Gene Expression Profiling Reveals Novel Candidate Markers of Ovarian Carcinoma Intraperitoneal Metastasis.

Epithelial ovarian cancer (EOC) has the highest mortality among gynecological carcinomas. The lack of specific markers for prognostic determination of EOC progression hinders the search for novel effective therapies. The aim of the present study was (i) to explore differences in expressions of ATP-binding cassette (ABC) and solute carrier (SLC) transporter genes, genes associated with drug metabolism and cell cycle regulation between control ovarian tissues (n = 14), primary EOCs (n = 44) and intraperitoneal metastases (n = 29); (ii) to investigate associations of gene expression levels with prognosis of patients with intraperitoneal metastases. In all tissue samples, transcript levels of the above target genes were assessed using quantitative real-time PCR. Gene expression levels were compared between particular tissue types and evaluated with regard to progression-free survival (PFS) and drug-resistance status of patients with metastases. Gene expression of ABCA7 significantly increased and that of ESR2 decreased in the order control ovarian tissues - primary EOCs - metastases. High expressions of ABCA2/8/9/10, ABCB1, ABCC9, ABCG2, ATP7A, SLC16A14, and SOD3 genes were significantly associated with longer progression-free survival of patients. In intraperitoneal metastases, expression of all of these genes highly correlated and indicated prognostic profile. Transporters from the ABCA family, ABCG2, and ESR2 are involved mainly in lipid metabolism, membrane transport, and cell proliferation. These processes are thus probably the most important for EOC progression. Based on these results, we have proposed novel markers of ovarian carcinoma progression and metastatic spread which might be potentially useful as therapeutic targets. Their significance should be further explored on a larger independent set of patients.

Genetic and functional analyses do not explain the association of high PRC1 expression with poor survival of breast carcinoma patients.

Microtubules are vitally important for eukaryotic cell division. Therefore, we evaluated the relevance of mitotic kinesin KIF14, protein-regulating cytokinesis 1 (PRC1), and citron kinase (CIT) for the prognosis of breast carcinoma patients. Transcript levels were assessed by quantitative real-time PCR in tissues from two independent groups of breast carcinoma patients and compared with clinical data. Tissue PRC1 protein levels were estimated using immunoblotting, and the PRC1 tagged haplotype was analyzed in genomic DNA. A functional study was performed in MDA-MB-231 cells in vitro. KIF14, PRC1, and CIT transcripts were overexpressed in tumors compared with control tissues. Tumors without expression of hormonal receptors or high-grade tumors expressed significantly higher KIF14 and PRC1 levels than hormonally-positive or low-grade tumors. Patients with high intra-tumoral PRC1 levels had significantly worse disease-free survival than patients with low levels. PRC1 rs10520699 and rs11852999 polymorphisms were associated with PRC1 transcript levels, but not with patients survival. Paclitaxel-induced PRC1 expression, but PRC1 knockdown did not modify the paclitaxel cytotoxicity in vitro. PRC1 overexpression predicts poor disease-free survival of patients with breast carcinomas. Genetic variability of PRC1 and the protein interaction with paclitaxel cytotoxicity do not explain this association.

Gene expression of membrane transporters: Importance for prognosis and progression of ovarian carcinoma.

Membrane transporters (such as ABCs, SLCs and ATPases) act in carcinogenesis and chemoresistance development, but their relevance for prognosis of epithelial ovarian cancer (EOC) remains poorly understood. We evaluated the gene expression profile of 39 ABC and 12 SLC transporters and three ATPases in EOC tissues and addressed their putative role in prognosis and clinical course of EOC patients. Relative gene expression in a set of primary EOC (n=57) and in control ovarian tissues (n=14) was estimated and compared with clinical data and survival of patients. Obtained data were validated in an independent set of patients (n=60). Six ABCs and SLC22A18 gene were significantly overexpressed in carcinomas when compared with controls, while expression of 12 ABCs, five SLCs, ATP7A and ATP11B was decreased. Expression of ABCA12, ABCC3, ABCC6, ABCD3, ABCG1 and SLC22A5 was higher in high grade serous carcinoma compared with other subtypes. ABCA2 gene expression significantly associated with EOC grade in both sets of patients. Notably, expression level of ABCA9, ABCA10, ABCC9 and SLC16A14 significantly associated with progression-free survival (PFS) of the disease in either pilot or validation sets. ABCG2 level associated with PFS in the pooled set of patients. In conclusion, ABCA2, ABCA9, ABCA10, ABCC9, ABCG2 and SLC16A14 present novel putative markers of EOC progression and together with the revealed relationship between ABCA12, ABCC3, ABCC6, ABCD3, ABCG1 and SLC22A5 expression, and high grade serous type of EOC should be further examined by larger follow-up study.

The association of taxane resistance genes with the clinical course of ovarian carcinoma.

Taxane and platinum-based chemotherapy regimens are standard treatment for advanced ovarian carcinoma. Expression levels of putative markers of taxane resistance in carcinoma tissues and paired peritoneal samples (n=55) and in 16 samples of ovaries without signs of carcinoma were compared with clinical data and the patients' time to progression. KIF14, PRC1, CIT and ABCC1 genes were significantly overexpressed in carcinomas when compared with normal ovarian tissues, while ABCB1 and CASP9 expression was decreased. Associations of protein expression of the proliferation marker Ki-67 with KIF14, PRC1, ABCB1 and CASP2 were found. Lastly, it was discovered that ABCB1 and CASP2 levels associated with FIGO stage and that the CIT level associated with the time to progression of ovarian carcinoma patients (P<0.0001). In conclusion, ABCB1, CASP2, KIF14, PRC1 and CIT genes seem to associate with surrogate markers of ovarian carcinoma progression and CIT gene associates with therapy outcome.

The expression profile of ATP-binding cassette transporter genes in breast carcinoma.

AIM: ATP-binding cassette (ABC) transporters contribute to development of resistance to anticancer drugs via ATP-dependent drug efflux. A major goal of our study was to investigate associations between the expression of ABC transporters and outcome of breast carcinoma patients. PATIENTS & METHODS: Transcript levels of all 49 human ABC transporters were determined in post-treatment tumor and non-neoplastic tissue samples from 68 breast carcinoma patients treated by neoadjuvant chemotherapy. Six ABC transporters were then evaluated in independent series of 100 pretreatment patients. RESULTS: ABCA5/6/8/9/10, ABCB1/5/11, ABCC6/9, ABCD2/4, ABCG5 and ABCG8 were significantly downregulated and ABCA2/3/7/12, ABCB2/3/8/9/10, ABCC1/4/5/10/11/12, ABCD1/3, ABCE1, ABCF1/2/3 and ABCG1 were upregulated in post-treatment tumors compared with non-neoplastic tissues. Significant associations of intratumoral levels of ABCC1 and ABCC8 with grade and expression of hormonal receptors were found in both sets of patients. ABCA12, ABCA13 and ABCD2 levels were significantly associated with the response to neoadjuvant chemotherapy in post-treatment patients. Protein expression of ABCA12, ABCC8 and ABCD2 in tumor tissues of patients with breast carcinoma was observed by immunoblotting for the first time. CONCLUSION: ABCA12, ABCA13, ABCC1, ABCC8 and ABCD2 present potential modifiers of progression and response to the chemotherapy of breast carcinoma.

Detection of frequent ABCB1 polymorphisms by high-resolution melting curve analysis and their effect on breast carcinoma prognosis.

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.

Transport, metabolism, cytotoxicity and effects of novel taxanes on the cell cycle in MDA-MB-435 and NCI/ADR-RES cells.

Resistance of tumours to taxanes causes chemotherapy failure in numerous patients. Resistance is partly due to the low tumour uptake of taxanes and their rapid metabolism. Structural modifications of taxanes can reduce their P-glycoprotein-related efflux or decrease metabolism and consequently increase taxane efficiency. This study compared cytotoxicity and effects of the cell cycle, transport and metabolism of novel taxanes SB-T-1102, SB-T-1103, SB-T-1214 and SB-T-1216, fluorinated SB-T-12851, SB-T-12852, SB-T-12853, SB-T-12854 and IDN5109 with paclitaxel in paclitaxel-sensitive (MDA-MB-435) and paclitaxel-resistant (NCI/ADR-RES) human cancer cells. We have shown before that NCI/ADR-RES cells were 1,000-fold less sensitive to paclitaxel than MDA-MB-435 cells in correspondence to P-glycoprotein overexpression and up to 20-fold lower uptake of the drug in the resistant cells. The uptake of novel taxanes was 1.2 to 3.8 times lower than that of paclitaxel in the MDA-MB-435 cells, but 1.5 to 6.5 times higher in NCI/ADR-RES cells. NCI/ADR-RES cells were correspondingly only 2- to 6.6-fold less sensitive than the MDA-MB-435 cells to novel taxanes. Both cell lines showed minimal metabolism of the novel taxanes which was therefore not responsible for their different sensitivity, the observed differences in their individual efficiency and higher effects than paclitaxel. All novel taxanes caused G(2)/M block of the cell cycle similar to paclitaxel, but lower at concentrations by order of magnitude. Thus, structural modifications of taxanes resulting in their decreased P-glycoprotein-related transport probably caused their higher efficiency than paclitaxel in multidrug-resistant NCI/ADR-RES tumour cells.

Association of superoxide dismutases and NAD(P)H quinone oxidoreductases with prognosis of patients with breast carcinomas.

Associations of transcript levels of oxidative stress-modifying genes SOD2, SOD3, NQO1 and NQO2 and their functional single nucleotide polymorphisms (SNPs) rs4880, rs1799895, rs2536512, rs699473, rs1800566 and rs1143684 with prognosis of breast cancer patients were studied. SNPs were assessed by allelic discrimination in a cohort of 321 breast cancer patients from the Czech Republic. Transcript levels were determined by real-time polymerase chain reaction (PCR) with absolute quantification in tumor and adjacent non-neoplastic control tissues. Both genotypes and transcript levels were then compared with available clinical data on patients. Patients carrying low activity allele Leu in NQO2 rs1143684 had a greater incidence of stage 0 or I disease (i.e., better prognosis) than patients with the Phe/Phe genotype. This association was more evident in patients without expression of progesterone receptors (p = 0.031). Patients carrying the Thr allele in SOD3 rs2536512 SNP had a significantly greater incidence of tumors expressing estrogen receptors than patients carrying the Ala/Ala genotype (p = 0.007). SOD3 transcript level was significantly higher in grade 1 or 2 tumors than in grade 3 tumors (p = 0.006). Patients carrying T allele in SOD3 rs699473 SNP had significantly poorer progression-free survival (PFS) than patients carrying the CC genotype (p = 0.038). The same applied to the subgroup of patients treated by hormonal regimens (p = 0.021). Patients carrying the high activity Ala/Ala genotype in SOD2 (rs4880) had significantly poorer PFS than Val allele carriers in the group treated by cyclophosphamide but not hormonal regimens (p = 0.004). Our results suggest that NQO2, SOD2 and SOD3 may significantly modify prognosis of breast cancer patients and that their significance should be further characterized.

Second-generation taxanes effectively suppress subcutaneous rat lymphoma: role of disposition, transport, metabolism, in vitro potency and expression of angiogenesis genes.

The study investigated possible mechanisms by which second-generation taxanes, established as significantly more effective than paclitaxel in vitro, suppress a rat lymphoma model in vivo. The studied mechanisms included taxane pharmacokinetics, expression of genes dominating their metabolism (Cyp3a1/2) and transport (Abcb1) and genes controlling tumour angiogenesis (growth factors and receptors). SB-T-1214, SB-T-12854 and IDN5109 suppressed rat lymphoma more effectively than paclitaxel, SB-T-12851, SB-T-12852, SB-T-12853 or IDN5390 as well as P388D1 leukaemia cells in vitro. The greater anti-lymphoma effects of SB-T-1214 in rats corresponded to a higher bioavailability than with SB-T-12854, and lower systemic toxicity of SB-T-1214 for rats reflected its lower cytotoxicity for P388D1 cells in vitro. Suppression of Abcb1 and CYP3a1 expression by SB-T-1214 and IDN5109 could partly explain their anti-lymphoma effects, but not that of SB-T-12854. Growth factors genes Egf, Fgf, Pdgf, and Vegf associated with tumour angiogenesis had significantly lower expression following treatment with anti-lymphoma effective IDN5109 and their receptors were unaffected, whereas inefficient IDN5390 increased expression of the most important Vegf. The effective SB-T-12854 inhibited Egf, Egfr, Fgfr and Pdgfr expression, while the ineffective SB-T-12851, SB-T-12852 and SB-T-12853 inhibited only Egf or Egfr expression. Vegfr expression was inhibited significantly by SB-T-12851 and SB-T-12854, but effect of SB-T-12851 was compromised by induced Vegf expression. The very effective SB-T-1214 decreased the expression of Vegf, Egf and all receptors most prominently indicating the possible supporting role of these genes in anti-lymphoma effects. In conclusion, SB-T-1214, SB-T-12854 and IDN5109 are good candidates for further study.

New model system for testing effects of flavonoids on doxorubicin-related formation of hydroxyl radicals.

Doxorubicin belongs to anthracycline cytotoxic drugs and it is widely used as a major therapeutic agent in the treatment of various types of tumors. However,its therapeutic use is limited by the development of myelosuppression and cardiotoxicity after a specific cumulative dose is reached. The aim of this study was to investigate the effect of flavonoids, either natural or synthetic on doxorubicin-mediated formation of oxidative stress implicated in doxorubicin toxicity. Doxorubicin caused a concentration-dependent increase in the formation of hydroxyl radicals in minipig liver microsomes used as an in-vitro model system. When bacterial membranes heterologously expressing human NADPH cytochrome-P450 oxidoreductase were incubated with doxorubicin, formation of the superoxide radical under aerobic conditions and the doxorubicin­semiquinone radical under anaerobic conditions was detected. Forty different flavonoids were tested for their potency to prevent NADPH-induced or Fe2+-induced peroxidation of lipids in the microsomal system. According to the results, seven flavonoids were selected for evaluation of their potency to inhibit doxorubicin-dependent formation of hydroxyl radicals assessed by electron spin resonance. Myricetin, fisetin, and kaempferol were found to produce a significant protective effect against hydroxyl radicals in the minipig liver microsomal system. In conclusion, this study shows the use of a novel cost-effective in-vitro model system for preselection of antioxidants for testing of their protective effects against toxicity of anthracyclines and potentially other oxidative stress-inducing chemicals.

Cell death induced by novel fluorinated taxanes in drug-sensitive and drug-resistant cancer cells.

The aim of this study is to compare the effects of new fluorinated taxanes SB-T-12851, SB-T-12852, SB-T-12853, and SB-T-12854 with those of the classical taxane paclitaxel and novel non-fluorinated taxane SB-T-1216 on cancer cells. Paclitaxel-sensitive MDA-MB-435 and paclitaxel-resistant NCI/ADR-RES human cancer cell lines were used. Cell growth and survival evaluation, colorimetric assessment of caspases activities, flow cytometric analyses of the cell cycle and the assessment of mitochondrial membrane potential, reactive oxygen species (ROS) and the release of cytochrome c from mitochondria were employed. Fluorinated taxanes have similar effects on cell growth and survival. For MDA-MB-435 cells, the C(50) of SB-T-12851, SB-T-12852, SB-T-12853 and SB-T-12854 was 3 nM, 4 nM, 3 nM and 5 nM, respectively. For NCI/ADR-RES cells, the C(50) of SB-T-12851, SB-T-12852, SB-T-12853, and SB-T-12854 was 20 nM, 20 nM, 10 nM and 10 nM, respectively. Selected fluorinated taxanes, SB-T-12853 and SB-T-12854, at the death-inducing concentrations (30 nM for MDA-MB-435 and 300 nM for NCI/ADR-RES) were shown to activate significantly caspase-3, caspase-9, caspase-2 and also slightly caspase-8. Cell death was associated with significant accumulation of cells in the G(2)/M phase. Cytochrome c was not released from mitochondria and other mitochondrial functions were not significantly impaired. The new fluorinated taxanes appear to use the same or similar mechanisms of cell death induction as compared with SB-T-1216 and paclitaxel. New fluorinated and non-fluorinated taxanes are more effective against drug-resistant cancer cells than paclitaxel. Therefore, new generation of taxanes, either non-fluorinated or fluorinated, are excellent candidates for further and detailed studies.

Comparison of cell death-inducing effect of novel taxane SB-T-1216 and paclitaxel in breast cancer cells.

BACKGROUND: In this study, the effect of novel taxane SB-T-1216 and paclitaxel on sensitive MDA-MB-435 and resistant NCI/ADR-RES human breast cancer cells was compared. MATERIALS AND METHODS: Cell growth and survival were evaluated after 96-hour incubation with tested concentrations of taxanes. The effect on the formation of microtubule bundles was assessed employing fluorescence microscopy and on the cell cycle employing flow cytometric analysis. The activity of caspases was assessed employing commercial colorimetric kits. RESULTS: The IC(50) (concentration resulting in 50% of living cells in comparison with the control) of SB-T-1216 in sensitive cells was 0.6 nM versus 1 nM for paclitaxel. However, the IC(50) of SB-T-1216 in resistant cells was 1.8 nM versus 300 nM for paclitaxel. Both SB-T-1216 and paclitaxel at death-inducing concentrations induced the formation of microtubule bundles in sensitive as well as resistant cells. Cell death induced in sensitive and resistant cells by paclitaxel was associated with the accumulation of cells in the G(2)/M phase. On the contrary, cell death induced by SB-T-1216 took place without the accumulation of cells in the G(2)/M phase but with a decreased number of G(1) cells and the accumulation of hypodiploid cells. Both SB-T-1216 and paclitaxel activated caspase-3, caspase-9, caspase-2 and caspase-8 in sensitive as well as resistant cells. CONCLUSION: Cell death induced by both paclitaxel and novel taxane SB-T-1216 in breast cancer cells is associated with caspase activation and with the formation of interphase microtubule bundles. Novel taxane SB-T-1216, but not paclitaxel, seems to be capable of inducing cell death without the accumulation of cells in the G(2)/M phase.

NAD(P)H:quinone oxidoreductase 1 Pro187Ser polymorphism and expression do not cosegregate with clinico-pathological characteristics of human mammary tumors.

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.

Single nucleotide polymorphisms in the multidrug resistance gene 1 (ABCB1): effects on its expression and clinicopathological characteristics in breast cancer patients.

OBJECTIVES: Resistance of tumor cells to multiple cytostatic agents is one of the major impediments of successful cancer chemotherapy. A large part of resistance of tumors to chemotherapy is caused by the ABC transporter P-glycoprotein encoded by the ABCB1 gene. The main aim of this study was to assess the prognostic value of ABCB1 genotype and phenotype in breast cancer. METHODS: Six ABCB1 single nucleotide polymorphisms (SNPs) were determined in 90 Czech breast cancer patients by a novel method that allows simultaneous assessment of multiple polymorphisms on a single electronic microarray. Expression levels of ABCB1 were quantified in tumor and nontumor samples of breast cancer patients by real-time PCR. T-test, analysis of variance and Fisher's exact test were used to analyze the effect of ABCB1 polymorphisms on ABCB1 expression levels and for the analysis of associations between ABCB1 expression, genotype and clinical and pathological characteristics. RESULTS: ABCB1 was expressed in 98.9% of the tumor and in 97.5% of the nontumor samples. ABCB1 was downregulated in 79.5% of tumors compared with the nontumor samples. No significant correlation was observed between ABCB1 mRNA expression levels and clinical and pathological characteristics. High frequencies of the variant alleles in ABCB1 exon 12 (1236C>T, 38.3%) and exon 26 (3435C>T, 54.0%) were observed. Individuals with variant alleles in exons 12 and 26 had significantly lower ABCB1 expression levels in their tumors. SNPs in exons 12 and 26 also correlated with estrogen receptor status of patients. CONCLUSION: ABCB1 SNPs may affect function of P-glycoprotein by influencing the expression level and modify breast cancer prognosis.

The effect of flavonoid derivatives on doxorubicin transport and metabolism.

This study investigated the effect of naturally occurring flavonoids and synthetic aurone derivatives on the formation of cardiotoxic doxorubicinol and transport of doxorubicin in breast cancer cells. Quercetin significantly inhibited the formation of doxorubicinol. Quercetin and aurones did not significantly affect transport of [14C]doxorubicin in human resistant breast cancer cells. In conclusion, quercetin should be further tested for its potency to decrease doxorubicin-mediated toxicity.

RNA expression of cytochrome P450 in breast cancer patients.

BACKGROUND: The expression pattern of cytochrome P450 genes (CYPs) affected by tumorigenesis may have an important role in the progression of cancer and in the metabolism of anticancer drugs. The aim of the study was to determine the expression patterns of four cytochrome P450 genes (CYP1B1, 2C9, 2E1 and 3A4) in breast cancer patients. PATIENTS AND METHODS: mRNA expression was quantified by real-time PCR. Analyses of 40 sets of human breast tumors, adjacent non-tumor tissues and of 18 peripheral blood lymphocyte samples were performed. Expression levels were tested for correlation with clinical and pathological data of patients. RESULTS: Expression levels of CYP2C9 and CYP3A4 were negligible. CYP1B1 expression was on average 50-fold higher than that of CYP2E1 with overexpression detected in one third of the tumors. Correlation of CYP1B1 expression in lymphocytes with that in non-tumor tissues was found. Significantly higher CYP2E1 expression was associated with an invasive lobular type of tumor, locally advanced disease as well as with non-tumor tissue of progesterone receptor-negative patients. CONCLUSION: CYP2E1 expression has a potential role as a breast cancer prognosis marker. The observed high CYP1B1 expression in tumor cells may evoke changes in their response to drugs which are substrates of P450 1B1 and influence metabolism or activation of environmental carcinogens.

Effects of paclitaxel, docetaxel and their combinations on subcutaneous lymphomas in inbred Sprague-Dawley/Cub rats.

We investigated, whether the effects on paclitaxel, docetaxel or their combinations on T-cell lymphomas in Sprague-Dawley/Cub rats were mainly caused by their different efficiency or combination of different mechanism of action, or limited by metabolic inactivation by P450 enzymes or drug efflux caused by P-glycoprotein (P-gp). Docetaxel most effectively prolonged the survival of rats and the time of lymphoma appearance, inhibited their intravital size and weight after sacrifice. Paclitaxel was poorly effective and combined administration had intermediate effects. Blood levels of both drugs were similar. Repeated administration of paclitaxel, but not docetaxel, decreased its area under concentration, but the effect disappeared 6h after dosing and was not sufficient to explain lower effects of paclitaxel. The faster metabolism of docetaxel than paclitaxel in vitro did not limit its higher efficiency and repeated administration of paclitaxel did not induce its metabolism to decrease its blood levels sufficiently. Likewise, undetectable expression of P-gp protein in tumours could not explain lower effects of paclitaxel, which is a better substrate of P-gp. Docetaxel was three-fold more effective than paclitaxel against P388D1 lymphoma cell line, used as a model of the T-cell lymphoma and combined action was dominated by the effects of docetaxel. Thus, docetaxel was effective against T-cell lymphomas and may be a potential anticancer drug in similar indications.

Modulation of paclitaxel transport by flavonoid derivatives in human breast cancer cells. Is there a correlation between binding affinity to NBD of P-gp and modulation of transport?

We have investigated the effect of 13 flavonoid derivatives on [(14)C]paclitaxel transport in two human breast cancer cell lines, the adriamycin-resistant NCI/ADR-RES and sensitive MDA-MB-435. For this study, we selected representatives of aurones, chalcones, flavones, flavonols, chromones, and isoflavones with known binding affinity toward nucleotide-binding domain (NBD2) of P-glycoprotein and for which no reported work is available regarding paclitaxel transport. Aurones CB-284, CB-285, CB-287, and ML-50 most effectively inhibited P-gp related transport in the resistant line in comparison with chalcones, flavones, flavonols, chromones, and isoflavone derivatives and accordingly increased the accumulation of [(14)C]paclitaxel and decreased its efflux. Those agents efficiently modulated paclitaxel transport in P-gp highly expressing resistant human breast cancer cells and they could increase the efficiency of chemotherapy in paclitaxel-resistant tumors. In contrast, the sensitive cell line responded reversely in that CB-284, CB-285, CB-287, and ML-50 significantly inhibited accumulation of [(14)C]paclitaxel and especially CB-287, which significantly stimulated its efflux. Some, but not all, of the data correlated with the binding of flavonoid derivatives to P-gp, and indicated that even in the P-gp highly expressing NCI/ADR-RES cells, the binding was not the only factor influencing the transport of [(14)C]paclitaxel. Opposite effects of flavonoid derivatives on the P-gp highly expressing and MDA-MB-435 non-expressing cell lines indicate that paclitaxel is not only transported by P-gp and let us assume that Mrp2 or ABCC5 seem to be good transport-candidates in these cells. The inhibition of paclitaxel accumulation and stimulation of its efflux are potentially unfavorable for drug therapy and since they could be due to modulation of drug transporters other than P-gp, their expression in tumors is of great significance for efficient chemotherapy.

Transport and cytotoxicity of paclitaxel, docetaxel, and novel taxanes in human breast cancer cells.

The resistance of tumors to classic taxanes (paclitaxel and docetaxel) presents problems in chemotherapy. Thus, new taxanes with higher antitumor activity in resistant tumors are synthesized. This study compared cytotoxicity and transport of paclitaxel and docetaxel with novel taxanes SB-T-1103, SB-T-1214, and SB-T-1216 in adriamycin-sensitive (MDA-MB-435) and -resistant (NCI/ADR-RES) human breast cancer cells. The cell lines examined differ in adriamycin transport, suggesting different expression of ABC membrane transporters. Reverse transcription-polymerase chain reaction revealed that NCI/ADR-RES cells expressed high levels of P-glycoprotein mRNA, which was absent in MDA-MB-435 cells, while the opposite was true for MRP2 mRNA. Both cell lines shared or differently expressed eight other ABC transporters and LRP. NCI/ADR-RES cells were 1,000-fold more resistant to paclitaxel and 600-fold more resistant to docetaxel in MTT assay than MDA-MB-435 cells, but almost equally sensitive to SB-T-1103, SB-T-1214, and SB-T-1216. This complied with the fact that NCI/ADR-RES cells absorbed almost 20-fold less [14C]paclitaxel, about 7-fold less docetaxel, and almost equal amounts of SB-T-1103, SB-T-1214, and SB-T-1216 as the MDA-MB-435 cells. Verapamil increased uptake of [14C]paclitaxel by NCI/ADR-RES cells 7-fold and decreased its efflux 2.5-fold; in contrast, it weakly influenced uptake and increased the efflux in MDA-MB-435 cells. SB-T-1103 and SB-T-1216 did not influence transport of paclitaxel, but SB-T-1214 decreased [14C]paclitaxel uptake in both cell lines indicating inhibition of uptake. This suggests that the novel taxanes are not inhibitors of P-glycoprotein. However, novel taxanes exert much higher activity on resistant tumor cells than classic taxanes and seem to be potential drugs for therapy in taxane-resistant tumors.