Development of taxane resistance has become clinically very important issue. The molecular mechanisms underlying the resistance are still unclear. To address this issue, we established ...paclitaxel-resistant sublines of the SK-BR-3 and MCF-7 breast cancer cell lines that are capable of long-term proliferation in 100nM and 300nM paclitaxel, respectively. Application of these concentrations leads to cell death in the original counterpart cells. Both sublines are cross-resistant to doxorubicin, indicating the presence of the MDR phenotype. Interestingly, resistance in both paclitaxel-resistant sublines is circumvented by the second-generation taxane SB-T-1216. Moreover, we demonstrated that it was not possible to establish sublines of SK-BR-3 and MCF-7 cells resistant to this taxane. It means that at least the tested breast cancer cells are unable to develop resistance to some taxanes.
Employing mRNA expression profiling of all known human ABC transporters and subsequent Western blot analysis of the expression of selected transporters, we demonstrated that only the ABCB1/PgP and ABCC3/MRP3 proteins were up-regulated in both paclitaxel-resistant sublines. We found up-regulation of ABCG2/BCRP and ABCC4 proteins only in paclitaxel-resistant SK-BR-3 cells. In paclitaxel-resistant MCF-7 cells, ABCB4/MDR3 and ABCC2/MRP2 proteins were up-regulated. Silencing of ABCB1 expression using specific siRNA increased significantly, but did not completely restore full sensitivity to both paclitaxel and doxorubicin. Thus we showed a key, but not exclusive, role for ABCB1 in mechanisms of paclitaxel resistance. It suggests the involvement of multiple mechanisms in paclitaxel resistance in tested breast cancer cells.
•Expression of all ABC transporters in paclitaxel-resistant sublines of SK-BR-3 and MCF-7 cells was analyzed.•SK-BR-3 and MCF-7 cells are unable to develop resistance to some taxanes.•Some taxanes are able to overcome developed resistance to paclitaxel.•Paclitaxel resistance was associated with increased levels of ABCB1 and ABCC3 protein.•ABCB1 silencing increased significantly sensitivity to both paclitaxel and doxorubicin.
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 main problem precluding successful therapy with conventional taxanes is de novo or acquired resistance to taxanes. Therefore, novel experimental taxane derivatives (Stony Brook taxanes; SB-Ts) ...are synthesized and tested as potential drugs against resistant solid tumors. Recently, we reported alterations in
,
, and
gene expression in a breast cancer cell line resistant to paclitaxel. The present study aimed to investigate gene expression changes of these three candidate molecules in the highly resistant ovarian carcinoma cells in vitro and corresponding in vivo models treated with paclitaxel and new experimental Stony Brook taxanes of the third generation (SB-T-121605 and SB-T-121606). We also addressed their prognostic meaning in ovarian carcinoma patients treated with taxanes. We estimated and observed changes in mRNA and protein profiles of ABCC3, CPS1, and TRIP6 in resistant and sensitive ovarian cancer cells and after the treatment of resistant ovarian cancer models with paclitaxel and Stony Brook taxanes in vitro and in vivo. Combining Stony Brook taxanes with paclitaxel caused downregulation of CPS1 in the paclitaxel-resistant mouse xenograft tumor model in vivo. Moreover, CPS1 overexpression seems to play a role of a prognostic biomarker of epithelial ovarian carcinoma patients' poor survival. ABCC3 was overexpressed in EOC tumors, but after the treatment with taxanes, its up-regulation disappeared. Based on our results, we can suggest ABCC3 and CPS1 for further investigations as potential therapeutic targets in human cancers.
The most commonly used flow cytometric (FCM) analysis of cellular DNA content relies on ethanol fixation followed by RNA digestion and propidium iodide (PI) intercalation into double‐stranded DNA. ...This is a laborious and time‐consuming procedure that is subject to systematic errors due to centrifugation and washing steps associated with sample preparation. It can adversely affect the reliability of the results. Here, we present a modified concept of DNA quantification in adherent cell lines by FCM that involves neither ethanol fixation nor any washing and cell transferring steps. Our high throughput assay of adherent cell lines reduces sample‐processing time, requires minimal workload, provides a possibility for automation, and, if needed, also allows a significant reduction in the size of individual samples. Working with a well‐proven commercial tool—The BD Cycletest™ Plus DNA Reagent Kit—primarily designed for cell cycle analysis and aneuploidy determination in experimental and clinical samples, we suggest a novel, very efficient, and robust approach for DNA research in adherent cell cultures.
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.
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.
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.
ABCA12, ABCA13, ABCC1, ABCC8 and ABCD2 present potential modifiers of progression and response to the chemotherapy of breast carcinoma.
Taxanes are widely used in the treatment of ovarian carcinomas. One of the main problems with conventional taxanes is the risk of development of multidrug resistance. New-generation synthetic ...experimental taxoids (Stony Brook Taxanes; SB-T) have shown promising effects against various resistant tumor models. The aim of our study was to compare the
in vitro
efficacy, intracellular content, and
in vivo
antitumor effect of clinically used paclitaxel (PTX) and SB-Ts from the previously tested second (SB-T-1214, SB-T-1216) and the newly synthesized third (SB-T-121402, SB-T-121605, and SB-T-121606) generation in PTX resistant ovarian carcinoma cells NCI/ADR-RES. The efficacy of the new SB-Ts was up to 50-times higher compared to PTX in NCI/ADR-RES cells
in vitro
. SB-T-121605 and SB-T-121606 induced cell cycle arrest in the G2/M phase much more effectively and their intracellular content was 10–15-times higher, when compared to PTX. Incorporation of SB-T-121605 and SB-T-121606 into therapeutic regimens containing PTX were effective in suppressing tumor growth
in vivo
in NCI/ADR-RES based mice xenografts at small doses (≤3 mg/kg), where their adverse effects were eliminated. In conclusion, new SB-T-121605 and SB-T-121606 analogs are promising candidates for the next phase of preclinical testing of their combination therapy with conventional taxanes in resistant ovarian carcinomas.
Pancreatic cancer is a severe malignancy with increasing incidence and high mortality due to late diagnosis and low sensitivity to treatments. Search for the most appropriate drugs and therapeutic ...regimens is the most promising way to improve the treatment outcomes of the patients. This study aimed to compare (1) in vitro efficacy and (2) in vivo antitumor effects of conventional paclitaxel and the newly synthesized second (SB-T-1216) and third (SB-T-121605 and SB-T-121606) generation taxanes in KRAS wild type BxPC-3 and more aggressive KRAS G12V mutated Paca-44 pancreatic cancer cell line models. In vitro, paclitaxel efficacy was 27.6 ± 1.7 nM, while SB-Ts showed 1.7–7.4 times higher efficacy. Incorporation of SB-T-121605 and SB-T-121606 into in vivo therapeutic regimens containing paclitaxel was effective in suppressing tumor growth in Paca-44 tumor-bearing mice at small doses (≤3 mg/kg). SB-T-121605 and SB-T-121606 in combination with paclitaxel are promising candidates for the next phase of preclinical testing.
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•Study explored effects of experimental taxanes in pancreatic cancer models•Stony Brook taxanes SB-T-1216, 121605, and 121606 were cytostatic in vitro•Mechanism was comparable in Paca-44 KRAS G12V mutated and BxPC-3 wild type in vitro•The combination of SB-T-121606 with paclitaxel had the best antitumor effect in vivo
Pharmacology; Cell biology; Cancer
Metabolism of anticancer drugs affects their antitumor effects. This study has investigated the associations of gene expression of enzymes metabolizing anticancer drugs with therapy response and ...survival of breast carcinoma patients. Gene expression of 13 aldo-keto reductases (AKRs), carbonyl reductase 1, and 10 cytochromes P450 (CYPs) was assessed using quantitative real-time polymerase chain reaction in tumors and paired adjacent nonneoplastic tissues from 68 posttreatment breast carcinoma patients. Eleven candidate genes were then evaluated in an independent series of 50 pretreatment patients. Protein expression of the most significant genes was confirmed by immunoblotting. AKR1A1 was significantly overexpressed and AKR1C1-4, KCNAB1, CYP2C19, CYP3A4, and CYP3A5 downregulated in tumors compared with control nonneoplastic tissues after correction for multiple testing. Significant association of CYP2B6 transcript levels in tumors with expression of hormonal receptors was found in the posttreatment set and replicated in the pretreatment set of patients. Significantly higher intratumoral levels of AKR1C1, AKR1C2, or CYP2W1 were found in responders to neoadjuvant chemotherapy compared with nonresponders. Patients with high AKR7A3 or CYP2B6 levels in the pretreatment set had significantly longer disease-free survival than patients with low levels. Protein products of AKR1C1, AKR1C2, AKR7A3, CYP3A4, and carbonyl reductase (CBR1) were found in tumors and those of AKR1C1, AKR7A3, and CBR1 correlated with their transcript levels. Small interfering RNA-directed knockdown of AKR1C2 or vector-mediated upregulation of CYP3A4 in MDA-MB-231 model cell line had no effect on cell proliferation after paclitaxel treatment in vitro. Prognostic and predictive roles of drug-metabolizing enzymes strikingly differ between posttreatment and pretreatment breast carcinoma patients. Mechanisms of action of AKR1C2, AKR7A3, CYP2B6, CYP3A4, and CBR1 should continue to be further followed in breast carcinoma patients and models.
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.
Oxysterols play significant roles in many physiological and pathological processes including cancer. They modulate some of the cancer hallmarks pathways, influence the efficacy of anti-cancer drugs, ...and associate with patient survival. In this study, we aimed to analyze the role of 7-ketocholesterol (7-KC) in breast carcinoma cells and its potential modulation of the tamoxifen effect. 7-KC effects were studied in two estrogen receptor (ER)-positive (MCF-7 and T47D) and one ER-negative (BT-20) breast cancer cell lines. First, we tested the viability of cells in the presence of 7-KC. Next, we co-incubated cells with tamoxifen and sublethal concentrations of 7-KC. We also tested changes in caspase 3/7 activity, deregulation of the cell cycle, and changes in expression of selected genes/proteins in the presence of tamoxifen, 7-KC, or their combination. Finally, we analyzed the effect of 7-KC on cellular migration and invasion. We found that the presence of 7-KC slightly decreases the efficacy of tamoxifen in MCF-7 cells, while an increased effect of tamoxifen and higher caspase 3/7 activity was observed in the BT-20 cell line. In the T47D cell line, we did not find any modulation of tamoxifen efficacy by the presence of 7-KC. Expression analysis showed the deregulation in CYP1A1 and CYP1B1 with the opposite trend in MCF-7 and BT-20 cells. Moreover, 7-KC increased cellular migration and invasion potential regardless of the ER status. This study shows that 7-KC can modulate tamoxifen efficacy as well as cellular migration and invasion, making 7-KC a promising candidate for future studies.
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•We studied effects of 7-ketocholesterol in tamoxifen-treated breast cancer cells.•Tamoxifen efficacy modulation by 7-KC is estrogen receptor dependent.•Synergy between tamoxifen and 7-KC is limited to ER-negative cells.•CYP1B1 expression is induced by TAM in ER-positive and reduced by 7-KC in ER-negative cells.•7-KC increases migration and invasion of cells regardless of the ER status.
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