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.
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.
Resistance of cancer cells to chemotherapeutic agents is one of the main causes of treatment failure. In order to detect proteins potentially involved in the mechanism of resistance to taxanes, we ...assessed differences in protein expression in MCF-7 breast cancer cells that are sensitive to paclitaxel and in the same cells with acquired resistance to paclitaxel (established in our lab). Proteins were separated using two-dimensional electrophoresis. Changes in their expression were determined and proteins with altered expression were identified using mass spectrometry. Changes in their expression were confirmed using western blot analysis. With these techniques, we found three proteins expressed differently in resistant MCF-7 cells, i.e., thyroid hormone-interacting protein 6 (TRIP6; upregulated to 650%), heat shock protein 27 (HSP27; downregulated to 50%) and cathepsin D (downregulated to 28%). Silencing of TRIP6 expression by specific siRNA leads to decreased number of grown resistant MCF-7 cells. In the present study we have pointed at some new directions in the studies of the mechanism of resistance to paclitaxel in breast cancer cells.
•MCF-7 cells sensitive and resistant to paclitaxel were compared.•2D-electrophoresis was employed to compare their total protein expression.•Protein levels of HSP27 and cathepsin D were decreased in resistant cells.•Protein level of TRIP6 was increased in resistant cells.•Silencing of TRIP6 led to decreased growth of resistant cells.
We studied the role of caspase-2 in apoptosis induction by taxanes (paclitaxel, novel taxane SB-T-1216) in breast cancer cells using SK-BR-3 (nonfunctional p53, functional caspase-3) and MCF-7 ...(functional p53, nonfunctional caspase-3) cell lines.
Both taxanes induced apoptosis in SK-BR-3 as well as MCF-7 cells. Caspase-2 activity in SK-BR-3 cells increased approximately 15-fold within 48 h after the application of both taxanes at the death-inducing concentration (100 nM). In MCF-7 cells, caspase-2 activity increased approximately 11-fold within 60 h after the application of taxanes (300 nM). Caspase-2 activation was confirmed by decreasing levels of procaspase-2, increasing levels of cleaved caspase-2 and the cleavage of caspase-2 substrate golgin-160. The inhibition of caspase-2 expression using siRNA increased the number of surviving cells more than 2-fold in MCF-7 cells, and at least 4-fold in SK-BR-3 cells, 96 h after the application of death-inducing concentration of taxanes. The inhibition of caspase-2 expression also resulted in decreased cleavage of initiator caspases (caspase-8, caspase-9) as well as executioner caspases (caspase-3, caspase-7) in both cell lines after the application of taxanes. In control cells, caspase-2 seemed to be mainly localized in the nucleus. After the application of taxanes, it was released from the nucleus to the cytosol, due to the long-term disintegration of the nuclear envelope, in both cell lines. Taxane application led to some formation of PIDDosome complex in both cell lines within 24 h after the application. After taxane application, p21WAF1/CIP1 expression was only induced in MCF-7 cells with functional p53. However, taxane application did not result in a significant increase of PIDD expression in either SK-BR-3 or MCF-7 cells. The inhibition of RAIDD expression using siRNA did not affect the number of surviving SK-BR-3 and MCF-7 cells after taxane application at all.
Caspase-2 is required, at least partially, for apoptosis induction by taxanes in tested breast cancer cells. We suggest that caspase-2 plays the role of an apical caspase in these cells. Caspase-2 seems to be activated via other mechanism than PIDDosome formation. It follows the release of caspase-2 from the nucleus to the cytosol.
Resistance to chemotherapeutics is a widespread phenomenon in cancer cells that may counteract the successful therapy of many patients. In resistant cells, higher level of ABC transporters, among ...others, often can be detected. This high level of ABC transporters represents a suspected mechanism of acquired cancer resistance. We studied the molecular mechanism of resistance to taxanes in cancer cells using SK-BR-3 and MCF-7 breast cancer cell lines. We analyzed the effect of paclitaxel on apoptosis induction in the originally sensitive cells of these lines as compared to their counterpart resistant cells, developed by gradual adaptation to paclitaxel. In resistant cells of the SK-BR-3 and MCF-7 lines, we did not detected ongoing induction of apoptosis but we did detect significantly increased expression of ABCB1 transporter after paclitaxel application. By silencing the expression of the transport via employment of small interfering RNA (siRNA), we tested the role of the ABCB1 transporter in cells resistant to paclitaxel. We found that resistant cells with silenced expression of the ABCB1 transporter had a statistically significant increase of sensitivity to paclitaxel as compared to control resistant cells with high expression of this transporter. Along with increased sensitivity, we demonstrated...
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