Amadeo M. Parissenti

Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance

BackgroundAnthracyclines and taxanes are commonly used in the treatment of breast cancer. However, tumor resistance to these drugs often develops, possibly due to overexpression of drug transporters. It remains unclear whether drug resistance in vitro occurs at clinically relevant doses of chemotherapy drugs and whether both the onset and magnitude of drug resistance can be temporally and causally correlated with the enhanced expression and activity of specific drug transporters. To address these issues, MCF-7 cells were selected for survival in increasing concentrations of doxorubicin (MCF-7DOX-2), epirubicin (MCF-7EPI), paclitaxel (MCF-7TAX-2), or docetaxel (MCF-7TXT). During selection cells were assessed for drug sensitivity, drug uptake, and the expression of various drug transporters.ResultsIn all cases, resistance was only achieved when selection reached a specific threshold dose, which was well within the clinical range. A reduction in drug uptake was temporally correlated with the acquisition of drug resistance for all cell lines, but further increases in drug resistance at doses above threshold were unrelated to changes in cellular drug uptake. Elevated expression of one or more drug transporters was seen at or above the threshold dose, but the identity, number, and temporal pattern of drug transporter induction varied with the drug used as selection agent. The pan drug transporter inhibitor cyclosporin A was able to partially or completely restore drug accumulation in the drug-resistant cell lines, but had only partial to no effect on drug sensitivity. The inability of cyclosporin A to restore drug sensitivity suggests the presence of additional mechanisms of drug resistance.ConclusionThis study indicates that drug resistance is achieved in breast tumour cells only upon exposure to concentrations of drug at or above a specific selection dose. While changes in drug accumulation and the expression of drug transporters does occur at the threshold dose, the magnitude of resistance cannot be attributed solely to changes in drug accumulation or the activity of drug transporters. The identities of these additional drug-transporter-independent mechanisms are discussed, including their likely clinical relevance.

cDNA microarray analysis of isogenic paclitaxel- and doxorubicin-resistant breast tumor cell lines reveals distinct drug-specific genetic signatures of resistance.

SummarycDNA microarray analysis is a highly useful tool for the classification of tumors and for prediction of patient prognosis to specific cancers based on this classification. However, to date, there is little evidence that microarray approaches can be used to reliably predict patient response to specific chemotherapy drugs or regimens. This is likely due to an inability to differentiate between genes affecting patient prognosis and genes that play a role in response to specific drugs. Thus, it would be highly useful to identify genes whose expression correlates with tumor cell sensitivity to specific chemotherapy agents in a drug-specific manner. Using cDNA microarray analysis of wildtype MCF-7 breast tumor cells and isogenic paclitaxel-resistant (MCF-7TAX) or doxorubicin-resistant (MCF-7DOX) derivative cell lines, we have uncovered drug-specific changes in gene expression that accompany the establishment of paclitaxel or doxorubicin resistance. These changes in gene expression were confirmed by quantitative reverse transcription polymerase chain reaction and immunoblotting experiments, with a confirmation rate of approximately 91–95%. The genes identified may prove highly useful for prediction of response to paclitaxel or doxorubicin in patients with breast cancer. To our knowledge this is the first report of drug-specific genetic signatures of resistance to paclitaxel or doxorubicin, based on a comparison of gene expression between isogenic wildtype and drug-resistant tumor cell lines. Moreover, this study provides significant insight into the wide variety of mechanisms through which resistance to these agents may be acquired in breast cancer.

Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization

BackgroundSince proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients.MethodsTo investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes.ResultsOf 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the “hit list” was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected.ConclusionsThese findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify highly relevant genes associated with doxorubicin resistance. The induction of one or more of these genes was found to be correlated with changes in the drug’s properties, while inhibiting one specific class of these genes (the AKRs) increased cellular doxorubicin content and restored drug DNA binding, cytotoxicity, and subcellular localization.

Induction of 1C aldoketoreductases and other drug dose-dependent genes upon acquisition of anthracycline resistance.

Objectives Recent studies suggest that tumor cells overexpressing aldoketoreductases (AKRs) exhibit increased resistance to DNA damaging agents such as anthracyclines. AKRs may induce resistance to the anthracycline doxorubicin by catalyzing its conversion to the less toxic 13-hydroxy metabolite doxorubicinol. However, it has not been established whether during selection for anthracycline resistance, AKR overexpression in tumor cells can be correlated with the onset or magnitude of drug resistance and with appreciable conversion of anthracyclines to 13-hydroxy metabolites. Methods and findings Through microarray and quantitative polymerase chain reaction studies involving rigid selection criteria and both correlative discriminate statistics and time-course models, we have identified several genes whose expression can be correlated with the onset and/or magnitude of anthracycline resistance, including AKR1C2 and AKR1C3. Also associated with the onset or magnitude of anthracycline resistance were genes involved in drug transport (ABCB1, ABCC1), cell signaling and transcription (RDC1, CXCR4), cell proliferation or apoptosis (BMP7, CAV1), protection from reactive oxygen species (AKR1C2, AKR1C3, FTL, FTH, TXNRD1, MT2A), and structural or immune system proteins (IFI30, STMN1). As expected, doxorubicin-resistant and epirubicin-resistant cells exhibited higher levels of doxorubicinol than wild-type cells, although at insufficient levels to account for significant drug resistance. Nevertheless, an inhibitor of Akr1c2 (5&bgr;-cholanic acid) almost completely restored sensitivity to doxorubicin in ABCB1-deficient doxorubicin-resistant cells, while having no effect on ABCB1-expressing epirubicin-resistant cells. Conclusion Taken together, we show for the first time that a variety of genes (particularly redox genes such as AKR1C2 and AKR1C3) can be temporally and causally correlated with the acquisition of anthracycline resistance in breast tumor cells.

Hypermethylation of the ABCB1 downstream gene promoter accompanies ABCB1 gene amplification and increased expression in docetaxel-resistant MCF-7 breast tumor cells.

Drug transporters have been implicated in resistance of solid and non-solid tumors to a variety of chemotherapeutic agents. Higher expression of the ABCB1 drug transporter is often observed in drug-resistant tumor cells, although the precise mechanism remains unclear. During selection of MCF-7 cells for survival in increasing concentrations of docetaxel (MCF-7TXT cells), we observed in this study a temporal correlation between the acquisition of docetaxel resistance at selection dose 9 and the increased expression of ABCB1. Both the magnitude of docetaxel resistance and the level of ABCB1 expression then rose as the selection dose was further elevated. We also observed through bisulfite sequencing experiments that the ABCB1 downstream promoter became increasingly methylated following the acquisition of drug resistance (selection doses 10-12). Transcription was solely attributed to the upstream ABCB1 promoter within MCF-7TXT cells at the highest selection dose suggesting that hypermethylation caused a shift in promoter usage. The hypermethylation was also accompanied by regional amplification of chromosome 7 containing the ABCB1 gene and its neighbor ABCB4 but not DBF-4. The amplification of the ABCB1 gene correlated positively both with the hypermethylation of the ABCB1 downstream promoter (r=0.90) and the increased expression of ABCB1 (r=0.78). Moreover demethylation of the ABCB1 downstream promoter induced by 5-aza-2Â’deoxycytidine treatment decreased the expression of ABCB1 mRNA in MCF-7TXT cells. Taken together, our findings suggest that the increased expression of ABCB1 upon acquisition of docetaxel resistance in breast tumor cells can be multifactorial, involving both epigenetic changes in promoter usage and regional chromosome amplification.

The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance

Induced expression of the Abcb1 drug transporter often occurs in tumors in response to chemotherapy. The role that epigenetic modifications within the ABCB1 promoter play in Abcb1 expression remains unclear. We selected MCF-7 cells for survival in increasing doses of chemotherapy drugs, and assessed the methylation status of 66 CpG sites within the ABCB1 promoter preceding, accompanying and following the onset of drug resistance. Increased ABCB1 transcript expression coincident with acquisition of resistance to epirubicin or paclitaxel was temporally associated with hypomethylation of the ABCB1 downstream promoter in the absence of gene amplifications or changes in mRNA stability. Treatment of control MCF-7 cells with demethylating and/or acetylating agents increased ABCB1 transcript expression. In addition to broad promoter hypomethylation, dramatic reductions in the methylation of specific CpG sites within the promoter were observed, suggesting that these sites may play a predominant role in transcriptional activation through promoter hypomethylation. Furthermore, our data suggest that allele-specific reductions in ABCB1 promoter methylation regulate promoter usage within paclitaxel-resistant cells. This study provides strong evidence that changes in ABCB1 promoter methylation, ABCB1 promoter usage and ABCB1 transcript expression can be temporally and causally correlated with the acquisition of drug resistance in breast tumor cells.

Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxel-resistant MCF-7 breast cancer cells

BackgroundChemoresistance is a major factor involved in a poor response and reduced overall survival in patients with advanced breast cancer. Although extensive studies have been carried out to understand the mechanisms of chemoresistance, many questions remain unanswered.MethodsIn this research, we used two isogenic MCF-7 breast cancer cell lines selected for resistance to doxorubicin (MCF-7DOX) or docetaxel (MCF-7TXT) and the wild type parental cell line (MCF-7CC) to study mechanisms underlying acquired resistance to taxanes in MCF-7TXT cells. Cytotoxicity assay, immunoblotting, indirect immunofluorescence and live imaging were used to study the drug resistance, the expression levels of drug transporters and various tubulin isoforms, apoptosis, microtubule formation, and microtubule dynamics.ResultsMCF-7TXT cells were cross resistant to paclitaxel, but not to doxorubicin. MCF-7DOX cells were not cross-resistant to taxanes. We also showed that multiple mechanisms are involved in the resistance to taxanes in MCF-7TXT cells. Firstly, MCF-7TXT cells express higher level of ABCB1. Secondly, the microtubule dynamics of MCF-7TXT cells are weak and insensitive to the docetaxel treatment, which may partially explain why docetaxel is less effective in inducing M-phase arrest and apoptosis in MCF-7TXT cells in comparison with MCF-7CC cells. Moreover, MCF-7TXT cells express relatively higher levels of β2- and β4-tubulin and relatively lower levels of β3-tubulin than both MCF-7CC and MCF-7DOX cells. The subcellular localization of various β-tubulin isoforms in MCF-7TXT cells is also different from that in MCF-7CC and MCF-7DOX cells.ConclusionMultiple mechanisms are involved in the resistance to taxanes in MCF-7TXT cells. The high expression level of ABCB1, the specific composition and localization of β-tubulin isoforms, the weak microtubule dynamics and its insensitivity to docetaxel may all contribute to the acquired resistance of MCF-7TXT cells to taxanes.

Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells

IntroductionThe taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance.MethodsMCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR).ResultsMCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways.ConclusionsWe report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.

Distinct genetic alterations occur in ovarian tumor cells selected for combined resistance to carboplatin and docetaxel

BackgroundCurrent protocols for the treatment of ovarian cancer include combination chemotherapy with a platinating agent and a taxane. However, many patients experience relapse of their cancer and the development of drug resistance is not uncommon, making successful second line therapy difficult to achieve. The objective of this study was to develop and characterize a cell line resistant to both carboplatin and docetaxel (dual drug resistant ovarian cell line) and to compare this cell line to cells resistant to either carboplatin or docetaxel.MethodsThe A2780 epithelial endometrioid ovarian cancer cell line was used to select for isogenic carboplatin, docetaxel and dual drug resistant cell lines. A selection method of gradually increasing drug doses was implemented to avoid clonal selection. Resistance was confirmed using a clonogenic assay. Changes in gene expression associated with the development of drug resistance were determined by microarray analysis. Changes in the expression of selected genes were validated by Quantitative Real-Time Polymerase Chain Reaction (QPCR) and immunoblotting.ResultsThree isogenic cell lines were developed and resistance to each drug or the combination of drugs was confirmed. Development of resistance was accompanied by a reduced growth rate. The microarray and QPCR analyses showed that unique changes in gene expression occurred in the dual drug resistant cell line and that genes known to be involved in resistance could be identified in all cell lines.ConclusionsOvarian tumor cells can acquire resistance to both carboplatin and docetaxel when selected in the presence of both agents. Distinct changes in gene expression occur in the dual resistant cell line indicating that dual resistance is not a simple combination of the changes observed in cell lines exhibiting single agent resistance.

Role of autophagy and lysosomal drug sequestration in acquired resistance to doxorubicin in MCF-7 cells

BackgroundThe roles and mechanisms involved in starvation-induced autophagy in mammalian cells have been extensively studied. However, less is known about the potential role for autophagy as a survival pathway in acquired drug resistance in cancer cells under nutrient-rich conditions.MethodsWe selected MCF-7 breast tumor cells for survival in increasing concentrations of doxorubicin and assessed whether the acquisition of doxorubicin resistance was accompanied by changes in doxorubicin and lysosome localization and the activation of autophagy, as assessed by laser scanning confocal microscopy with or without immunohistochemical approaches. The ultrastructure of cells was also viewed using transmission electron microscopy. Cellular levels of autophagy and apoptosis-related proteins were assessed by immunoblotting techniques, while protein turnover was quantified using a flux assay.ResultsAs cells acquired resistance to doxorubicin, the subcellular location of the drug moved from the nucleus to the perinuclear region. The location of lysosomes and autophagosomes also changed from being equally distributed throughout the cytoplasm to co-localizing with doxorubicin in the perinuclear region. There was an apparent temporal correlation between the acquisition of doxorubicin resistance and autophagy induction, as measured by increases in monodansylcadaverine staining, LC3-II production, and co-localization of LAMP1 and LC3-II immunofluorescence. Electron microscopy revealed an increase in cytoplasmic vacuoles containing mitochondria and other cellular organelles, also suggestive of autophagy. Consistent with this view, a known autophagy inhibitor (chloroquine) was highly effective in restoring doxorubicin sensitivity in doxorubicin-resistant cells. Moreover, this induction of autophagy correlated temporally with increased expression of the selective cargo receptor p62, which facilitates the delivery of doxorubicin-damaged mitochondria and other organelles to autophagosomes. Finally, we suggest that autophagy associated with doxorubicin resistance may be distinct from classical starvation-induced autophagy, since Beclin 1 and Atg7 expression did not change upon acquisition of doxorubicin resistance, nor did recombinant Bcl2 overexpression or an Atg7 knockdown alter doxorubicin cytotoxicity.ConclusionTaken together, our findings suggest that doxorubicin resistance in MCF-7 breast cancer cells is mediated, at least in part, by the activation of autophagy, which may be distinct from starvation-induced autophagy.