Doxorubicin-resistant Subline Research Articles

MDR1 Inhibition Reverses Doxorubicin-Resistance in Six Doxorubicin-Resistant Canine Prostate and Bladder Cancer Cell Lines.

Acquired chemoresistance during chemotherapy, often accompanied by cross- and multi-resistance, limits therapeutic outcomes and leads to recurrence. In order to create in vitro model systems to understand acquired doxorubicin-resistance, we generated doxorubicin-resistant sublines of canine prostate adenocarcinoma and urothelial cell carcinoma cell lines. Chemoresistance to doxorubicin, cross-resistance to carboplatin, and the reversibility of the acquired resistance by the specific MDR1-inhibitor tariquidar were quantified in metabolic assays. Resistance mechanisms were characterized by expression of the efflux transporters MDR1 and RALBP1, as well as the molecular target of doxorubicin, TOP2A, with qPCR and Western blotting. Six out of nine cell lines established stable resistance to 2 µM doxorubicin. Drug efflux via massive MDR1 overexpression was identified as common, driving resistance mechanism in all sublines. MDR1 inhibition with tariquidar extensively reduced or reversed the acquired, and also partly the parental resistance. Three cell lines developed additional, non-MDR1-dependent resistance. RALBP1 was upregulated in one resistant subline at the protein level, while TOP2A expression was not altered. Combination therapies aiming to inhibit MDR1 activity can now be screened for synergistic effects using our resistant sublines. Nevertheless, detailed resistance mechanisms and maintained molecular target expression in the resistant sublines are still to be examined.

Mitochondrial dysfunction and chromatin changes with autophagy-mediated survival in doxorubicin resistant cancer cell lines.

Acquired chemoresistance against doxorubicin remains an obstacle in long-term treatment. The comprehensive molecular mechanism underlying the acquirement of doxorubicin resistance has not been reported. The objective of the present study is to understand the survival strategies and investigate alternate treatments for doxorubicin-resistant cervical and liver cancer cells. In this study, doxorubicin-resistant sublines were established by continuous incremental exposure of the drug to parental cervical and liver cancer cells. The transcriptome data in drug-resistant model revealed downregulated energy production pathways like glycolysis, oxidative phosphorylation, and mTOR signalling. This resulted in slow proliferation and altered mitochondrial changes in doxorubicin-resistant cells. The altered metabolic state of the resistant cells was associated with hypo-acetylation of chromatin. Pre-treatment with HDACi sensitized the drug-resistant cells to doxorubicin by increased drug accumulation in the cells, thereby leading to apoptosis. Additionally, we demonstrated that autophagy gets activated in doxorubicin-resistant cervical and liver cancer cells. Autophagy acts as pro-survival mechanism in resistant cells, as inhibition of autophagy leads to cell death. In conclusion, the data highlights survival ability of resistant cells with mitochondrial dysfunction, altered chromatin state, and pro-survival autophagy. The study proposes targeting chromatin alteration with the combinatorial treatment of HDACi with doxorubicin or survival mechanism through autophagy inhibitor against doxorubicin-resistant cancer phenotype.

Curcumin Rescues Doxorubicin Responsiveness via Regulating Aurora a Signaling Network in Breast Cancer Cells.

Background:Insensitivity towards anthracycline drugs like doxorubicin poses a significant challenge in the treatment of breast cancer. Among several factors, Aurora A (a mitotic serine threonine kinase) plays crucial roles in acquiring non-responsiveness towards doxorubicin. However, the mechanisms underlying need to be elucidated. The present study was therefore designed to evaluate the underlying mechanisms of Aurora A mediated doxorubicin insensitivity in MCF-7Dox/R, an isolated resistant-subline of MCF-7 (breast adenocarcinoma cell line). Effect of curcumin, a natural phytochemical in restoring doxorubicin sensitivity by targeting Aurora A was assessed furthermore. Methods:A doxorubicin resistant subline (MCF-7Dox/R) was isolated from the parental MCF-7 cells by treating the cell with gradual step-wise increasing concentration of the drug. Expressions of Aurora A and its target proteins (Akt, IκBα and NFκB) were assessed in both parental and MCF-7Dox/R cells. Both the cell lines were pretreated with curcumin prior to doxorubicin treatment. Cellular proliferation rate was measured using BrdU (5-bromo-2’-deoxyuridine) assay kit. Intracellular doxorubicin accumulation was estimated spectrofluorimetrically. Cellular uptake of curcumin (spectrophotometric and spectrofluorimetric method) and its nuclear localization was confirmed by confocal microscopic study. Protein expressions were determined by western blot analysis. Localization of Aurora A was ascertained by immunofluorescence assay. To explore the possible outcome of impact of curcumin on Aurora A, cell-cycle distribution and apoptosis were performed subsequently.Results:Higher expressions of Aurora A in MCF-7Dox/R cells led to phosphorylation of Akt as well as IκBα. Phosphorylated IκBα preceded release of NFκB. Phospho-Akt, NFκB consequentially decreased doxorubicin accumulation by enhancing the expressions of ABCG2 and Pgp1 respectively. Curcumin by regulating Aurora A and its target molecules sensitized resistant subline towards doxorubicin mediated G2/M-arrest and apoptosis. Conclusion:Molecular targeting of Aurora A by curcumin restores chemosensitivity by increasing the efficacy of doxorubicin in breast cancer.

Synthesis of novel phytol-derived \u03b3-butyrolactones and evaluation of their biological activity

The synthesis of phytol-derived γ-butyrolactones as well as their evaluation for deterrent activity towards peach-potato aphid Myzus persicae and antiproliferative activity against four selected cancer cell lines are reported. Products were obtained in good yields (19–96%) and their structures were fully characterized by spectroscopic data (NMR, HRMS). Four synthesized δ-halo-γ-lactones (4–7) are new and have not been previously described in the literature. In the choice test phytol (1) appeared deterrent to M. persicae, whereas modifications of its structure did not cause the avoidance of the treated leaves by the aphids. In contrast, aphids were attracted to the leaves treated with the new trans-δ-chloro-γ-lactone (6). Electrical Penetration Graph (EPG) technique applied to explore the aphid probing and feeding activity revealed that neither phytol nor lactone 6 affected aphid probing and the consumption of phloem sap, which means that both phytol and the lactone 6 might have acted as postingestive modifiers of aphid behavior. The results of in vitro antitumor assays showed that obtained phytol derivatives exhibit cytotoxic activity against studied cancer cell lines (leukemia, lung and colon carcinoma and its doxorubicin resistant subline). Halolactones 4–6 were identified as the compounds, which arrest cell cycle of leukemia cells mainly in G2/M and S phases.

ABCA1/ABCB1 Ratio Determines Chemo- and Immune-Sensitivity in Human Osteosarcoma.

The ATP Binding Cassette transporter B1 (ABCB1) induces chemoresistance in osteosarcoma, because it effluxes doxorubicin, reducing the intracellular accumulation, toxicity, and immunogenic cell death induced by the drug. The ATP Binding Cassette transporter A1 (ABCA1) effluxes isopentenyl pyrophosphate (IPP), a strong activator of anti-tumor Vγ9Vδ2 T-cells. Recruiting this population may represent an alternative strategy to rescue doxorubicin efficacy in ABCB1-expressing osteosarcoma. In this work, we analyzed how ABCA1 and ABCB1 are regulated in osteosarcoma, and if increasing the ABCA1-dependent activation of Vγ9Vδ2 T-cells could be an effective strategy against ABCB1-expressing osteosarcoma. We used 2D-cultured doxorubicin-sensitive human U-2OS and Saos-2 cells, their doxorubicin-resistant sublines (U-2OS/DX580 and Saos-2/DX580), and 3D cultures of U-2OS and Saos-2 cells. DX580-sublines and 3D cultures had higher levels of ABCB1 and higher resistance to doxorubicin than parental cells. Surprisingly, they had reduced ABCA1 levels, IPP efflux, and Vγ9Vδ2 T-cell-induced killing. In these chemo-immune-resistant cells, the Ras/Akt/mTOR axis inhibits the ABCA1-transcription induced by Liver X Receptor α (LXRα); Ras/ERK1/2/HIF-1α axis up-regulates ABCB1. Targeting the farnesylation of Ras with self-assembling nanoparticles encapsulating zoledronic acid (NZ) simultaneously inhibited both axes. In humanized mice, NZ reduced the growth of chemo-immune-resistant osteosarcomas, increased intratumor necro-apoptosis, and ABCA1/ABCB1 ratio and Vγ9Vδ2 T-cell infiltration. We suggest that the ABCB1highABCA1low phenotype is indicative of chemo-immune-resistance. We propose aminobisphosphonates as new chemo-immune-sensitizing tools against drug-resistant osteosarcomas.

Tricarbonylrhenium(I) complexes with heterodentate ligands based on functionalized amides: Synthesis, structural features, and cytotoxic activity

The reactions of 2-diphenylphosphorylaniline with 2-methylsulfanylbenzoyl chloride or 2-diphenylphosphinobenzoic acid in the presence of a coupling agent afforded new potentially tridentate ligands with different donor sets. Upon interaction with Re(CO)5Br, the compounds obtained formed either bidentately bound molecular or pincer-type tricarbonylrhenium(I) complexes depending on the reaction conditions. The resulting complexes were fully characterized in solution and in the solid state by multinuclear NMR and IR spectroscopy and, in most cases, by single-crystal X-ray diffraction. The preliminary evaluation of cytotoxicity of the complexes obtained against human colon (HCT116), breast (MCF7), and prostate (PC3) cancer cell lines revealed the high activity of κ3-S,N,O-pincer-type complex with ancillary S-donor group. The latter demonstrated considerable effect also on transformed breast cell line HBL100 and its doxorubicin-resistant subline HBL100/Dox.

Increased lipid peroxidation, apoptosis and selective cytotoxicity in colon cancer cell line LoVo and its doxorubicin-resistant subline LoVo/Dx in the presence of newly synthesized phenothiazine derivatives

Cancer cells often develop the resistance to pro-apoptotic signaling that makes them invulnerable to conventional treatment. Therapeutic strategies that make cancer cells enter the path of apoptosis are desirable due to the avoidance of inflammatory reaction that usually accompanies necrosis. In the present study phenothiazines (fluphenazine and four recently synthesized derivatives) were investigated in order to identify compounds with a potent anticancer activity. Since phenothiazines are known as multidrug resistance modulators the sensitive human colorectal adenocarcinoma cell line (LoVo) and its doxorubicin-resistant, ABCB1 overexpressing, subline (LoVo/Dx) have been employed as a model system. In studied cancer cells cytotoxic effect of the phenothiazine derivatives was accompanied by apoptosis and autophagy induction as well as by the increase of cellular lipid peroxidation and intracellular reactive oxygen species generation. Molecular modelling revealed that reactivity of phenothazines (manifested by their low energy gap) but not lipophilicity was positively correlated with their anticancer potency, pro-oxidant properties and apoptosis induction ability. Additionally, some of the studied compounds turned out to be more potent cytotoxic and pro-apoptotic agents in doxorubicin-resistant (LoVo/Dx) cells than in sensitive ones (LoVo). The hypothesis was assumed that studied phenothiazine derivatives induced apoptotic cell death by increasing the production of reactive oxygen species.

Phosphorus-containing isothiocyanate-derived mercapturic acids as a useful alternative for parental isothiocyanates in experimental oncology

A series of phosphonates, phosphinates and phosphine oxides isothiocyanate-derived mercapturic acids were synthesized. A temperature dependence dynamic proton decoupled 31P NMR studies indicated that in most cases the compounds were obtained as a mixture of rotamers. Moreover, biologically relevant reversibility of mercapturic acids synthesis from the parental isothiocyanates was confirmed. All compounds were evaluated ashighly active antiproliferative agents in vitro in human colon cancer cell lines (LoVo and its doxorubicin-resistant subline LoVo/DX). The cell cycle progression and caspase-3 activity analyses revealed compounds moderate activity as apoptosis inducers and their poor influence on cell cycle progression in the LoVo cells. Our results confirm that isothiocyanate-derived mercapturic acids present a reasonable alternative for the parental compounds, and can replace them in the future studies on isothiocyanates potential as anticancer agents.

A newly synthesized nickel chelate can selectively target and overcome multidrug resistance in cancer through redox imbalance both in vivo and in vitro.

Induction of undesired toxicity and emergence of multidrug resistance (MDR) are the major obstacles for cancer treatment. Moreover, aggressive cancers are less sensitive towards existing chemotherapeutics. Therefore, selective targeting of cancers without inducing undesired side effects and designing proper strategies to overcome MDR has utmost importance in modern chemotherapy. Previously we revealed the anticancer properties of some transition metal chelates of Schiff base, but the effectiveness of nickel complex is still unrevealed. Herein, we synthesized and characterized a Schiff base nickel chelate, nickel-(II) N-(2-hydroxyacetophenone) glycinate (NiNG), through different spectroscopic means. NiNG proves to be a broad spectrum anticancer agent with considerable efficacy to overcome MDR in cancer. Antiproliferative effects of NiNG was evaluated using drug-resistant (CEM/ADR5000; NIH-MDR-G185; EAC/Dox), drug-sensitive aggressive (Hct116; CCRF-CEM; EAC/S) and normal (NIH-3T3) cells that reveal the selective nature of NiNG towards drug resistant and sensitive cancer cells without inducing any significant toxicity in normal cells. Moreover, NiNG involves reactive oxygen species (ROS)-mediated redox imbalance for induction of caspase 3-dependent apoptosis in aggressive drug-sensitive Hct116 and drug-resistant NIH-MDR-G185 cells through disruption of mitochondrial membrane potential. Moreover, intraperitoneal (i.p.) application of NiNG at non-toxic doses caused significant increase in the life-span of Swiss albino mice bearing sensitive and doxorubicin-resistant subline of Ehrlich ascites carcinoma cells. It is noteworthy that, in vitro NiNG can only overcome P-glycoprotein-mediated MDR while in vivo NiNG can overcome MRP1-mediated MDR in cancer. Therefore, NiNG has therapeutic potential to target and overcome MDR in cancer.

Genomic and transcriptomic profiling of resistant CEM/ADR-5000 and sensitive CCRF-CEM leukaemia cells for unravelling the full complexity of multi-factorial multidrug resistance

We systematically characterised multifactorial multidrug resistance (MDR) in CEM/ADR5000 cells, a doxorubicin-resistant sub-line derived from drug-sensitive, parental CCRF-CEM cells developed in vitro. RNA sequencing and network analyses (Ingenuity Pathway Analysis) were performed. Chromosomal aberrations were identified by array-comparative genomic hybridisation (aCGH) and multicolour fluorescence in situ hybridisation (mFISH). Fifteen ATP-binding cassette transporters and numerous new genes were overexpressed in CEM/ADR5000 cells. The basic karyotype in CCRF-CEM cells consisted of 47, XX, der(5)t(5;14) (q35.33;q32.3), del(9) (p14.1), +20. CEM/ADR5000 cells acquired additional aberrations, including X-chromosome loss, 4q and 14q deletion, chromosome 7 inversion, balanced and unbalanced two and three way translocations: t(3;10), der(3)t(3;13), der(5)t(18;5;14), t(10;16), der(18)t(7;18), der(18)t(21;18;5), der(21;21;18;5) and der(22)t(9;22). CCRF-CEM consisted of two and CEM/ADR5000 of five major sub-clones, indicating genetic tumor heterogeneity. Loss of 3q27.1 in CEM/ADR5000 caused down-regulation of ABCC5 and ABCF3 expression, Xq28 loss down-regulated ABCD1 expression. ABCB1, the most well-known MDR gene, was 448-fold up-regulated due to 7q21.12 amplification. In addition to well-known drug resistance genes, numerous novel genes and genomic aberrations were identified. Transcriptomics and genetics in CEM/AD5000 cells unravelled a range of MDR mechanisms, which is much more complex than estimated thus far. This may have important implications for future treatment strategies.

Abstract 3848: Subtype-specific DNA damage dependent and independent functions of damage repair proteins confer chemo-resistance to leukemia

Abstract Resistance to chemotherapy is a major challenge in the field of leukemia. Since, cancer cells can acquire resistance to DNA damage based therapy by modulating DNA Damage Repair (DDR) pathways, an in-depth understanding of DDR is important for novel drug discoveries. Hence, to understand relation between DDR and resistance we used doxorubicin resistant sub-line (K562-R) of K562 and Mitoxantrone resistant sub-line (HL60/MX) of HL60 as a model system. We show that lethal doses of Mitoxantrone to HL60 and HL60/MX do not induce DNA DSBs (double strand breaks) and accordingly there is no activation of Chk2, although there is equal uptake of drug in both, sensitive and resistant cell types. Since Mitoxantrone produce DSBs by targeting TOP2B, we checked the expression of TOPB2 and found significant decrease in the levels of topoisomerase 2 B (TOP2B). Moreover, negative regulator of TOPB2, mir 21-5P is upregulation in untreated HL60/MX explaining the absence of DSBs in HL-60/MX with Mitoxantrone treatment. Surprisingly, although there are no DSBs induced, we find high levels of DNA-PKcs, MRE11 and Rad50 in HL-60/MX. Furthermore, inhibition of DNA-PKcs re-sensitizes HL-60/MX cells to Mitoxantrone, suggesting a DNA damage independent role for DNA-PK in HL-60/MX cells. Recently DNA-PKcs is shown to play key role in fat metabolism. Indeed we find significant differences in the metabolism of HL60 and HL-60/MX where HL-60 cells use glucose but HL-60/MX cells show reduced glucose and glutamine metabolism and an over expression of fatty acid synthase. However link between DNA-PKcs over expression and differential metabolism in these cells still need to be explored. Interestingly, in contrast to HL60 model, Doxorubicin does induce equal DSBs in K562 and K562-R cells. However K562-R cells hyperactivate ATM and Chk2 to repair their DNA and survive. Importantly, we show that K562 start to show elevated activation of ATM and Chk-2 as early as two rounds of drug treatment. Accordingly, ATM inhibition leads to induction of apoptosis in resistant K562 cells. In conclusion, we show that K562 and HL60 cells use the DNA double strand break repair proteins in DNA damage dependent and independent pathways to acquire resistance to DSB inducing chemo therapeutics. Our data also highlight the importance of considering subtype specific therapeutics in leukemia. Note: This abstract was not presented at the meeting. Citation Format: Sameer Salunkhe, Ekjot Kaur, Ashwin Ramaswamy, Ketaki Patkar, Shilpee Dutt. Subtype-specific DNA damage dependent and independent functions of damage repair proteins confer chemo-resistance to leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3848. doi:10.1158/1538-7445.AM2015-3848

Synthesis of poly[N-(2-hydroxypropyl)methacrylamide] conjugates of inhibitors of the ABC transporter that overcome multidrug resistance in doxorubicin-resistant P388 cells in vitro.

The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 μM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.

Vimentin silencing effect on invasive and migration characteristics of doxorubicin resistant MCF-7 cells

Chemotherapy is one of the well-known treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). One of the major mechanisms of MDR is the activity of several ATP binding cassette (ABC) transporters that pump drugs out of the cells. Doxorubicin intercalates and inhibits DNA replication; it is a powerful chemotherapeutic agent. However, it causes development of MDR in tumor cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness of tumor cells. In this study, MCF-7 cell line which is a model for human mammary carcinoma, and a doxorubicin resistant subline (MCF-7/Dox) were used. The resistant subline was previously obtained by stepwise selection in our laboratory. In the resistant cells, high levels of vimentin expression were observed. The main purpose of this study was to investigate changes in invasive and migration characteristics of MCF-7/Dox cell line, after transient silencing of vimentin gene by specific siRNA.

A novel manganese complex, Mn-(II) N-(2-hydroxy acetophenone) glycinate overcomes multidrug-resistance in cancer

Multidrug resistance (MDR) remains a significant problem for effective cancer chemotherapy. In spite of considerable advances in drug discovery, most of the cancer cases still stay incurable because of resistance to chemotherapy. We synthesized a novel, Mn (II) complex (chelate), viz., manganese N-(2-hydroxy acetophenone) glycinate (MnNG) that exhibits considerable efficacy to overcome drug resistant cancer. The antiproliferative activity of MnNG was studied on doxorubicin resistant and sensitive human T lymphoblastic leukemia cells (CEM/ADR 5000 and CCRF/CEM). MnNG induced apoptosis significantly in CEM/ADR 5000 cells probably through generation of reactive oxygen species. Moreover, intraperitoneal (i.p.) application of MnNG at non-toxic doses caused significant increase in the life-span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of Ehrlich ascites carcinoma cells.

Increased glutathione and mitogen‐activated protein kinase phosphorylation are involved in the induction of doxorubicin resistance in hepatocellular carcinoma cells

The human hepatocellular carcinoma (HCC) cell line HepG2 can easily acquire resistance to doxorubicin. However, the mechanism of action is unclear. In the present study, we used confocal microscopy, flow cytometry and other methods to reveal the mechanisms by which HepG2 cells acquire doxorubicin resistance. Our results showed that R-HepG2 cells, a doxorubicin-resistant sub-line of HepG2, exhibited decreased intracellular accumulation of doxorubicin and increased expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 when compared with HepG2 cells. R-HepG2 cells also harbored higher levels of glutathione and increased expression of glutathione peroxidase. Furthermore, we demonstrated that the phosphorylation of mitogen-activated protein kinases (p38 and c-jun-N-terminal kinases), IkBα and CREB were increased in R-HepG2 cells. Specific p38 inhibitor SB203580 decreased P-gp expression. The multi-kinase inhibitor sorafenib tosylate also significantly suppressed the phosphorylation of these proteins and inhibited the expression of P-gp. These findings reveal that the drug resistance could be acquired through mitogen-activated protein kinase-dependent upregulation of P-gp. This mechanism protects R-HepG2 cells from the anticancer action of doxorubicin.

Overcoming multidrug resistance (MDR) in cancer in vitro and in vivo by a quinoline derivative

Multidrug resistance (MDR) mediated by the over expression of drug efflux protein P-glycoprotein (P-gp) is one of the major impediments to successful treatment of cancer. P-gp acts as an energy-dependent drug efflux pump and reduces the intracellular concentration of structurally unrelated drugs inside the cells. Therefore, there is an urgent need for development of new compound that are less toxic and effective against drug resistance in cancer. Preclinical studies have shown that quinoline derivatives possess anticancer activities. Here, we report the antitumor potential of quinoline derivative, 2-(2-Methyl-quinolin-4ylamino)-N-phenyl acetamide (S4). To evaluate the cytotoxic potential of S4, we used four different cell lines (Hela, HCT-116, CCRF-CEM, and CEM/ADR 5000) in vitro, and showed that S4 kills doxorubicin resistant T lymphoblastic leukemia cell, CEM/ADR 5000 in a concentration dependent manner while others remains unaffected. Moreover, S4 induces apoptosis in CEM/ADR 5000 cells through generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) completely blocks ROS generation and, subsequently, abrogates S4 induced apoptosis. Furthermore, in vivo treatment with S4 significantly increases the life span of swiss albino mice bearing sensitive and doxorubicin resistant subline of Ehrlich ascites carcinoma. In addition, intraperitoneal application of S4 in mice does not show any systemic toxicity at concentrations that in preliminary trials in a mice Ehrlich ascites carcinoma model. Therefore, present report provides evidence that S4, a quinoline derivative, may be a promising new therapeutic agent against drug resistant cancers.

Upregulation of mdr1 gene is related to activation of the MAPK/ERK signal transduction pathway and YB-1 nuclear translocation in B-cell lymphoma

Multidrug resistance (MDR) in human B-cell lymphoma constitutes a major obstacle to the effectiveness of chemotherapy. The aim of this study was to investigate the molecular mechanism of MDR in B-cell lymphoma. The B-cell lymphoma MDR sublines were developed by exposing the parental Daudi cells to stepwise increasing concentrations of doxorubicin. Interaction of Y-box binding protein-1 (YB-1) with the Y-box motif of the mdr1 gene promoters was studied by electrophoretic mobility shift assay. The effects of YB-1 on mdr1 promoter activity were examined by luciferase assay. After silencing of YB-1 gene by shRNA, the role of YB-1 nuclear translocation in the formation of induced MDR was examined. Expression of mdr1 and YB-1 was examined further after Daudi cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitor PD98059 for 1 hour. Doxorubicin-resistant sublines was generated from the Daudi cell line by stepwise selection in doxorubicin. We found that acquisition of MDR is associated with enhanced YB-1 nuclear translocation and MAPK/extracellular signal-regulated kinase (ERK) activity. Electrophoretic mobility shift assay revealed that doxorubicin increased binding of YB-1 to the Y-box of mdr1 promoter. Luciferase reporter assays demonstrated that the Y-box region is essential for YB-1 regulation of mdr1 expression. The introduction of exogenous YB-1 shRNA into Daudi cells resulted in decreased levels of the expression of mdr1 gene and P-glycoprotein induced by doxorubicin. When Daudi cells were pretreated with MAPK inhibitor PD98059, the phosphorylation of ERK was effectively inhibited as well as the nuclear translocation of YB-1 and the expression of mdr1 gene. Doxorubicin can increase expression of mdr1/P-glycoprotein through activating MAPK/ERK transduction pathway, then increasing expression of YB-1, inducing YB-1 nuclear translocation, and enhancing DNA-binding activity of YB-1.

An in vitro and in vivo study of a novel zinc complex, zinc N-(2-hydroxyacetophenone)glycinate to overcome multidrug resistance in cancer

Multiple drug resistance (MDR) remains a major clinical challenge for cancer treatment. P-glycoprotein is the major contributor and they exceed their role in the chemotherapy resistance of most of the malignancies. Attempts in several preclinical and clinical studies to reverse the MDR phenomenon by using MDR modulators have not yet generated promising results. In the present study, a co-ordination complex of zinc viz., Zn N-(2-hydroxyacetophenone)glycinate (ZnNG) has been synthesized, characterized and its antitumour activity was tested in vitro against drug sensitive and resistant human T-lymphoblastic leukemic cell lines (CCRF/CEM and CEM/ADR5000 respectively) and in vivo against Ehrlich ascites carcinoma (EAC) implanted in female Swiss albino mice. To evaluate the cytotoxic potential of ZnNG, we used sensitive CCRF/CEM and drug resistant CEM/ADR 5000 cell lines in vitro. Moreover, ZnNG also has the potential ability to reverse the multidrug resistance phenotype in drug resistant CEM/ADR 5000 cell line and induces apoptosis in combination with vinblastine. ZnNG remarkably increases the life span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of EAC in presence and in absence of doxorubicin. In addition, intraperitoneal application of ZnNG in mice does not show any systemic toxicity in preliminary trials in normal mice. To conclude, a novel metal chelate of zinc viz., ZnNG, may be a promising therapeutic agent against sensitive as well as drug resistant cancers.

Drug resistant MCF-7 cells exhibit epithelial-mesenchymal transition gene expression pattern

Purpose Multidrug resistance is resistance to structurally unrelated anticancer agents. Large-scale expression analysis by using high-density oligonucleotide microarrays may provide information about new candidate genes contributing to MDR. This study demonstrates alterations in expression levels of several genes related to epithelial-mesenchymal transition (EMT) in paclitaxel, docetaxel, and doxorubicin resistant MCF-7 cells. Methods Resistant sublines were developed from sensitive cells by selective paclitaxel, docetaxel, and doxorubicin applications in dose increments. cDNA microarray analysis was performed for sensitive and resistant cells. Genes having statistically significantly altered expression levels more than two-folds compared to the sensitive MCF-7 cells were considered. Genes encoding the determinants of the EMT were evaluated. Immunostaining was performed for relevant protein expressions. Results Key elements of EMT were transcriptionally activated in paclitaxel, docetaxel and doxorubicin resistant sublines. One of the upregulated genes was Slug, a transcription factor of E-cadherin, occludin repression, and N-cadherin, vimentin activation. Decreased estrogen receptor-α (ER) levels in cells might have stimulated Slug expression. Increased expression levels of TGF-beta receptor2 (TGFBR2) together with SMAD3 might have stimulated EMT in resistant cells. Immunocytochemistry results confirmed loss of ER and E-cadherin, together with high vimentin levels. Conclusions EMT was induced in multidrug resistant MCF-7 cells indicating a relationship of this process and drug resistance. However, the relationship of each specific component of EMT with drug resistance requires further analysis.

Abstract 5431: Histone deacetylase inhibitor (HDAC) panobinostat (LBH589) enhances the antiproliferative effect of a topoisomerase II (topo II) inhibitor in doxorubicin-resistant HL-60 cells, despite high MDR expression

Abstract Acute myeloid leukemia (AML) is difficult to treat, particularly in the relapsed or refractory setting due to drug resistance. Topo II inhibitors are effectively used to treat AML, and histone deacetylase (HDAC) inhibitors have been used pre-clinically and clinically with some success. We previously reported that vorinostat (SAHA) was effective in combination with topo II inhibitors in AML cells (Proc. AACR, 2009, Abstract 4567). In the present study we evaluate LBH589, a novel class I/II HDAC inhibitor. Since resistance to HDAC inhibitors has not been well described, we hypothesized that LBH589 would be effective in combination with a topo II inhibitor in a doxorubicin-resistant AML cell line that expresses MDR and resistant by MDR-independent mechanism. A sensitive HL60 parent line (S) and the doxorubicin-resistant sub-line (R) were used. Early studies revealed that vorinostat (SAHA) had comparable anti-proliferative effects in S & R cells but the R cells were relatively more resistant to LBH589. This resistance was likely MDR-mediated. We then tested whether sub-lethal doses of SAHA or LBH589, used in combination with sub-lethal doses of etoposide (VP-16), would be effective. S and R cells were treated with VP-16 for 1 h, washed, and re-incubated with SAHA or LBH, or drug-free media, for a total of 144 hours. Cell viability and apoptosis were measured at 72 h and 144 h following treatment. At 72 h and at 144 h, the combination of VP-16 and LBH was significantly more effective (p<0.01) in reducing proliferation in HL60/S and HL60/R cells, compared to either agent alone. At both 72 h and 144 h the combination of VP-16 and LBH also led to significantly (p<0.05) greater apoptosis in both S & R cells. Western blotting for phospho-histone H2AX, a marker of DNA damage, showed increased DNA damage in the R cells treated with VP-16 and LBH589 compared to either drug alone. Overall, the effects of LBH589 in combination with VP-16 were significantly (p<0.05) more effective than SAHA in the S & R cells. In summary, our results demonstrate that MDR may mediate resistance to LBH589. Further, in spite of MDR expression, LBH589 is a useful adjunct to VP-16 treatment and enhances the anti-proliferative effect of the topo II inhibitor via increased apoptosis, even in the doxorubicin-resistant cells. Thus, post-treatment with LBH589 in combination with a topo II inhibitor may be useful treatment strategy in relapsed / refractory AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5431.