Background:The most aggressive forms of breast cancer are characterized by independence from steroid hormones but a strong dependence on growth factors.In such cancer cells,oncogenic receptors,including human epiderma...Background:The most aggressive forms of breast cancer are characterized by independence from steroid hormones but a strong dependence on growth factors.In such cancer cells,oncogenic receptors,including human epidermal growth factor receptor 2(HER2),are activated,and their targeted inhibition represents an attractive therapeutic strategy.The study aimed to develop small-molecule potential dual heat shock protein 90(HSP90)-HER2 inhibitors and evaluate them as anticancer agents in HER2-positive cells.Methods:The research project involved obtaining a series of compounds with potential dual inhibitory activity against HSP90 and HER2 by targeted organic synthesis,which was preliminarily assessed using molecular modelling and calculation of key parameters of molecular dynamics.The potential therapeutic benefit of the obtained molecules was studied using basic molecular biological methods,including assessment of cytotoxic activity in vitro using the MTT test,as well as determination of a possible mechanism of action based on the expression of key participants in intracellular signaling(western blotting).Additionally,therapeutic combinations were developed and tested on a cellularmodel of the disease,including a lead compound and chemotherapeutic drugs used in clinical practice,in order to find synergistic pairs and improve the effectiveness of the treatment.Results:In this work,novel dual HSP90-HER2 inhibitors,based on the fused thiazole-dihydrobenzisoxazole polycyclic scaffold,were designed and synthesized.The resulting compounds exhibited strong antiproliferative activity against HER2-positive breast cancer cells with high selectivity.Among them,ATF-2 demonstrated antiproliferative activity comparable to HER2 inhibitor lapatinib and significantly suppressed HER2 expression and activity,epidermal growth factor receptor(EGFR)activity,and cyclin-dependent kinase 6(CDK6)expression in HCC1954 breast cancer cells.Conclusion:These findings highlight ATF-2 as a promising dual HSP90-HER2 inhibitor with broader inhibitory effects on the HER2,EGFR,and CDK6 pathways.展开更多
Aim:The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen(HT)on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods:The anal...Aim:The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen(HT)on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods:The analysis of cell viability was performed by the MTT method.The expression of signaling proteins was analyzed by immunoblotting and flow cytometry.ERαactivity was evaluated by gene reporter assay.To establish hormone-resistant subline MCF7,breast cancer cells were treated with 4-hydroxytamoxifen for 12 months.Results:The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen,and the resistance index was 2.Increased Akt activity(2.2-fold)and decreased ERαexpression(1.5-fold)were revealed in MCF7/HT cells.The activity of the estrogen receptorαwas reduced(1.5-fold)in MCF7/HT.Evaluation of class Ⅲβ-tubulin expression(TUBB3),a marker associated with metastasis,revealed the following trends:higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells(P<0.05).The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells(MCF7/HT<MCF7<MDA-MB-231,approximately 1:2:4).High TUBB3 expression strongly correlated with docetaxel resistance:IC_(50)value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells,whereas resistant MCF7/HT cells were the most sensitive to the drug.The accumulation of cleaved PARP(a 1.6-fold increase)and Bcl-2 downregulation(1.8-fold)were more pronounced in docetaxel-treated resistant cells(P<0.05).The expression of cyclin D1 decreased(2.8-fold)only in resistant cells after 4 nM docetaxel treatment,while this marker was unchanged in parental MCF7 breast cancer cells.Conclusion:Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising,especially for cancers with low TUBB3 expression.展开更多
Aim:Resistance to hormonal and targeted therapies in breast cancer limits treatment efficacy.Epigenetic alterations,including changes mediated by DNA methyltransferases,play a key role in this process.Previously,we id...Aim:Resistance to hormonal and targeted therapies in breast cancer limits treatment efficacy.Epigenetic alterations,including changes mediated by DNA methyltransferases,play a key role in this process.Previously,we identified that resistance to tamoxifen and rapamycin is associated with the suppression of DNMT3A.This study aims to further explore the mechanisms underlying this suppression,with a focus on identifying NR6A1 as a novel regulatory factor.Methods:Acquisition of resistant breast cancer cell sublines,MTT-test,immunoblotting,transient transfection and reporter analysis,lentiviral infection,qRT-PCR,and analysis of methylation using bisulfite pyrosequencing.Results:Our findings indicate that the development of cross-resistance in breast cancer cells to hormonal and targeted therapies involves a shift in cell signaling to alternative AKT pathways,marked by a localized suppression of the NR6A1/DNMT3A axis and associated DNA methylation changes.We demonstrated the critical role of NR6A1 downregulation in resistance development.Additionally,we observed activation of Snail-a key regulator in the epithelial-mesenchymal transition-as a mediator of the effects of NR6A1 depletion,establishing a direct link between Snail expression and resistance formation.Conclusion:The coordinated suppression of NR6A1 and DNMT3A may contribute to sustaining the resistant phenotype in breast cancer cells.This pathway could serve as a predictive marker,helping guide the selection of optimal therapeutic strategies for breast cancer treatment in the future.展开更多
基金funded by the Belarusian Republican Foundation for Fundamental Research(BRFFR project X22MC-030,chemical synthesis)the Russian Science Foundation(grant number 24-15-00273,in vitro investigation of HSP90 signaling).
文摘Background:The most aggressive forms of breast cancer are characterized by independence from steroid hormones but a strong dependence on growth factors.In such cancer cells,oncogenic receptors,including human epidermal growth factor receptor 2(HER2),are activated,and their targeted inhibition represents an attractive therapeutic strategy.The study aimed to develop small-molecule potential dual heat shock protein 90(HSP90)-HER2 inhibitors and evaluate them as anticancer agents in HER2-positive cells.Methods:The research project involved obtaining a series of compounds with potential dual inhibitory activity against HSP90 and HER2 by targeted organic synthesis,which was preliminarily assessed using molecular modelling and calculation of key parameters of molecular dynamics.The potential therapeutic benefit of the obtained molecules was studied using basic molecular biological methods,including assessment of cytotoxic activity in vitro using the MTT test,as well as determination of a possible mechanism of action based on the expression of key participants in intracellular signaling(western blotting).Additionally,therapeutic combinations were developed and tested on a cellularmodel of the disease,including a lead compound and chemotherapeutic drugs used in clinical practice,in order to find synergistic pairs and improve the effectiveness of the treatment.Results:In this work,novel dual HSP90-HER2 inhibitors,based on the fused thiazole-dihydrobenzisoxazole polycyclic scaffold,were designed and synthesized.The resulting compounds exhibited strong antiproliferative activity against HER2-positive breast cancer cells with high selectivity.Among them,ATF-2 demonstrated antiproliferative activity comparable to HER2 inhibitor lapatinib and significantly suppressed HER2 expression and activity,epidermal growth factor receptor(EGFR)activity,and cyclin-dependent kinase 6(CDK6)expression in HCC1954 breast cancer cells.Conclusion:These findings highlight ATF-2 as a promising dual HSP90-HER2 inhibitor with broader inhibitory effects on the HER2,EGFR,and CDK6 pathways.
基金supported by the Ministry of Science and Higher Education of the Russian Federation(agreement No.075-15-2020-789).
文摘Aim:The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen(HT)on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods:The analysis of cell viability was performed by the MTT method.The expression of signaling proteins was analyzed by immunoblotting and flow cytometry.ERαactivity was evaluated by gene reporter assay.To establish hormone-resistant subline MCF7,breast cancer cells were treated with 4-hydroxytamoxifen for 12 months.Results:The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen,and the resistance index was 2.Increased Akt activity(2.2-fold)and decreased ERαexpression(1.5-fold)were revealed in MCF7/HT cells.The activity of the estrogen receptorαwas reduced(1.5-fold)in MCF7/HT.Evaluation of class Ⅲβ-tubulin expression(TUBB3),a marker associated with metastasis,revealed the following trends:higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells(P<0.05).The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells(MCF7/HT<MCF7<MDA-MB-231,approximately 1:2:4).High TUBB3 expression strongly correlated with docetaxel resistance:IC_(50)value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells,whereas resistant MCF7/HT cells were the most sensitive to the drug.The accumulation of cleaved PARP(a 1.6-fold increase)and Bcl-2 downregulation(1.8-fold)were more pronounced in docetaxel-treated resistant cells(P<0.05).The expression of cyclin D1 decreased(2.8-fold)only in resistant cells after 4 nM docetaxel treatment,while this marker was unchanged in parental MCF7 breast cancer cells.Conclusion:Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising,especially for cancers with low TUBB3 expression.
基金supported by the Russian Science Foundation(project No.24-15-00173,https://rscf.ru/project/24-15-00173/).
文摘Aim:Resistance to hormonal and targeted therapies in breast cancer limits treatment efficacy.Epigenetic alterations,including changes mediated by DNA methyltransferases,play a key role in this process.Previously,we identified that resistance to tamoxifen and rapamycin is associated with the suppression of DNMT3A.This study aims to further explore the mechanisms underlying this suppression,with a focus on identifying NR6A1 as a novel regulatory factor.Methods:Acquisition of resistant breast cancer cell sublines,MTT-test,immunoblotting,transient transfection and reporter analysis,lentiviral infection,qRT-PCR,and analysis of methylation using bisulfite pyrosequencing.Results:Our findings indicate that the development of cross-resistance in breast cancer cells to hormonal and targeted therapies involves a shift in cell signaling to alternative AKT pathways,marked by a localized suppression of the NR6A1/DNMT3A axis and associated DNA methylation changes.We demonstrated the critical role of NR6A1 downregulation in resistance development.Additionally,we observed activation of Snail-a key regulator in the epithelial-mesenchymal transition-as a mediator of the effects of NR6A1 depletion,establishing a direct link between Snail expression and resistance formation.Conclusion:The coordinated suppression of NR6A1 and DNMT3A may contribute to sustaining the resistant phenotype in breast cancer cells.This pathway could serve as a predictive marker,helping guide the selection of optimal therapeutic strategies for breast cancer treatment in the future.
