhibition of cell motility in metastatic DU145. In CWR22 murine mouse models treatment with EL102 resulted in decreased tumor volume compared to control. A docetaxel and EL102 combination arm demonstrated the greater inhibition of tumor growth than EL102 or docetaxel alone. The lung cancer cell line DLKP, its drug resistant variants DLKPA (MDR1 overexpressing) and DLKPMitox (BCRP overexpressing) were equally sensitive to EL102 indicating that EL102 is not a substrate for MDR1 or BCRP. Conclusions: EL102 is a potential therapeutic for the treatment of prostate cancer, in particular in combination with docetaxel, and exhbibits the potential to overcome drug resistane. Future studies will include the efficacy of this drug in prostate cancer metastatic mouse models.

Chebulagic Acid Synergizes the Cytotoxicity of Doxorubicin in Human Hepatocellular Carcinoma Through COX-2 Dependant Modulation of MDR-1

Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) specific inhibitors are anti-inflammatory agents that have also shown to be useful in anticancer therapy. The effects of chebulagic acid (CA), a benzopyran tannin from Terminalia chebula having COX-2/5-LOX dual inhibitory properties, on the sensitivity of doxorubicin (Dox) in human hepatocellular carcinoma cell line HepG2 were studied in the present investigation. CA increased the accumulation of Dox in a concentration dependant manner and also enhanced the cytotoxicity of Dox in HepG2 cells by 20 folds. Quantitation of interaction by calculating Combination Index (CI) showed a strong synergistic interaction between CA and Dox in terms of cell growth inhibition. Calculation of dose reduction index (DRI) for CA-Dox combinations also showed a significant decrease in the dosage of Dox in the presence of CA. The induction of MDR1 expression by PGE(2), a metabolite of COX-2, and its downregulation by COX-2 knockdown or CA implies that the enhanced sensitivity of HepG2 cells to doxorubicin by CA is mediated by the downregulation of MDR1 expression, via COX-2-dependent mechanism. Further studies reveal the inactivation of signal transduction pathways involving Akt, ERK, JNK and p38 and the transcription factor NF-κB in the CA induced down regulation of MDR1. The present study shows the efficacy of CA to overcome MDR-1 mediated drug resistance in HepG2 cells through COX-2 dependant modulation of MDR-1.

Human AP endonuclease (APE1/Ref-1) and its acetylation regulate YB-1-p300 recruitment and RNA polymerase II loading in the drug-induced activation of multidrug resistance gene MDR1

Overexpression of human AP-endonuclease (APE1/Ref-1), a key enzyme in the DNA base excision repair (BER) pathway, is often associated with tumor cell resistance to various anticancer drugs. In this study, we examined the molecular basis of transcriptional regulatory (non repair) function of APE1 in promoting resistance to certain types of drugs. We have recently shown that APE1 stably interacts with Y-box-binding protein 1 (YB-1), and acts as its coactivator for the expression of multidrug resistance gene MDR1, thereby causing drug-resistance. Here we show for the first time that APE1 is stably associated with the basic transcription factor RNA polymerase II (RNA pol II) and the coactivator p300 on the endogenous MDR1 promoter. APE1’s depletion significantly reduces YB-1/p300 recruitment to the promoter, resulting in reduced RNA pol II loading. Drug-induced APE1 acetylation which is mediated by p300 enhances formation of acetylated APE1 (AcAPE1)/YB-1/p300 complex on the MDR1 promoter. Enhanced recruitment of this complex increases MDR1 promoter dependent luciferase activity and its endogenous expression. Using APE1 downregulated cells and cells overexpressing wild type APE1 or its nonacetylable mutant we have demonstrated that the loss of APE1’s acetylation impaired MDR1 activation and sensitizes the cells to cisplatin or etoposide. We have thus established the basis for APE1’s acetylation-dependent regulatory function in inducing MDR1-mediated drug resistance.

Xenopus laevis oocytes expressing human P-glycoprotein: Probing trans- and cis-inhibitory effects on [ 3H]vinblastine and [ 3H]digoxin efflux

P-glycoprotein (P-gp; MDR1) recognizes and actively transports many structurally diverse compounds (hydrophobic neutral and cationic). We studied MDR1-mediated drug transport using a high-throughput (96-well) oocyte expression system. MDR1-expressing oocytes contained sufficient ATP levels to conduct fundamental efflux studies; the optimal experimental temperature was 25 °C. [ 3H]Vinblastine efflux by MDR1-expressing oocytes was detectable and afforded a K m of 145.5 ± 25.4 μM. [ 3H]Vinblastine (5.6 ± 0.3 μM) and [ 3H]digoxin (1.0 ± 0.1 μM) were individually injected into MDR1-expressing oocytes and their efflux monitored. Quinidine and verapamil, known MDR1 substrates/inhibitors, showed trans-inhibition on MDR1-mediated [ 3H]vinblastine and [ 3H]digoxin efflux. Conversely, doxorubicin demonstrated cis-inhibition without trans-inhibition on MDR1-mediated [ 3H]vinblastine efflux. The MDR1-expressing oocyte system offers researchers with an alternative in vitro method to screen compounds and may allow one to probe P-gp drug–drug and/or drug–inhibitor interactions.

99mTc‐MIBI imaging for prediction of therapeutic effects of second‐generation MDR1 inhibitors in malignant brain tumors

The aim of this study was to explore whether (99m)Tc-methoxyisobutylisonitrile ((99m)Tc-MIBI) is suitable to elucidate multidrug resistance and prediction of potentiation of antitumor agents by second-generation MDR1 inhibitors (PSC833, MS-209) in malignant brain tumors in rat. Malignant tumor cells (RG2 and C6 gliomas, Walker 256 carcinoma) were incubated with low dose vincristine (VCR) to induce multidrug resistance. MTT assay demonstrated a significant increase of surviving fractions in VCR-resistant sublines compared to those of drug-naive cells. Reverse transcriptase polymerase chain reaction revealed higher expression of MDR1 mRNA in VCR-resistant cells than drug-naive cells in each line. Volume distribution (V(d)) of (99m)Tc-MIBI was negatively correlated with MDR1 mRNA expression among drug-naive and VCR-resistant cells. MDR1 inhibitors decreased surviving fractions and increased V(d) of (99m)Tc-MIBI significantly in VCR-resistant sublines, whereas MDR1 mRNA expression was unchanged. These findings indicate that (99m)Tc-MIBI efflux was functionally suppressed by MDR1 inhibitors. Autoradiographic images of (99m)Tc-MIBI revealed higher uptake in drug-naive cells at basal ganglia compared with VCR-resistant cells at the opposite basal ganglia of rats. Oral administration of the second-generation MDR1 inhibitors significantly increased (99m)Tc-MIBI accumulation of both tumors. Therapeutic effects of VCR with or without the MDR1 inhibitors were also evaluated autoradiographically using (14)C-methyl-L-methionine ((14)C-Met) and MIB-5 index. (14)C-Met uptake and MIB-5 index of both tumors treated with VCR following the MDR1 inhibitor treatment significantly decreased compared with tumors treated with VCR alone. Analysis of (99m)Tc-MIBI accumulation is considered informative for detecting MDR1-mediated drug resistance and for monitoring the therapeutic effects of MDR1 inhibitors in malignant brain tumors.

C-JUN-NH2-Terminal Kinase Promotes Apoptosis, Suppresses P-Glycoprotein Mediated Multidrug Resistance but Enhances MRP Mediated Efflux in AML Cells.

JNK has been implicated in distinct cellular events, as proliferation, cellular transformation and apoptosis. JNK has also been recently reported to reverse MDR1 mediated drug resistance and increase sensitivity to chemotherapeutic agents in non-hematopoietic cancer cells. As acquired drug resistance represents a major obstacle in successful therapy of Acute Myeloid Leukemia (AML), the significance of JNK activation in relation to apoptosis induction and drug resistance in AML was sought. JNK is active in U937 leukemia cells and undergoes further activation upon treatment with therapeutically relevant concentrations of daunomycin. Daunomycin induces massive apoptosis in these cells, which is significantly reduced if U937 cells are preincubated with JNK inhibitor 420116. MDR1 and MRP expression levels are low in U937 cells. URD cells, a U937 deritive cell line characterized by resistance to adriamycin, was tested. Interestingly, no basal JNK activity was detected in URD cells and daunomycin treatment did not activate JNK nor induced apoptosis, albeit abundant JNK protein expression. JNK has been proposed to be prerequisite for mediating drug resistance in HL60 cells, by up regulating GSTP1 and MRP efflux pump. GSTP1 was strongly expressed in URD cells in the absence of JNK activation, but MRP expression was insignificant. Moreover, GSTP1 contributed to JNK inactivation in these cells, as immunoprecipitation experiments revealed a complex of GSTP1 with JNK. MDR1 activity in URD cells was 90%, as detected by the calcein AM efflux assay. Transfection of URD cells with SEKED plasmid expressing JNK decreased MDR1 activity by 35% while MRP activity exhibited >3 fold increase, from 5% to 18,4%. 29% of URD cells underwent apoptosis after 24hr of treatment with 1μM daunomycin compared to 5% of cells transfected with empty vector, as measured by annexin V/ PI and DAPI staining. To validate our results to primary AML cells, 25 AML bone marrow samples were tested for JNK activation, apoptosis induction after daunomycin treatment and GSTP1, MDR1 and MRP expresion levels. Basal JNK activity was detected in 58% of patients and did not correlate with apoptosis susceptibility or MDR1 and MRP expression levels. JNK activation within 1 hr of treatment occurred in 9 samples (36%) and strongly correlated with statistically significant apoptosis (p=0,0044< 0,05) after 12hr of continuous drug exposure. In 16 samples (64%), JNK activation was not observed upon daunomycin treatment and apoptosis did not reach a statistically significant level (p=0,3>0,05). Chemotherapy induced JNK activation was irrelevant to basal JNK activity status. GSTP1 was strongly expressed in all samples examined. Our experimental data indicate that JNK activation is implicated in distinct cellular processes in response to chemotherapy. Activation of JNK correlated with apoptosis induction in U937 cells and in primary leukemia samples. Moreover, JNK activity was lost in resistant URD cells, perhaps due to GSTP1 inhibition, and when expressed induced MDR1 downregulation and apoptosis. The proapoptotic function of JNK in URD cells can not be attributed only to MDR1 downregulation, since it is also observed in U937 cells, characterized by low MDR1 activity. Interestingly, JNK expression in URD cells also upregulated the MRP efflux pump. Further studies are needed to elucidate whether JNK represents a possible drug target to overcome drug resistance in AML.

Significance of MDR1 and multiple drug resistance in refractory human epileptic brain.

BackgroundThe multiple drug resistance protein (MDR1/P-glycoprotein) is overexpressed in glia and blood-brain barrier (BBB) endothelium in drug refractory human epileptic tissue. Since various antiepileptic drugs (AEDs) can act as substrates for MDR1, the enhanced expression/function of this protein may increase their active extrusion from the brain, resulting in decreased responsiveness to AEDs.MethodsHuman drug resistant epileptic brain tissues were collected after surgical resection. Astrocyte cell cultures were established from these tissues, and commercially available normal human astrocytes were used as controls. Uptake of fluorescent doxorubicin and radioactive-labeled Phenytoin was measured in the two cell populations, and the effect of MDR1 blockers was evaluated.Frozen human epileptic brain tissue slices were double immunostained to locate MDR1 in neurons and glia. Other slices were exposed to toxic concentrations of Phenytoin to study cell viability in the presence or absence of a specific MDR1 blocker.ResultsMDR1 was overexpressed in blood vessels, astrocytes and neurons in human epileptic drug-resistant brain. In addition, MDR1-mediated cellular drug extrusion was increased in human 'epileptic' astrocytes compared to 'normal' ones. Concomitantly, cell viability in the presence of cytotoxic compounds was increased.ConclusionsOverexpression of MDR1 in different cell types in drug-resistant epileptic human brain leads to functional alterations, not all of which are linked to drug pharmacokinetics. In particular, the modulation of glioneuronal MDR1 function in epileptic brain in the presence of toxic concentrations of xenobiotics may constitute a novel cytoprotective mechanism.

Protein kinase C isozymes as potential targets for anticancer therapy.

Protein kinase C (PKC) comprises a family of isozymes (alpha, betaI, betaII, gamma, delta, epsilon, theta, eta, lambda/iota [mouse/human], and zeta) which are involved in signal transduction from membrane receptors to the nucleus. Activation of PKC by phorbol esters promotes tumor formation, and from that it was concluded that inhibitors of PKC might prevent carcinogenesis or inhibit tumor proliferation. However, the situation is more complicated because the exact function of the different PKC isozymes is not known at present. They have been shown to be involved in synaptic transmissions, the activation of ion fluxes, secretion, cell cycle control, differentiation, proliferation, tumorigenesis, metastasis and apoptosis. Modulators such as bryostatin-1, phospholipid analogues, PKC-activating adriamycin derivatives, CGP41251, UCN-01, and antisense oligonucleotides directed against PKCalpha, have shown antitumor activity in cancer patients. PKC inhibitors are not specific to PKC, but also interact with other signaling molecules, which may contribute to the antitumor effects. Modulators of PKC have also been shown to influence non-MDR1-mediated and MDR1-mediated antitumor drug resistance. This review is focussed on the role of PKC isozymes in human cell proliferation, apoptosis and antitumor drug resistance, and on the use of PKC modulators as antitumor agents.

Inhibition of organic anion transporting polypeptide-mediated hepatic uptake is the major determinant in the pharmacokinetic interaction between bosentan and cyclosporin A in the rat.

In clinical trials, a significant interaction between the endothelin receptor antagonist bosentan and the immunosuppressant cyclosporin A was observed, which could not be rationalized in terms of inhibition of drug-metabolizing enzymes. We present here a study performed in rats investigating the mechanisms underlying this interaction, including the inhibition of active drug transport processes as part of the gastrointestinal absorption and disposition into the liver. In vitro, the majority of bosentan uptake into liver cells was shown to depend on active transport and to be efficiently inhibited by cyclosporin A. All known members of the organic anion transporting polypeptide (oatp) transport protein family expressed in rat liver, i.e., oatp1, oatp2, and oatp4, were shown to be involved in the uptake of bosentan. Results from both series of experiments point to inhibition of active bosentan uptake into the liver by cyclosporin A as the major underlying mechanism for this pharmacokinetic interaction that is in line with reports on other oatp-transported drugs. Significant contributions of other mechanisms such as inhibition of mdr1-mediated drug efflux during gastrointestinal absorption, inhibition of bosentan metabolism, or inhibition of hepatobiliary excretion seemed to be unlikely. The interaction between bosentan and cyclosporin A is a rare example of a pharmacokinetic interaction, which can mostly be attributed to the inhibition of transport processes in the liver. It also demonstrates that inhibition of uptake into the liver might become rate-limiting in the overall elimination process even for compounds whose clearance is dependent on metabolism. The relevance of these findings in the rat for clinical use remains to be explored. It is, however, clear that inhibition of CYP3A4-mediated metabolism by cyclosporin A alone is insufficient to explain the increased bosentan concentrations and that inhibition of hepatocellular uptake offers an attractive mechanistic alternative also in human.

Treatment of acute myeloid leukemia: State-of-the-art and future directions

Despite major recent advances in the understanding of the molecular biology of the disease, the treatment of acute myeloid leukemia (AML) in adults remains challenging. For the 75% of AML patients older than 60 years, currently available treatments produce significant toxicity with poor overall response rates and survival. In younger patients, standard regimens using cytarabine and an anthracycline for induction followed by some form of intensive postremission therapy can produce response rates of 70% with 5-year relapse-free survival rates of 25% to 40%. Chromosomal analyses define three prognostic categories with favorable, intermediate, and unfavorable risk. In older adults, AML appears to be an intrinsically resistant disorder of proximal pluripotent hematopoietic stem cells. A variety of targeted therapies currently in development include modulators of MDR1-mediated drug resistance, immunotherapeutics, angiogenesis inhibitors, proapoptotic antisense oligonucleotides, and specific small molecule inhibitors of tyrosine kinase and farnesyltransferase. For example, oral farnesyltransferase inhibitors have demonstrated activity and tolerability in patients with refractory AML and are now in phase II testing. Such targeted therapeutics offer the promise of novel antileukemic activity combined with an improved therapeutic index. Semin Hematol 39(suppl 2):4-10. Copyright 2002, Elsevier Science (USA). All rights reserved.