Multidrug-resistant Subline Research Articles

Formation of Tissue Factor-Factor VIIa-Factor Xa Complex Promotes Survival of Human Breast Cancer Cells by Inhibition of Apoptosis.

Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa). TF is constitutively expressed by a variety of tumor cells. Recently, TF has been shown to induce cellular signaling and promote tumor growth, angiogenesis, and metastasis. We showed previously that formation of TF-FVIIa-Factor Xa (FXa) complex induces cellular signaling in the Adr-MCF-7 cell line, a multidrug-resistant subline of the human breast cancer cell line, MCF-7 (Jiang et al. J. Thromb Haemost 2: 2004; 93–101). This cell line has high endogenous expression of TF. Treatment of the Adr-MCF-7 cells with the combination of FVIIa (10 nM) and FX (150 nM) induces phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and protein kinase B (PKB), whereas no increase in the basal level of phosphorylation of these proteins occurs with treatment of the cells with FVIIa (10 nM) alone. In the present study, we investigated whether TF-FVIIa-induced signaling might alter apoptosis in human breast cancer cells. Apoptosis of the Adr-MCF-7 cells was induced by serum starvation for 5–7 days. Treatment of the cells with the combination of FVIIa (10 nM) and FX (150 nM), reduced apoptosis of the cells by nearly 50% compared with untreated, control cells using an ELISA that detects histone-DNA fragments. In contrast, FVIIa (10 nM) alone did not significantly prevent apoptosis. Pre-treatment of the Adr-MCF-7 cells with hirudin did not inhibit the anti-apoptotic effect of the combination of FVIIa and FX, whereas this effect could be completely blocked by either U0126 (10 μM), which inhibits p44/42 MAPK phosphorylation, or LY294002 (50 μM), which inhibits PKB phosphorylation. In addition, treatment of the Adr-MCF cells with the combination of FVIIa and FX led to a 50% increase in the level of the anti-apoptotic protein, survivin, compared with untreated cells by Western blot analysis. Results from this study indicate that formation of TF-FVIIa-FXa complex prevents apoptosis of breast cancer cells by a thrombin-independent pathway. Moreover, the anti-apoptotic effect of this signaling pathway involves both phosphorylation of p44/42 MAPK and PKB and might be mediated in part by an increase in cell survivin levels.

ABCChips as New Diagnostic Tool to Monitoring Multidrug Resistance in Human Tumor Cells to Chemotherapeutics Agents by Expression Profiling of ATP-Binding Cassette Transporter Genes.

A major problem in the treatment of tumors represents the development of resistance to chemotherapy. Many mechanisms are responsible for the failure of treatment, the main one being the activation of the ABC transporters. In the present investigation, we developed a low density DNA microarray which contains 38 ABC transporter genes. This tool has been validated with three different characterized multidrug-resistant sublines (CEM/ADR5000, HL60/AR, MCF7-CH1000) and their corresponding drug-sensitive parental cell lines (CCRF-CEM, HL60, MCF7). The multidrug-resistant cell lines used are known to overexpress either the MDR1, MRP1, or BCRP genes. Interestingly, we found not only the overexpression of these genes but also of other ABC transporter genes by using low density microarrays. These results were corroborated by quantitative real time RT-PCR. The microarray allowed the determination of the expression profile of ABC transporters in a single hybridization experiment and may, hence harbor the potential as diagnostic tool to detect drug resistance in the clinic.

Induction of Stem Cell Factor/c-Kit/Slug Signal Transduction in Multidrug-resistant Malignant Mesothelioma Cells

Malignant mesothelioma (MM) is strongly resistant to conventional chemotherapy by unclear mechanisms. We and others have previously reported that cytokine- and growth factor-mediated signal transduction is involved in the growth and progression of MM. Here, we identified a pathway that involves stem cell factor (SCF)/c-Kit/Slug in mediating multidrug resistance of MM cells. When we compared gene expression profiles between five MM cells and their multidrug-resistant (MM DX) sublines, we found that MM DX cells expressed both SCF and c-Kit and had higher mRNA levels of Slug. Knockdown of c-Kit or Slug expression with their respective small interfering RNA sensitized MM DX cells to the induction of apoptosis by different chemotherapeutic agents, including doxorubicin, paclitaxel, and vincristine. Transfection of c-Kit in parental MM cells in the presence of SCF up-regulated Slug and increased resistance to the chemotherapeutic agents. Moreover, MM cells expressing Slug showed a similar increased resistance to the chemotherapeutic agents. These results indicate that induction of Slug by autocrine production of SCF and c-Kit activation plays a key role in conferring a broad spectrum chemoresistance on MM cells and reveal a novel signal transduction pathway for pharmacological or genetic intervention of MM patients.

629 Acquired irofulven-resistance is mediated by a novel and drug-specific resistance mechanism associated with overexpression of ABCA12

In an endeavor to improve responsiveness of tumor cells to drug combination treatments, we analyzed the effect of 5-azacytidine (5AC) as a model compound for a new class of drugs, DNA-demethylating agents. We used parental K562/WT chronic myelogenous leukemia cells and a multidrug-resistant subline thereof, K562/ADM. Multidrug-resistant cells were more resistant to daunorubicin, but more sensitive to cisplatin than parental K562 cells as measured by growth inhibition and apoptosis assays. Resistance to daunorubicin can be explained by amplification of the MDR1 drug transporter gene. Cisplatin induced more DNA damage in specific genes and in the entire genome of K562/ADM cells compared to K562/WT cells using PCR stop assays and atomic absorption spectroscopy. Pretreatment with 5AC modulated the response of K562/ADM cells toward MDR-type drugs (daunorubicin, vincristine, etoposide) and reduced function and expression of MDR1 as analyzed by flow cytometry and RT-PCR. Analysis of CpG island methylation in the promotor region of the MDR1 gene by bisulfite sequencing and a methylation-sensitive HpaII-digestion/PCR approach revealed that methylation of the MDR1 promotor of K562/ADM cells was greater than in K562/WT cells. 5AC treatment completely abolished MDR1 promotor methylation. The unexpected observation that DNA demethylation by 5AC rather decreases than increases MDR1 expression in K5612/ADM cells points to still unexplored sequences in the MDR1 promotor whose transcriptional activity may be affected by the methylation status. 5AC pretreatment also modulated K562/WT and K562/ADM cells to non-MDR-type drugs such as cisplatin and increased cisplatin-induced DNA damage.

Mitochondria-mediated apoptosis operating irrespective of multidrug resistance in breast cancer cells by the anticancer agent prodigiosin

Prodigiosin (PG) is a red pigment produced by Serratia marcescens with pro-apoptotic activity in haematopoietic and gastrointestinal cancer cell lines, but no marked toxicity in non-malignant cells. Breast cancer is the most frequent malignancy among women in the European Union and better therapies are needed, especially for metastatic tumors. Moreover, multidrug resistance is a common phenomenon that appears during chemotherapy, necessitating more aggressive treatment as prognosis worsens. In this work, we extend our experiments on PG-induced apoptosis to breast cancer cells. PG was potently cytotoxic in both estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines. Cytochrome c release, activation of caspases-9, -8 and -7 and cleavage of poly (ADP-ribose) polymerase protein typified the apoptotic event and caspase inhibition revealed that PG acts via the mitochondrial pathway. In a multidrug-resistant subline of MCF-7 cells that over-expresses the breast cancer resistance protein, the cytotoxic activity of PG was slightly reduced. However, flow-cytometry analysis of PG accumulation and efflux in MCF-7 sublines showed that PG is not a substrate for this resistance protein. These results suggest that PG is an interesting and potent new pro-apoptotic agent for the treatment of breast cancer even when multidrug resistance transporter molecules are present.

Carminomycin, 14‐Hydroxycarminomycin and Its Novel Carbohydrate Derivatives Potently Kill Human Tumor Cells and Their Multidrug Resistant Variants.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Anomalies of deconvolution via discrete Fourier transform: a case study on assessing transport at live cell preparations

Deconvolution is one of the most frequently used mathematical operations in the evaluation of data in many branches of science and engineering. Analytical chemistry is no exception: deconvolution is part of routine methodologies in major instrumental techniques such as many modalities of spectroscopy and chromatography. The need for proper deconvolution, however, is often overlooked in the emerging field of bio-analytical chemistry as applied to live tissue in vivo, or cells in vitro. It is in this context that some of the major problems inherent to deconvolution, and to one of its most commonly used methodologies, deconvolution via discrete Fourier transform (DFT), are explored in this work. To illustrate the nature and severity of the difficulties involved and the resulting errors and potential for misinterpretation, the problem of assessing cellular processes with different microsensors is chosen as an example here. In particular, cancer multidrug resistance (MDR) modulated doxorubicin (DOX) efflux from drug sensitive cells derived from Chinese hamster ovary cancer, and their MDR subline, are discussed. DOX concentration monitored with a carbon fiber microelectrode at close proximity to a monolayer of these cells can be perceived as the result of convolution of cellular efflux with extracellular diffusion. Deconvolution via DFT to reconstruct the efflux process, however, leads to patterns that are very far from realistic. The observations and conclusions made using this example are relevant to other areas of analytical chemistry, and science and engineering in general that use deconvolution methods in processing experimental results of many types.

Amplification and overexpression of the mouse mdr 1a gene in nine independently derived multidrug-resistant SEWA murine cell lines.

Many different drugs may be used in selecting cells for multidrug resistance (MDR). Enhanced expression and/or gene amplification is known to cause overproduction of membrane-bound 170,000 P-glycoproteins, responsible for the MDR. In rodents, the P-glycoproteins are encoded by a small gene family: mdr 1a, mdr 1b, and mdr 2. To evaluate the relationship between the pattern of MDR and the selecting drug, nine MDR sublines were independently selected from a sensitive mouse tumor cell line, SEWATC13K, using three different drugs. Each MDR subline displayed amplification of one or more of the three mdr genes, but only one, mdr 1a, was consistently overexpressed. Thus, our results indicate that the pattern of mdr gene amplification and overexpression is independent of the selective agent. Furthermore, in four of the MDR sublines, where all three mdr genes had been originally amplified, pulsed field gel electrophoresis (PFGE) revealed that amplification of mdr 1a, only, was a second step of gene amplification. In addition, the gene for the calcium-binding protein, sorcin, was coamplified in eight of the nine MDR sublines. The sorcin gene was overexpressed in seven of these eight sublines. Finally, hybridizations with a probe homologous with a putative region of RFLP (restriction fragment length polymorphism), indicated that the amplified sequences originate from one or the other of the two homologous chromosomes with no preference.

Mitochondria in tumor cells studied by laser scanning confocal microscopy.

We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine. We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology. In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery. Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells. We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein. In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be seen in this cell line. Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology. Our results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity.

Carminomycin, 14-hydroxycarminomycin and its novel carbohydrate derivatives potently kill human tumor cells and their multidrug resistant variants.

The new hydrophilic derivatives of 14-hydroxycarminomycin were obtained using 13-dimethyl ketal of 14-bromocarminomycin (6) as the starting compound. The reductive alkylation of 6 with melibiose or D-galactose followed by hydrolysis of the corresponding intermediate bromoketals 9 and 11 produced 3'-N-[-alpha-D-(galactopyranosyl-(1 --> 6)-O-D-1-desoxyglucit-1-yl]-14-hydroxycarminomycin (10) and 3'-N-(1-desoxy-D-galactit-1-yl)-14-hydroxycarminomycin (12), respectively. These novel derivatives 10 and 12 were less toxic than carminomycin or 14-hydroxycarminomycin for leukemia (K562) and breast carcinoma (MCF-7) cells. Importantly, carminomycin, 14-hydroxycarminomycin and compounds 10 and 12 were similarly active for wild type cells and their multidrug resistant (MDR) sublines, K562i/S9 and MCF-7Dox.

Effect of 5-aminolevulinic acid-mediated photodynamic therapy on MCF-7 and MCF-7/ADR cells.

Photodynamic therapy (PDT) has been proposed as an alternative approach in overcoming multidrug resistance (MDR) phenotype. To verify whether 5-aminolevulinic acid (ALA)-mediated PDT is effective in MDR cells, we studied the protoporphyrin IX (PpIX) content, intracellular localization, and phototoxicity in human breast cancer cells MCF-7 and derived MDR subline, MCF-7/ADR. The fluorescence kinetics of ALA-induced PpIX was evaluated by spectrofluorometer. The phototoxicity of MCF-7 and MCF-7/ADR cells was determined by tetrazolium (MTT) assays and clonogenic assay. Furthermore, Annexin V and propidium iodide (PI) binding assays were performed to analyze the characteristics of cell death after ALA-PDT. MCF-7/ADR accumulated a lower level of PpIX as compared to parental MCF-7 cells. Significant phototoxicity was observed in MCF-7 and increased in a fluence-dependent manner with LD(50) around 8 J/cm(2). Compared to its parental counterpart, MCF-7/ADR cells were less sensitive to ALA photodynamic treatment and PDT-induced cytotoxicity did not increase in a dose responsive manner as the concentration of ALA increased or the fluence of light increased. ALA-PDT was less effective for MCF-7/ADR cells than MCF-7 cells even under the condition when these two cell lines contained the similar amounts of PpIX. These results indicate that, except for the MDR related characteristics, MCF-7/ADR cells might possess intrinsic mechanisms that render them less sensitive to ALA-PDT induced phototoxicity.

Chromosomal imbalances associated with drug resistance and thermoresistance in human pancreatic carcinoma cells

Resistance to therapeutic treatment is the major obstacle to advances in the successful management of pancreatic cancer. To characterize chromosomal alterations associated with different phenotypes of acquired multidrug resistance (MDR) and thermoresistance, comparative genomic hybridization (CGH) was applied to compare human pancreatic carcinoma-derived cells. This panel of cell lines consists of the parental, drug- and thermosensitive pancreatic carcinoma cell line EPP85 - 181P, its atypical MDR variant EPP85-181RNOV, the classical MDR subline EPP85-181RDB, and their thermoresistant counterparts EPP85-181P-TR, EPP85-181RNOV-TR, and EPP85 - 181RDB-TR, respectively. CGH using genomic DNA prepared from these cell lines as probes successfully identified genomic gains and/or losses in chromosomal regions encoding putative genes associated with drug resistance and/or thermoresistance. These genes included 23 members of the family of ABC transporters, 27 members of the family of cytochrome P450 (CYP) monooxygenases, various molecular chaperones, DNA repair enzymes, and factors involved in the regulation of cell cycle and apoptosis. The importance of these cell variant-specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated.

Cytotoxicity, apoptosis induction and downregulation of MDR-1 expression by the anti-topoisomerase II agent, salvicine, in multidrug-resistant tumor cells.

Salvicine, a novel topoisomerase II inhibitor and a diterpenoid quinone compound, exerts potent in vitro and in vivo antitumor effects. In our study, we show that salvicine effectively kills multidrug-resistant (MDR) sublines, such as K562/A02, KB/VCR and MCF-7/ADR, and parental K562, KB and MCF-7 cell lines to an equivalent degree. These cytotoxic activities of salvicine were much more potent than those of several classical anticancer drugs (average resistance factor: 1.42 for salvicine vs. 344.35, 233.19 and 71.22 for vincristine, doxorubicin and etoposide, respectively). Flow cytometry and DNA agarose gel electrophoresis demonstrated that salvicine induced similar levels of apoptosis in MDR K562/A02 and parental cells. The compound activated caspase-1 and -3 (but not caspase-8) and increased the ratio of bax to bcl-2 mRNA via reduction of bcl-2 mRNA expression in the same cells. Furthermore, salvicine induced the downregulation of mdr-1 gene and P-gp expression but had no effect on MRP and LRP gene expression in MDR K562/A02 cells. These results suggest that the reduction of mdr-1 and bcl-2 expression by salvicine possibly contributes to its cytotoxicity and apoptotic induction in this system. The effectiveness, broad-spectrum activity and possibly novel mechanism of killing MDR tumor cells in vitro of salvicine signify promising in vivo and clinical activity. The novel chemical structure of this compound further implies a role for salvicine in future MDR tumor therapy.

Synthesis and Biological Evaluation of N-Heterocyclic Indolyl Glyoxylamides as Orally Active Anticancer Agents

A series of N-heterocyclic indolyl glyoxylamides were synthesized and evaluated for in vitro and in vivo anticancer activities. They exhibited a broad spectrum of anticancer activity not only in murine leukemic cancer cells but also in human gastric, breast, and uterus cancer cells as well as their multidrug resistant sublines with a wide range of IC(50) values. They also induced apoptosis and caused DNA fragmentation in human gastric cancer cells. Among the compounds studied, 7 showed the most potent activity of growth inhibition (IC(50) = 17-1711 nM) in several human cancer cells. Given orally, compounds 7 and 13 dose-dependently prolonged the survival of animals inoculated with P388 leukemic cancer cells. N-Heterocyclic indolyl glyoxylamides may be useful as orally active chemotherapeutic agents against cancer and refractory cancerous diseases of multidrug resistance phenotype.

Kinetic analysis of fluorescein and dihydrofluorescein effluxes in tumour cells expressing the multidrug resistance protein, MRP1

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane. A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport. The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU). We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1. The measurements were made in real time using intact cells. The kinetics parameters, k a= V M/ K m, which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues. They were highly comparable with values for k a of other negatively charged substrates, such as GSH and calcein. The active efflux of both FLU derivatives was inhibited by GSH depletion.

New insights into the P-glycoprotein-mediated effluxes of rhodamines.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.

Progesterone: a novel adjunct to intravesical chemotherapy.

To investigate the effect of progesterone on multidrug-resistant urothelial cell lines, as the failure of intravesical chemotherapeutic drugs is often caused by multidrug resistance (MDR), mediated by the drug efflux pump P-glycoprotein (PGP), the function of which can be down-regulated by various compounds including steroid hormones. Two urothelial cell lines (RT112S and MGH-U1S) and their MDR sublines (RT112R, to cisplatin; and MGH-U1R, a cell line expressing PGP) were used to assess the cytotoxic effects of progesterone, epirubicin and their combination. Cytotoxicity was assessed using a tetrazolium-based assay and in situ confocal microscopy. Cell lines sensitive to epirubicin (MGH-U1S, RT112S and RT112R) required a much lower dose of epirubicin to kill half the cells than did the MDR cell line. Progesterone was intrinsically cytotoxic to all cell lines with little difference among them. Combined therapy had no cumulative effect on epirubicin-sensitive cell lines, but reversed MDR in the MGHU1R cell line, both assessed by confocal microscopy and by the tetrazolium assay. Progesterone can reverse MDR in urothelial cells in vitro. This, combined with its effects on cell differentiation and apoptosis, together with its safety and tolerability compared to other MDR agents, suggests it may be a valuable adjunct to intravesical chemotherapy.

A diagnostic tool for monitoring multidrug resistance expression in human tumor tissues

Studies on multidrug resistance (MDR) require a sensitive and quantitative assay of mRNA expression in clinical tumor samples. Based on the small size, heterogenity, and the possibility of partial degradation of clinical specimens, unambiguous data are often difficult to obtain. The aim of the present study was to develop a multiplex polymerase chain reaction (PCR) in combination with nested PCR for quantitative analyses of mRNA expression of MDR1, MRP (multidrug resistance protein), and DNA topoisomerase IIα in small amounts of tumor tissue. RNA samples extracted from the human cell line RPMI 8226 and its MDR sublines 8226/Dox6 and DOXint40c, that overexpress MDR1 and MRP, respectively, were used as model substrates. In the first step, cDNAs of the three genes as well as of the housekeeping gene β-actin were simultaneously amplified in single tubes using 20 cycles of PCR after random-primed reverse transcription. When necessary, a second amplification step of the preamplified PCR products was employed using nested primer pairs. Primer competition was evaluated by analyses of serially diluted amounts of cDNA and at different numbers of PCR cycles. Based on the results obtained, this multiplex/nested PCR approach may provide a base for quantitative analyses of MDR1, MRP, and topoisomerase IIα mRNA expression in clinical tumor biopsies.

Nonaqueous Biocatalytic Synthesis of New Cytotoxic Doxorubicin Derivatives: Exploiting Unexpected Differences in the Regioselectivity of Salt-Activated and Solubilized Subtilisin

Two enzymes, Mucor javanicus lipase and subtilisin Carlsberg (SC), catalyzed the nonaqueous acylation of doxorubicin (DOX). Compared to the untreated enzyme the rate of DOX acylation at the C-14 position with vinyl butyrate in toluene was 25-fold higher by lipase ion-paired with Aerosol OT (AOT) and 5-fold higher by lipase activated by 98% (w/w) KCl co-lyophilization (3.21 and 0.67 mumol/min g-lipase, respectively, vs 0.13 mumol/min g-lipase). Particulate subtilisin Carlsberg (SC) was nearly incapable of DOX acylation, but ion-paired SC (AOT-SC) catalyzed acylation at a rate of 2.85 mumol/min g-protease. The M. javanicus formulations, AOT-SC, and SC exclusively acylated the C14 primary hydroxyl group of DOX. Co-lyophilization of SC with 98% (w/w) KCl expanded the enzyme's regiospecificity such that KCl-SC additionally acylated the C4' hydroxyl and C3' amine groups. The total rate of DOX conversion with KCl-SC was 56.7 mumol/min g-protease. The altered specificity of KCl-SC is a new property of the enzyme imparted by the salt activation, and represents the first report of unnatural regioselectivity exhibited by a salt-activated enzyme. Using AOT-SC catalysis, four unique selectively acylated DOX analogues were generated, and KCl-SC was used to prepare DOX derivatives acylated at the alternative sites. Cytotoxicities of select derivatives were evaluated against the MCF7 breast cancer cell line (DOX IC50 = 27 nM) and its multidrug-resistant sub-line, MCF7-ADR (DOX IC50 = 27 muM). The novel derivative 14-(2-thiophene acetate) DOX was relatively potent against both cell lines (IC50 of 65 nM and 8 muM, respectively) and the 14-(benzyl carbonate) DOX analogue was as potent as DOX against the MCF7 line (25 nM). Activated biocatalysts and their novel regioselectivity differences thus enabled single-step reaction pathways to an effective collection of doxorubicin analogues.

Alterations in the expression of cytochrome c oxidase subunits in doxorubicin-resistant leukemia K562 cells

Doxorubicin (DOX), a widely used antitumoral drug, induces numerous modifications in sensitive cells, interacting with nuclear and mitochondrial DNA. In previous studies achieved in two K562 DOX-resistant sublines (K562/0.2R and K562/0.5R), we have shown stable mitochondrial damage comparatively with sensitive parental cells, such as decrease of cytochrome c oxidase activity (COX; EC 1.9.3.1) and cytochrome aa3 content. In order to explain these data, we have studied several COX genes and their expression, in relationship with altered COX activity and multidrug resistance (MDR) phenotype. We have observed a lower expression of the catalytic subunits COX I and II in MDR sublines, which was neither related to mutations in the corresponding mitochondrial genes, nor to a reduced transcription rate. In contrast, we have noticed an increase in both MDR K562 variants, in the mRNA expression of the catalytic subunit COX III, related to an increase in the half-life of these transcripts. Moreover, the doxorubicin resistance phenotype in K562 cells was accompanied by modifications of the expression and steady-state mRNA levels of several nuclear-encoded regulatory COX subunits. Thus, doxorubicin-resistant K562 cells represent an interesting model to study stable modifications concomitant to MDR phenotype. Our results seem to indicate compensatory mechanisms which highlight the complexity of regulatory systems of COX enzyme, involving coordinate regulation of both nuclear and mitochondrial subunit expression.