Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estroge...Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.展开更多
To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly ...To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.展开更多
A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function ...A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.展开更多
The purpose of this study was to verify that a combination of mild hyperthermia and docetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach.The eff...The purpose of this study was to verify that a combination of mild hyperthermia and docetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach.The effects of docetaxel on the proliferation of cells from the estrogen receptor(ER)-positive human breast cancer cell line MCF-7 and the ER-negative human breast cancer cell line MDA-MB-453 were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,and effective experimental concentrations of docetaxel were determined.The effects of mild hyperthermia plus docetaxel therapy on apoptosis rate in the MCF-7 and MDA-MB-453 human breast cancer cell lines were analyzed by using flow cytometry with Annexin-V fluorescein isothiocyanate(FITC)/propidium iodide(PI)staining.The effects of these combined treatments on cell cycle progression in the MCF-7 and MDA-MB-453 human breast cancer cell lines were examined by using flow cytometry.The effects of these combined treatments on the expression of apoptosis-related proteins and proteins in the mitogen-activated protein kinase(MAPK)pathways were analyzed by using Western blotting.The effects of these combined treatments on the expression of the heat shock protein 70(HSP70)and the multi-drug resistance(MDR)gene product P-glycoprotein(Pgp)were examined by using Western blotting.The results showed that the half-maximal inhibitory concentration(IC50)of docetaxel for MCF-7 and MDA-MB-453 cells was 19.57±1.12 and 21.64±2.31μmol/L respectively.Mild hyperthermia with docetaxel therapy could increase apoptosis rate in the MCF-7 and MDA-MB-453cells.Apoptosis rate in MCF-7 and MDA-MB-453 cells was increased from(23.66±3.59)%and(18.51±3.17)%in docetaxel treatment group to(47.12±6.73)%and(55.16±7.42)%in mild hyperthermia plus docetaxel group,indicating that the mild hyperthermia and docetaxel therapeutic approaches exhibited significant synergistic antitumor effects.Treatments of mild hyperthermia plus docetaxel induced G2/M cell cycle arrest in the MCF-7 and MDA-MB-453 cells.Western blotting demonstrated that proteins in the MAPK pathway were expressed at higher levels in docetaxel-treated cells following mild hypothermia than those in cells treated with docetaxel alone.As compared with blank control group,cells from the mild hyperthermia plus docetaxel group exhibited significantly decreased B-cell lymphoma 2(Bcl-2)protein expression but slightly increased Bcl-2-associated X protein(Bax)expression.Western blotting results revealed that HSP70 and Pgp expression levels were significantly increased following mild hypothermia.It was concluded that treatments of mild hyperthermia plus docetaxel inhibited the proliferation of human breast cancer cells,promoted apoptosis of breast cancer cells,and produced synergistic antitumor effects.展开更多
文摘Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.
文摘To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.
基金This research was supported by the National Natural ScienceYouth Grant.
文摘A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.
文摘The purpose of this study was to verify that a combination of mild hyperthermia and docetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach.The effects of docetaxel on the proliferation of cells from the estrogen receptor(ER)-positive human breast cancer cell line MCF-7 and the ER-negative human breast cancer cell line MDA-MB-453 were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,and effective experimental concentrations of docetaxel were determined.The effects of mild hyperthermia plus docetaxel therapy on apoptosis rate in the MCF-7 and MDA-MB-453 human breast cancer cell lines were analyzed by using flow cytometry with Annexin-V fluorescein isothiocyanate(FITC)/propidium iodide(PI)staining.The effects of these combined treatments on cell cycle progression in the MCF-7 and MDA-MB-453 human breast cancer cell lines were examined by using flow cytometry.The effects of these combined treatments on the expression of apoptosis-related proteins and proteins in the mitogen-activated protein kinase(MAPK)pathways were analyzed by using Western blotting.The effects of these combined treatments on the expression of the heat shock protein 70(HSP70)and the multi-drug resistance(MDR)gene product P-glycoprotein(Pgp)were examined by using Western blotting.The results showed that the half-maximal inhibitory concentration(IC50)of docetaxel for MCF-7 and MDA-MB-453 cells was 19.57±1.12 and 21.64±2.31μmol/L respectively.Mild hyperthermia with docetaxel therapy could increase apoptosis rate in the MCF-7 and MDA-MB-453cells.Apoptosis rate in MCF-7 and MDA-MB-453 cells was increased from(23.66±3.59)%and(18.51±3.17)%in docetaxel treatment group to(47.12±6.73)%and(55.16±7.42)%in mild hyperthermia plus docetaxel group,indicating that the mild hyperthermia and docetaxel therapeutic approaches exhibited significant synergistic antitumor effects.Treatments of mild hyperthermia plus docetaxel induced G2/M cell cycle arrest in the MCF-7 and MDA-MB-453 cells.Western blotting demonstrated that proteins in the MAPK pathway were expressed at higher levels in docetaxel-treated cells following mild hypothermia than those in cells treated with docetaxel alone.As compared with blank control group,cells from the mild hyperthermia plus docetaxel group exhibited significantly decreased B-cell lymphoma 2(Bcl-2)protein expression but slightly increased Bcl-2-associated X protein(Bax)expression.Western blotting results revealed that HSP70 and Pgp expression levels were significantly increased following mild hypothermia.It was concluded that treatments of mild hyperthermia plus docetaxel inhibited the proliferation of human breast cancer cells,promoted apoptosis of breast cancer cells,and produced synergistic antitumor effects.
