Abstract Background: The ABCB1 gene encodes P-glycoprotein implicated in the development of cellular drug resistance. The aim of this study was to develop high-resolution melting (HRM) analysis for determination of ABCB1 polymorphisms and evaluate their associations with clinical data of breast carcinoma patients. Methods: HRM analysis was designed to assess five single nucleotide polymorphisms (SNPs) in ABCB1 (rs2214102, rs1128503, rs2032582, rs2032583 and rs1045642) in genomic DNA from 103 breast carcinoma patients. Results were confirmed by direct DNA sequencing. Results: HRM analysis revealed distinct patterns of melting curves for the respective genotypes of all followed SNPs. Sensitivity of HRM analysis compared with direct DNA sequencing was superior (97.1% vs. 93.9%). The overall accuracy of HRM was 97.6%. The coefficients of variation in replicate experiments encompassed the range 0.002%–0.038%. On the basis of the examined SNPs, one strong haplotype block containing rs2032582 and rs1128503 SNPs was identified. Significant associations of rs2032582 SNP with tumor size, negative HER-2/neu status, and family history of breast carcinoma were found. Patients carrying the ancestral homozygous genotype (GG) in rs2214102 had significantly worse progression-free survival in comparison with carriers of the non-ancestral allele (A) in the adjuvant set (p=0.005). Conclusions: A rapid, accurate, low-cost and time-effective method for screening ABCB1 SNPs was developed. Significant associations of ABCB1 rs2032582 and rs2214102 SNPs with prognostic factors and survival of patients were found.
med by the non-parametric Kruskal-Wallis test. The individual values of t0.5α of all patients significantly correlated with t0.5β (r = 0.587, p < 0.01), but there was no correlation between t0.5β and t0.5γ. The observed differences between patients with most rapid and slowest elimination of paclitaxel were 3.2-fold (AUC0-t), 3.6-fold (CL0-t and CL0-∞) and 3-fold (CL/kg). In accordance, the difference in rate of α phase in these patients was 3-fold and t0.5α was also shorter in the patient with the most rapid clearance. Similar differences were found in β phase. Moreover, the patient with the lowest clearance had 2.4-higher maximal concentration (Cmax). Conclusion: Pharmacokinetic parameters in the followed patients were significantly different and the differences remained unchanged during repeated infusions. These differences could potentially influence outcome of the therapy.
Abstract Increased expression of the ABCB1 gene in cancer cells is usually connected with occurrence of multidrug resistance (MDR) and poor prognosis. However, the correlation between ABCB1 expression and MDR phenotype is difficult to prove in clinical samples. Most of the researchers believe that these difficulties are due to the poor reliability and sensitivity of assays for detection of ABCB1 expression in clinical samples. However, the complexity of P‐gp mediated resistance cannot be reduced to the methodical difficulties only. Here, we addressed the question how widely used methods for detection of ABCB1 expression levels could predict its functional activity and thus its contribution to drug resistance in defined conditions in vitro . The ABCB1 expression was assessed at the mRNA level by quantitative real‐time polymerase chain reaction ( qRT ‐PCR), and at the protein level by flow cytometry using UIC2 antibody. The ABCB1 function was monitored using a calcein AM accumulation assay. We observed that K562 cells have approximately 320 times higher level of ABCB1 mRNA than HL‐60 cells without detectable function. In addition, resistant K562/Dox cells exhibited significantly higher ABCB1 mRNA expression than resistant K562/HHT cells. However, the functional tests clearly indicated opposite results. Flow cytometric assessment of P‐gp, although suggested as a reliable method, contradicted the functional test in K562/Dox and K562/HHT cells. We further used a set of MDR cells expressing various levels of P‐gp. Similarly here, flow cytometry not always corresponded to the functional analysis. Our results strongly suggest that an approach which exclusively relies on a simple correlation between ABCB1 expression, either at the mRNA level or protein level, and overall resistance may fail to predict actual contribution of P‐gp to overall resistance as the data indicating transporter expression reflect its function only roughly even in well‐defined in vitro conditions.
A81 Multidrug resistance of tumor cells to cytotoxic drugs is one of the major impediments in successful cancer chemotherapy. A significant part of tumor resistance to chemotherapy is caused by ABC transporter P-glycoprotein (P-gp, encoded by ABCB1 gene). High expression of ABCB1 and its genetic variations (e.g. SNPs) thus may have a great clinical impact. The aim of this study was to investigate the relationship between multiple ABCB1 SNPs, the expression level of the gene and prognosis of patients with breast carcinomas. Analyses of 7 ABCB1 SNPs and expression levels in 90 sets of samples from breast cancer patients were performed. ABCB1 genotype was assayed with a novel method that allows simultaneous assessment of multiple SNPs in numerous samples on a single Nanogen electronic microarray platform. The distribution of ABCB1 genotype in breast cancer cases was compared with the distribution in 100 healthy controls. ABCB1 expression was quantified in cDNA samples from tumor and non-tumor tissues of breast cancer patients by real-time PCR. Both ABCB1 SNPs and expression levels were correlated with clinico-pathological characteristics to answer the prognostic value of ABCB1 genotypes and/or phenotypes in breast cancer. ABCB1 was expressed in 87/88 (98.9 %) of tumor and in 39/40 (97.5%) of non-tumor samples. A striking inter-individual variability in expression of ABCB1 was found. ABCB1 was down-regulated in 89.7% of all tumors. No significant correlation between ABCB1 expression and any clinico-pathological dates was observed. High frequencies of variant alleles in ABCB1 exon 12 (1236C>T, qT = 38.3), exon 21 (2677G>T/A, qT = 40.0, qA = 6.7) and exon 26 (3435C>T, qT = 54.0) were revealed. Individuals with variant alleles in these three SNPs had a significantly lower ABCB1 expression in their tumors than patients with normal genotype. SNPs in exon 12, exon 21 and exon 26 also correlated with estrogen receptor status of patients. Thus these SNPs may affect function of P-gp and modify breast cancer prognosis. > This work was supported by grants of Grant Agency of the Czech Republic 305/07/P347, Internal Grant Agency of the Czech Ministry of Health, no.: 9426-3 and the National Program FUGE no.: 151924/150 and 15204/150, funded by The Research Council in Norway.
Objective The aim of this study was to further clarify the recently reported role of NAD(P)H:quinone oxidoreductase 1 (NQO1) as a strong prognostic and predictive factor in breast cancer. Methods NQO1 transcript levels were monitored in mammary tumors by real-time polymerase chain reaction. NQO1 protein levels were immunohistochemically determined in formalin-fixed paraffin-embedded tissues. NQO1 polymorphism (Pro187Ser, rs1800566) was also assessed. Evaluation (N=52) and validation (N=53) sets were analyzed subsequently. Results Carriers of variant NQO1-Ser allele had significantly more frequently NQO1-negative protein expression (P=0.001) in both sets. NQO1 transcript levels in samples with negative protein expression were significantly lower than in those with positive NQO1 protein expression (P=0.007) in both sets. Patients with stages 0/I/II had more often positive NQO1 protein expression than patients with stages III/IV (P=0.022) in the evaluation set. Significant association between NQO1 protein expression and TP53 protein status was also found (P=0.037). However, both associations were not replicated by analysis of the validation set. Analysis of both sets combined did not show significant association of NQO1 protein expression either with stage (P=0.231) or with TP53 protein status (P>0.999). Thus, the results observed in the evaluation set were effects of small sample size. Conclusion The role of NQO1 in human mammary gland carcinogenesis does not seem to be directly associated with classical clinico-pathological factors.
733 Background: The quinone oxidoreductases, NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH(dihydronicotinamide riboside):quinone oxidoreductase 2 (NQO2), are integral flavoproteins responsible for the detoxification and metabolism of quinones and quinoid compounds. NQO1 and NQO2 protect cells against accumulation of toxic quinoid compounds, as well as against mutagenicity and carcinogenicity. The aim of this study was to assess the importance of gene expression of NQO1 and NQO2 and breast cancer development and/or progression in line with known frequent polymorphisms. We focused on the NQO1 polymorphism causing exchange of Proline to Serine at position 187 which leading to a nonfunctional enzyme. In NQO2, rarely studied insertion/deletion in promotor and Phe47Leu exchange in exon 3 were addressed. Materials and Methods: Levels of NQO1 and NQO2 mRNAs were monitored in human carcinomas of the mammary gland, mammary tissue without presence of tumor cells and in peripheral blood lymphocytes. Real-time PCR using absolute quantification and normalization to the expression of control gene cyclophilin A was used. Polymorphisms in NQO1 and NQO2 were assessed in DNA samples from peripheral blood lymphocytes of the same patients by PCR-RFLP based methods and DNA sequencing. Results: Polymorphism in NQO1 was associated with increased risk of breast cancer in Czech population. Moreover, we observed high interindividual variability in NQO1 and NQO2 expression among patients and differences between tumor and non-tumor tissues. Different expression patterns in samples divided according to the presence of genetic polymorphisms in NQO1 and NQO2 were observed as well. By comparing these data with result of primary adjuvant therapy the clinical significance of NQO1 and NQO2 expression and genetic variability seems to be evident. Conclusions: First study on genetic profiles of NQO1 and NQO2 in Czech breast cancer patients suggested that variations may play a role in the development and progression of breast cancer. Multiplex analysis of NQO1 and NQO2 polymorphism and expressions on larger and multiethnic cohorts should refine possible use in prediction and modification of breast cancer therapy. This study was supported by grants of Internal Grant Agency of Ministry of Health of the Czech Republic, no.: 1A/8248, and 8563-5.
Oxysterols are oxidative derivatives of cholesterol that play many roles in human physiology and pathology, including cancer. For example, oxysterols modulate cell proliferation, apoptosis, or migration. This study aimed to analyze the role of important oxysterol, 7-ketocholesterol (7-KC), in response of breast carcinoma cell line models to treatment with tamoxifen.
Methods
Two estrogen receptor (ER) positive (MCF-7 and T47D) and one ER-negative (BT-20) breast carcinoma cell lines were employed. Cell lines were co-incubated with tamoxifen and 7-KC at different concentration ratios, and the viability of cells, proliferation, cell cycle, caspase activity, and gene expression changes were evaluated. Next, the ability of 7-KC to stimulate cell migration and invasivity was tested.
Results
7-KC slightly increased the IC50 value of tamoxifen in the MCF7 cell line, but decreased it in the BT-20 cell line. No significant difference was observed for T47D cells. In line with these data, caspase 3/7 activity was enhanced by 7-KC in BT-20 cells, but not in any ER-positive cell line. Gene expression analysis showed upregulation of tamoxifen metabolizing genes, e.g. CYP1A1 and CYP1B1 in MCF-7 while downregulation in BT-20 cells. Finally, we found that the presence of 7-KC potentiates cellular migration and invasivity.
Conclusions
7-KC seems to modulate the response of breast carcinoma cells to tamoxifen according to ER status in vitro, making it an interesting candidate for future studies. The study was supported by projects INTER-ACTION no. LTAUSA19032 and AZV no. NU20–09–00174.
Abstract SB-T-12854 is a novel taxane where fluorine in the molecule could decrease metabolism and increase activity over paclitaxel with efficiency limited by multidrug resistance due to ABCB1 transport, metabolism, and other factors. Purposes of the study: this study investigated metabolism of SB-T-12854 by human, pig and rat CYP enzymes, cytotoxicity of the drug and isolated metabolites, transport in drug-resistant NCI/ADR-RES cells and sensitive MDA-MB-435, and effects on the cell cycle. Experimental procedures: Metabolism of SB-T-12854 was studied in human, pig, rat and CYP3A1/2-induced rat microsomes and human cDNA-expressed CYP enzymes in vitro. The metabolites were determined by HPLC and MS analyses. Human tumor lines MDA-MB-435 and NCI-ADR-RES (NCI, Frederick MD) were kept in standard conditions. Viability was assessed by the MTT assay. Transport of SB-T-12854 by tumor cells was determined by measuring their cellular levels by HPLC. Progress in the cell cycle was determined by FACS. Summary of new and unpublished data: In human, pig and rat microsomes SB-T-12854 formed the same four products at similar rates, but CYP3A1/2-induced rat microsomes formed 13 metabolites at 16-fold higher rate indicating very high activity of CYP3A, the main human CYP form of varied expression. The cDNA-expressed CYP3A4 formed the same products, but CYP1A2, 1B1, 2A6, 2C9, or 2E1 were inactive. The time course of metabolism and further metabolism of isolated metabolites showed their mutual conversion and identified primary and secondary products. In rat microsomes, Km values for metabolites M5-6 and M9-12 were 14.01 and 6.89 (M), respectively, and Vmax (pmol/min/mg protein) were 70.4 (M5-6) and 370.4 (M9-12). In MDA-MB-435 cells some metabolites were inactive, but others were considerably more cytotoxic than SB-T-12854 itself in contrast to paclitaxel whose metabolites are less active. SB-T-12854 was 6-fold more cytotoxic than paclitaxel in MDA-MB-435 cells and 30-fold more potent in NCI/ADRRES cells. In both cell lines SB-T-12854 caused G2/M block in the same way as paclitaxel. Conclusions: SB-T-12854 is metabolized by human, pig and rat CYP3A enzymes to identical and to some markedly more active products in contrast to paclitaxel, which is only inactivated. SB-T-12854 is 30-fold more cytotoxic than paclitaxel in ABCB1 highly expressing NCI/ADR-RES cells, partly because SB-T-12854 is not transported by ABCB1 in contrast to paclitaxel. SB-T-12854 influenced the cell cycle of NCI/ADR-RES and MDA-MB-435 cells in the same way as paclitaxel causing G2/M block, but at substantially lower concentrations. Lack of ABCB1 transport and higher cytotoxicity suggest a potential advantage of this taxane over paclitaxel, which is ineffective against certain tumors, due to high ABCB1 expression. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A147.
The aim of the study was to contribute to our understanding of the mechanisms responsible for the resistance of breast cancer cells to taxanes.Cell cycle characteristics, DNA fragmentation, p53 and p21(WAF1/CIP1) expression, caspase-3 and caspase-9 activity, cytochrome c release from mitochondria during cell death induction by the taxanes paclitaxel and docetaxel in highly-sensitive MDA-MB-435 and highly-resistant NCI-ADR-RES human breast cancer cells were compared.Approximately 300-fold higher concentrations of the taxanes were required to induce death in resistant NCI-ADR-RES cells than in sensitive MDA-MB-435 cells. Cell death induced by the taxanes in both sensitive and resistant cells was preceded by the accumulation of cells in the G2/M-phase. Neither cell type produced any DNA fragmentation (DNA ladder) typical of regular apoptosis. The p53 and the p21(WAF1/CIP1) levels did not change in sensitive or in resistant cells during cell death induction by the taxanes. The activity of the executioner caspase-3 increased significantly (2 to 2.5-fold) and, similarly, the activity of caspase-9 increased significantly (2 to 3.5-fold) in both cell types. However, cytochrome c was found to be released from mitochondria into the cytosol only in the resistant NCI-ADR-RES cells, but not in the sensitive MDA-MB-435 cells.The death induced by the taxanes in the studied breast cancer cells can be characterized as an apoptosis-like death, including caspase-3 and caspase-9 activation but not oligonucleosomal DNA fragmentation. However, the mechanisms of death induction by the taxanes in sensitive MDA-MB-435 cells and resistant NCI-ADR-RES cells differ. Cytochrome c is released from the mitochondria in resistant but not in sensitive cells.
