The aim of this study is to investigate the anti-proliferative effects of Zebularine on caspase-3 and 9 genes by methylation and protein expression on SKBR3 cells. The SKBR3 cells were treated with Zebularine at diffe...The aim of this study is to investigate the anti-proliferative effects of Zebularine on caspase-3 and 9 genes by methylation and protein expression on SKBR3 cells. The SKBR3 cells were treated with Zebularine at different concentrations (0 - 140 μM) for 24 - 96 hours and the effects on the cell viability were shown. The effects on the cell migration and cell transformation were also demonstrated at the specified dose (IC50 = 40 μM). At the same time, the HRM method and western-blot analysis were used to understand the effects in the apoptotic mechanism. According to the obtained results, it was observed that Zebularine significantly decreased the cell proliferation, cell migration and the cell growth (p < 0.001). As a result of the methylation analyzes performed on SKBR-3 cells at the applied dose, it was observed that Zebularine caused a decrease in both caspase-3 and 9 methylation rates at the 72nd hour. However, both reductions in the methylation levels didn’t cause a significant change (p > 0.05). At the same time, it was detected by the western-blot analyses that there was a time dependent increase in caspase-3 genes while a decrease was detected in the caspase-9 gene in progress of time. The obtained results showed that the methylation changes occurring in the caspase genes weren’t related to the protein levels. According to these results, supportive results are obtained showing that the Zebularine can be used in chemotherapy and that this study is unique since it is the first study in the literature intending to investigate the effects of Zebularine on the SKBR3 cells.展开更多
Background and Aim: In this study, it was aimed to examine the cytotoxic effect of temozolomide (TMZ) treatment, on MCF-7 and SKBR3 cell lines, to study the methylation levels of MGMT gene expression and gene promoter...Background and Aim: In this study, it was aimed to examine the cytotoxic effect of temozolomide (TMZ) treatment, on MCF-7 and SKBR3 cell lines, to study the methylation levels of MGMT gene expression and gene promoter region. Methods: The MTT test was performed to determine the effective dose of TMZ. The time-dependent cell survival test was performed after the IC50 value was found. Western blotting was performed to determine MGMT gene expression levels. High Resolution Melting (HRM) technique was used to determine the methylation levels of MGMT gene promoter region. Results: TMZ has been shown to have a high cytotoxic effect on SKBR3 cell line and low cytotoxicity on MCF-7. When MGMT expression levels before and after TMZ treatment were observed by western blotting, the gene expression levels of TMZ treatment were shown to decrease in both cell lines. It was observed that MGMT gene promoter region was hypermethylated in two cell lines, and that the application of TMZ further increased the methylation levels in the promoter region. Conclusions: It was seen that TMZ could be used as a single agent in SKBR-3 cell line. With this study on breast cancer, it is expected that temozolomide treatment will lead future in vitro and in vivo studies for breast cancer.展开更多
OBJECTIVE： To test the synergistic effects of the aqueous extract of Tubeimu （Rhizoma Bolbosternmatis） and Fuzi （Radix Aconiti Lateralis Preparata） on MDA-MB-231 and SKBR3 breast cancer cells.METHODS： A combined...OBJECTIVE： To test the synergistic effects of the aqueous extract of Tubeimu （Rhizoma Bolbosternmatis） and Fuzi （Radix Aconiti Lateralis Preparata） on MDA-MB-231 and SKBR3 breast cancer cells.METHODS： A combined index was created for the effects of Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts. Cell proliferation was performed by trypan blue exclusion and 3-（4,5-dimethylthiazol-2-yl）-5-（3- carboxy- methoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） assays. Flow cytometry was used to assess cell cycle distribution and apoptosis. Cell migration was determined by wound-healing and transwell assays. Confocal microscopy was used to detect E-cadherin and actin filaments. RESULTS： The aqueous extract from Tubeimu （Rhizoma Bolbostemmatis）and Fuzi （Radix Aconiti lateralis Preparata） exerted synergetic effects on the growth of MDA-MB-231 cells and G1 phase arrest. When exposed to extracts at concentrations of 62.5 ：62.5 and 62.5：31.3 μg/mL, the combination index was 0.83 and 0.74, respectively. Interestingly, 62.5： 31.3 μg/mL of combined drugs enhanced the inhibitory effect of Tubeimu （Rhizoma Bolbostemmatis） on the migration of SKBR3 cells and reduced the stimulative effect of Fuzi （Radix Aconiti Lateralis Preparata） （P 〈 0.01）, in which cells showed an increased expression of E-cadherin and reorganization of actin filaments （P 〈 0.001）. 62.5：62.5 μg/mL extract also synergistically induced apoptosis of MDA-MB-231 cells （P 〈 0.05 and P 〈 0.001）. Acting as the main active ingredients in the extract, tubeimoside I and acetylbenzoylaconine at 10：10μg/ mL and 5：2.5 μg/mL also produced inhibitory effects on the proliferation and migration of cells （P 〈 0.01）. CONCLUSION： Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts had synergic effects on MDA-MB-231 and SKBR3 cells.展开更多
文摘The aim of this study is to investigate the anti-proliferative effects of Zebularine on caspase-3 and 9 genes by methylation and protein expression on SKBR3 cells. The SKBR3 cells were treated with Zebularine at different concentrations (0 - 140 μM) for 24 - 96 hours and the effects on the cell viability were shown. The effects on the cell migration and cell transformation were also demonstrated at the specified dose (IC50 = 40 μM). At the same time, the HRM method and western-blot analysis were used to understand the effects in the apoptotic mechanism. According to the obtained results, it was observed that Zebularine significantly decreased the cell proliferation, cell migration and the cell growth (p < 0.001). As a result of the methylation analyzes performed on SKBR-3 cells at the applied dose, it was observed that Zebularine caused a decrease in both caspase-3 and 9 methylation rates at the 72nd hour. However, both reductions in the methylation levels didn’t cause a significant change (p > 0.05). At the same time, it was detected by the western-blot analyses that there was a time dependent increase in caspase-3 genes while a decrease was detected in the caspase-9 gene in progress of time. The obtained results showed that the methylation changes occurring in the caspase genes weren’t related to the protein levels. According to these results, supportive results are obtained showing that the Zebularine can be used in chemotherapy and that this study is unique since it is the first study in the literature intending to investigate the effects of Zebularine on the SKBR3 cells.
基金supported by Scientific and Technological Research Council of Turkey(TUBITAK),“2209/A-University Students Research Projects Support Program”(Grant No.1919B011601865).
文摘Background and Aim: In this study, it was aimed to examine the cytotoxic effect of temozolomide (TMZ) treatment, on MCF-7 and SKBR3 cell lines, to study the methylation levels of MGMT gene expression and gene promoter region. Methods: The MTT test was performed to determine the effective dose of TMZ. The time-dependent cell survival test was performed after the IC50 value was found. Western blotting was performed to determine MGMT gene expression levels. High Resolution Melting (HRM) technique was used to determine the methylation levels of MGMT gene promoter region. Results: TMZ has been shown to have a high cytotoxic effect on SKBR3 cell line and low cytotoxicity on MCF-7. When MGMT expression levels before and after TMZ treatment were observed by western blotting, the gene expression levels of TMZ treatment were shown to decrease in both cell lines. It was observed that MGMT gene promoter region was hypermethylated in two cell lines, and that the application of TMZ further increased the methylation levels in the promoter region. Conclusions: It was seen that TMZ could be used as a single agent in SKBR-3 cell line. With this study on breast cancer, it is expected that temozolomide treatment will lead future in vitro and in vivo studies for breast cancer.
基金Supported by Funding Program of(National Science Foundation of China(Mechanism of Warming and Relieving Cold Phlegm Therapy Inhibiting Tumor Metastasis from the Field of Biomechanical,No.81503383)Science and Technology Development of Tianjin Municipal Higher Education(Compatibility Principle of Warming and Relieving Cold Phlegm Formula for the Treatment of Human Hepatocellular Carcinoma,No.20110105)Tianjin city application basis and cutting-edge technology research program(Effect of Herbs Formula Radix Aconiti Praeparata-Rhizoma Bolbostemmatis on Breast Cancer,No.13JCQNJC13800)
文摘OBJECTIVE： To test the synergistic effects of the aqueous extract of Tubeimu （Rhizoma Bolbosternmatis） and Fuzi （Radix Aconiti Lateralis Preparata） on MDA-MB-231 and SKBR3 breast cancer cells.METHODS： A combined index was created for the effects of Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts. Cell proliferation was performed by trypan blue exclusion and 3-（4,5-dimethylthiazol-2-yl）-5-（3- carboxy- methoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） assays. Flow cytometry was used to assess cell cycle distribution and apoptosis. Cell migration was determined by wound-healing and transwell assays. Confocal microscopy was used to detect E-cadherin and actin filaments. RESULTS： The aqueous extract from Tubeimu （Rhizoma Bolbostemmatis）and Fuzi （Radix Aconiti lateralis Preparata） exerted synergetic effects on the growth of MDA-MB-231 cells and G1 phase arrest. When exposed to extracts at concentrations of 62.5 ：62.5 and 62.5：31.3 μg/mL, the combination index was 0.83 and 0.74, respectively. Interestingly, 62.5： 31.3 μg/mL of combined drugs enhanced the inhibitory effect of Tubeimu （Rhizoma Bolbostemmatis） on the migration of SKBR3 cells and reduced the stimulative effect of Fuzi （Radix Aconiti Lateralis Preparata） （P 〈 0.01）, in which cells showed an increased expression of E-cadherin and reorganization of actin filaments （P 〈 0.001）. 62.5：62.5 μg/mL extract also synergistically induced apoptosis of MDA-MB-231 cells （P 〈 0.05 and P 〈 0.001）. Acting as the main active ingredients in the extract, tubeimoside I and acetylbenzoylaconine at 10：10μg/ mL and 5：2.5 μg/mL also produced inhibitory effects on the proliferation and migration of cells （P 〈 0.01）. CONCLUSION： Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts had synergic effects on MDA-MB-231 and SKBR3 cells.
