Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocreba...Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.展开更多
<span style="font-family:Verdana;">To synthesize, characterize and evaluate the antitumor potential derived from ruthenium compounds was generated in this study, from the precursor K[RuCl</span>&...<span style="font-family:Verdana;">To synthesize, characterize and evaluate the antitumor potential derived from ruthenium compounds was generated in this study, from the precursor K[RuCl</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">(bipy)] a route in a simple and reproducible synthesis for a novel compound of coordinating Ru</span><sup><span style="font-family:Verdana;">+3</span></sup><span style="font-family:Verdana;"> with bipy and L-trip. The spectroscopic characterization in the mi</span><span style="font-family:Verdana;">ddle infrared region (FTIR) shows the interactions between Ru-(L-trip), evidenced by the displacement of the carboxylate ion band for</span><span><span style="font-family:Verdana;"> higher energies, and also by the displacements of aliphatic amine bands, suggesting that bidentate coordination of the L-trip ligand occurred. Analysis of the results obtained with thermoanalytical techniques showed that the minimum formula of the compound, [RuCl</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;">(bipy)(L-trip)]1/2H</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;">O. Evaluation of the</span></span><span><span style="font-family:Verdana;"> antitumor potential of precursor K[RuCl</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">(bipy)] showed the toxic effects on MCF-7 cell line, but </span></span><span style="font-family:Verdana;">did not show selectivity and not reached PBMC cells to the same extent. The evaluation of the antitumor potential of the newly synthesized compound, [RuCl</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;">(bipy)(L-trip)], demonstrated that the insertion of an L-tryptophan molecule into the precursor coordination sphere made it selective when compared to PBMC cells, for MCF-7 type tumor cells.</span>展开更多
In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefo...In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.展开更多
Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear.In this study, the effect...Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear.In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay.Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.展开更多
Objective:To investigate whether dietary daidzein interact with endogenous 17β-Estradiol (E2) to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods:Cell cycle d...Objective:To investigate whether dietary daidzein interact with endogenous 17β-Estradiol (E2) to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods:Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein (1,5 μmol/L) and E2 (0.1-10 nmol/L) for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha (ERα),estrogen receptor beta (ERβ) and ERβ-estrogen response element (ERE) dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptotic p53,bax and antiapoptotic bcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot. Results:Daidzein enhanced the antiapoptotic effect in an E2 dose-dependent manner,but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition,daidzein only antagonized E2-upregulated expression of p53 and bax,but had no effect on E2-upregulated expression of bcl-2. Conclusion:Daidzein enhances the antiapoptotic effect of E2 on breast cancer cells by inhibiting E2-mediated p53-bax proapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2 in hormone-dependent breast cancer.展开更多
Objective To investigate the anticancer effect of xanthoceraside in vitro and the possible mechanisms involved in the potent antiproliferative effect on human breast cancer MCF-7 cell.Methods The inhibition rate of di...Objective To investigate the anticancer effect of xanthoceraside in vitro and the possible mechanisms involved in the potent antiproliferative effect on human breast cancer MCF-7 cell.Methods The inhibition rate of different tumor cells and human peripheral blood lymphocyte cells was investigated by MTT assay.AO/EB double fluorescent dye staining was used to investigate the morphology changes of MCF-7.The DNA agarose gel electrophoresis was further used to observe the DNA Fragmentation.Flow cytometry was employed to investigate the volume changes,the cell cycle distribution and the mitochondrial membrane potential of MCF-7.The antioxidant N-acetylcysteine(NAC)was chosen to detect the influence on oxidant-stress system of MCF-7 cells.Necrostatin-1 was next chosen to detect the influence on antiproliferative effect of xanthoceraside-treated MCF-7 cells.Results Xanthoceraside could inhibit the proliferation of tumor cells significantly in a dose-dependent manner and it has no cytotoxic effects on human peripheral blood lymphocyte cells in vitro.Cytoplasm vacuole was observed but no significant condense of nuclear chromatin was found,meanwhile,MCF-7 cells were bigger and smear was observed by agarose gel electrophoresis after MCF-7 cells were exposed to xanthoceraside.The cell cycle distribution of MCF-7 was greatly changed after exposure to xanthoceraside with an obvious G1 arrest.The mitochondrial membrane potential showed significant decrease.NAC attenuate the antiproliferative effect of xanthoceraside-treated MCF-7 cells but necrostatin-1 had no effects.Conclusions Xanthoceraside-induced necrosis might be dependent of mitochondria,meanwhile reactive oxygen species(ROS)participated in it.The xanthoceraside-induced MCF-7 cell death might not be the cell necrosis which initiated by Fas/TNFR and must be through RIP1 kinase.展开更多
Ekor naga’s leaf (Rhaphidophora pinnata (Lf) Schott) is a type of vines and climbing plant. The leaves are elongated round and hollowed inside. This plant had been using for the treatment of breast cancer. Extraction...Ekor naga’s leaf (Rhaphidophora pinnata (Lf) Schott) is a type of vines and climbing plant. The leaves are elongated round and hollowed inside. This plant had been using for the treatment of breast cancer. Extraction with percolation method has been done in ekor naga’s leaves with ethanol, and fractionated by nhexane, chloroform and ethyl acetate using liquid-liquid extraction (LLE). Cytotoxicity assay of ethanol extract, n-hexane fraction, chloroform fraction, ethyl acetate fraction and water fraction against MCF-7 cells were done using MTT method (3-(4,5-dimetiltiazol-2-il)-2,5-diphenyl tetrazolium bromide). Phytochemical screening results showed the presence of the compounds such as triterpenoida/steroid, alkaloid, flavonoid, tannin, and saponin. n-hexane fraction was positive for the presence of triterpenoida/steroid, chloroform fraction containing alkaloids, saponin and triterpenoid;ethyl acetate fraction contained, flavonoid, tannin, and the fraction of water indicated the presence of tannin and saponin. Secondary metabolite compounds in ethanol extract, chloroform fraction and ethyl acetate fraction gave positive results against MCF-7 cells. Cytotoxicity assay of MCF-7 cell line showed that crude ethanol extracts had 112.240 mcg/ml IC50 chloroform fraction IC50 was 59.082 mcg/ml, and ethyl acetate fraction IC50 was 812.663 mcg/ml.展开更多
[Objectives] To investigate the mechanism of DNA damage of cisplatin( DDP),a broad spectrum anticancer drug on breast cancer MCF-7 cells,and to study the mechanism of apoptosis induced by DDP.[Methods]MCF-7 cells were...[Objectives] To investigate the mechanism of DNA damage of cisplatin( DDP),a broad spectrum anticancer drug on breast cancer MCF-7 cells,and to study the mechanism of apoptosis induced by DDP.[Methods]MCF-7 cells were treated by DDP( 0 mg/L,2 mg/L,4 mg/L,6 mg/L,6 mg/L,and 10 mg/L) for 48 hours. MTT assay was used to detect the inhibitory effect of DDP on MCF-7 cells and IC50 value was calculated. Western blot was adopted to detect the expression of γ-H2 AX,which was the marker of DNA double stranded breaks( DSBs) and ATM( sensory molecules of DSBs),the apoptotic signal transduction molecule cleaved caspase-3,and the proteins associated with apoptosis calpain.[Results]DDP inhibited MCF-7 cell activity in a concentration-dependent manner and IC50 was 7. 57 mg/L. In contrast to the control group( without DDP treatment),MCF-7 cells with DDP treatment expressed more γ-H2 AX,ATM,cleaved caspase-3 and calpain.[Conclusions] DDP could inhibit the activity of breast cancer MCF-7 cells. Its mechanisms may be associated with inhibition of MCF-7 cell apoptosis,induction of DNA double strand breaking and the expression of pro-apoptotic protein up-regulation.展开更多
Activator protein-1(AP-1)transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to th...Activator protein-1(AP-1)transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus.The phorbol 12-myristate 13-acetate and Forskolin(Fo)are well-known kinase activators/stimulators of Protein Kinase C(PKC)and Protein Kinase A(PKA)respectively.Importantly,these kinases are found to be present in transitional points of many cell signaling pathways,especially those involved in proliferation.The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized.Hence,the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated.Where,cells treated with PMA showed increased cell proliferation,while Fo had no effect,but inhibited the PMA induced proliferation.The RT-PCR results showed the PMA induced c-Jun,c-Fos and Fra-1 expressions compared to control and Fo.However,Fo in combination with PMA,inhibit the PMA induced above mRNA expressions where Fo alone has no effect.Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells.Further,PMA increases the mRNA expression of Cyclin-E1,Cyclin-D1,and CDK-4,whereas Fo decreases their expressions.Thus,mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.展开更多
基金Natural Science Foundation of Hainan Province(No.820RC776)。
文摘Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.
文摘<span style="font-family:Verdana;">To synthesize, characterize and evaluate the antitumor potential derived from ruthenium compounds was generated in this study, from the precursor K[RuCl</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">(bipy)] a route in a simple and reproducible synthesis for a novel compound of coordinating Ru</span><sup><span style="font-family:Verdana;">+3</span></sup><span style="font-family:Verdana;"> with bipy and L-trip. The spectroscopic characterization in the mi</span><span style="font-family:Verdana;">ddle infrared region (FTIR) shows the interactions between Ru-(L-trip), evidenced by the displacement of the carboxylate ion band for</span><span><span style="font-family:Verdana;"> higher energies, and also by the displacements of aliphatic amine bands, suggesting that bidentate coordination of the L-trip ligand occurred. Analysis of the results obtained with thermoanalytical techniques showed that the minimum formula of the compound, [RuCl</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;">(bipy)(L-trip)]1/2H</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;">O. Evaluation of the</span></span><span><span style="font-family:Verdana;"> antitumor potential of precursor K[RuCl</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">(bipy)] showed the toxic effects on MCF-7 cell line, but </span></span><span style="font-family:Verdana;">did not show selectivity and not reached PBMC cells to the same extent. The evaluation of the antitumor potential of the newly synthesized compound, [RuCl</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;">(bipy)(L-trip)], demonstrated that the insertion of an L-tryptophan molecule into the precursor coordination sphere made it selective when compared to PBMC cells, for MCF-7 type tumor cells.</span>
文摘In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.
基金supported by grants from The National Maritime Bureau Public Science and Technology Research Funds Projects of Ocean(No.201005013)the Wuhan Municipal Science and Technology Research Project of China(No.201260523185)
文摘Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear.In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay.Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.
基金supported by the National Natural Science Foundation of China (No.30671508)by State Key Laboratory for Agrobiotechnology of China (No.2009SKLAB07-5)
文摘Objective:To investigate whether dietary daidzein interact with endogenous 17β-Estradiol (E2) to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods:Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein (1,5 μmol/L) and E2 (0.1-10 nmol/L) for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha (ERα),estrogen receptor beta (ERβ) and ERβ-estrogen response element (ERE) dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptotic p53,bax and antiapoptotic bcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot. Results:Daidzein enhanced the antiapoptotic effect in an E2 dose-dependent manner,but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition,daidzein only antagonized E2-upregulated expression of p53 and bax,but had no effect on E2-upregulated expression of bcl-2. Conclusion:Daidzein enhances the antiapoptotic effect of E2 on breast cancer cells by inhibiting E2-mediated p53-bax proapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2 in hormone-dependent breast cancer.
文摘Objective To investigate the anticancer effect of xanthoceraside in vitro and the possible mechanisms involved in the potent antiproliferative effect on human breast cancer MCF-7 cell.Methods The inhibition rate of different tumor cells and human peripheral blood lymphocyte cells was investigated by MTT assay.AO/EB double fluorescent dye staining was used to investigate the morphology changes of MCF-7.The DNA agarose gel electrophoresis was further used to observe the DNA Fragmentation.Flow cytometry was employed to investigate the volume changes,the cell cycle distribution and the mitochondrial membrane potential of MCF-7.The antioxidant N-acetylcysteine(NAC)was chosen to detect the influence on oxidant-stress system of MCF-7 cells.Necrostatin-1 was next chosen to detect the influence on antiproliferative effect of xanthoceraside-treated MCF-7 cells.Results Xanthoceraside could inhibit the proliferation of tumor cells significantly in a dose-dependent manner and it has no cytotoxic effects on human peripheral blood lymphocyte cells in vitro.Cytoplasm vacuole was observed but no significant condense of nuclear chromatin was found,meanwhile,MCF-7 cells were bigger and smear was observed by agarose gel electrophoresis after MCF-7 cells were exposed to xanthoceraside.The cell cycle distribution of MCF-7 was greatly changed after exposure to xanthoceraside with an obvious G1 arrest.The mitochondrial membrane potential showed significant decrease.NAC attenuate the antiproliferative effect of xanthoceraside-treated MCF-7 cells but necrostatin-1 had no effects.Conclusions Xanthoceraside-induced necrosis might be dependent of mitochondria,meanwhile reactive oxygen species(ROS)participated in it.The xanthoceraside-induced MCF-7 cell death might not be the cell necrosis which initiated by Fas/TNFR and must be through RIP1 kinase.
文摘Ekor naga’s leaf (Rhaphidophora pinnata (Lf) Schott) is a type of vines and climbing plant. The leaves are elongated round and hollowed inside. This plant had been using for the treatment of breast cancer. Extraction with percolation method has been done in ekor naga’s leaves with ethanol, and fractionated by nhexane, chloroform and ethyl acetate using liquid-liquid extraction (LLE). Cytotoxicity assay of ethanol extract, n-hexane fraction, chloroform fraction, ethyl acetate fraction and water fraction against MCF-7 cells were done using MTT method (3-(4,5-dimetiltiazol-2-il)-2,5-diphenyl tetrazolium bromide). Phytochemical screening results showed the presence of the compounds such as triterpenoida/steroid, alkaloid, flavonoid, tannin, and saponin. n-hexane fraction was positive for the presence of triterpenoida/steroid, chloroform fraction containing alkaloids, saponin and triterpenoid;ethyl acetate fraction contained, flavonoid, tannin, and the fraction of water indicated the presence of tannin and saponin. Secondary metabolite compounds in ethanol extract, chloroform fraction and ethyl acetate fraction gave positive results against MCF-7 cells. Cytotoxicity assay of MCF-7 cell line showed that crude ethanol extracts had 112.240 mcg/ml IC50 chloroform fraction IC50 was 59.082 mcg/ml, and ethyl acetate fraction IC50 was 812.663 mcg/ml.
基金Supported by Project of National Natural Science Foundation(30600753&81172154)Hunan Provincial Natural Science Foundation of China(2016JJ2088)+1 种基金Project of Hunan Provincial Department of Education(13C541)Project of Hunan Administration of Traditional Chinese Medicine(2015123)
文摘[Objectives] To investigate the mechanism of DNA damage of cisplatin( DDP),a broad spectrum anticancer drug on breast cancer MCF-7 cells,and to study the mechanism of apoptosis induced by DDP.[Methods]MCF-7 cells were treated by DDP( 0 mg/L,2 mg/L,4 mg/L,6 mg/L,6 mg/L,and 10 mg/L) for 48 hours. MTT assay was used to detect the inhibitory effect of DDP on MCF-7 cells and IC50 value was calculated. Western blot was adopted to detect the expression of γ-H2 AX,which was the marker of DNA double stranded breaks( DSBs) and ATM( sensory molecules of DSBs),the apoptotic signal transduction molecule cleaved caspase-3,and the proteins associated with apoptosis calpain.[Results]DDP inhibited MCF-7 cell activity in a concentration-dependent manner and IC50 was 7. 57 mg/L. In contrast to the control group( without DDP treatment),MCF-7 cells with DDP treatment expressed more γ-H2 AX,ATM,cleaved caspase-3 and calpain.[Conclusions] DDP could inhibit the activity of breast cancer MCF-7 cells. Its mechanisms may be associated with inhibition of MCF-7 cell apoptosis,induction of DNA double strand breaking and the expression of pro-apoptotic protein up-regulation.
基金Grants Commission(UGC),New Delhi for providing Major Research Project financial Grant(F.No.34-250/2008(SR))to SCS.
文摘Activator protein-1(AP-1)transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus.The phorbol 12-myristate 13-acetate and Forskolin(Fo)are well-known kinase activators/stimulators of Protein Kinase C(PKC)and Protein Kinase A(PKA)respectively.Importantly,these kinases are found to be present in transitional points of many cell signaling pathways,especially those involved in proliferation.The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized.Hence,the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated.Where,cells treated with PMA showed increased cell proliferation,while Fo had no effect,but inhibited the PMA induced proliferation.The RT-PCR results showed the PMA induced c-Jun,c-Fos and Fra-1 expressions compared to control and Fo.However,Fo in combination with PMA,inhibit the PMA induced above mRNA expressions where Fo alone has no effect.Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells.Further,PMA increases the mRNA expression of Cyclin-E1,Cyclin-D1,and CDK-4,whereas Fo decreases their expressions.Thus,mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.
