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OBJECTIVE To study in vitro inhibitory effects of realgar nanoparticles on breast cancer stem cells. METHODS Human breast cancer MCF- 7 parent cells were selected as subjects and cultured by serum-free culture to obtain breast cancer stem cells. Using adriamycin （1 mg/L）as positive control ，same concentration of water-processed realgar as reference ，the effects of realgar nanoparticles on the proliferation of MCF- 7 parent cells and stem cells were detected by CCK- 8 method. The effects of realgar nanoparticles on the formation of mammosphere ，the ability of differentiation ，migration and invasion ，the proportion of CD44+/CD24－ subgroup in breast cancer stem cells were detected by mammosphere formation and differentiation experiment ， scratch experiment ，Transwell invasion experiment and flow cytometry. Western blot assay was used to detect the expression of proteins related to epithelial mesenchymal transformation pathway （E-cadherin and vimentin ） in breast cancer stem cells. RESULTS The survival rates of MCF- 7 parent cells and stem cells （except for breast cancer stem cells in both 1 mg/mL groups ）in 1，5，10，40，60，80 mg/L groups of water-processed realgar and realgar nanoparticles were significantly lower than blank control group（P＜0.01）. The number of mammosphere （＞20 stem cells ）in 1，2.5，5，10 mg/L groups of water-processed realgar and realgar nanoparticles was significantly lower than blank control group （P＜0.01）；the volume of mammosphere decreased and the differentiated adherent cells decreased ；the healing rate of wound ，relative invasion rate （except for water-processed realgar 1 mg/L group）and the proportion of CD 44+/CD24－ subgroup were significantly lower than blank control group （P＜0.01）. The expressions of E-cadherin in 2.5，10 mg/L groups of water-processed realgar and realgar nanoparticles was significantly higher than blank control group ，and the expressions of vimentin was significantly lower than those in blank control group （P＜0.01）. The above effects of realgar nanoparticles were generally better than those of water-processed realgar with the same mass concentration （P＜ 0.01）. CONCLUSIONS Compared with water-processed realgar with the same mass concentration ，realgar nanoparti cles can significantly inhibit the proliferation of breast cancer stem cells， the formulation and differential ability of mammo- sphere，and reduce the proportion of CD 44+/CD24－ subgroup. The effect may be associated with the inhibition of migration and invasion of breast cancer stem cells by inhibiting the expression of proteins related to epithelial mesenchymal transformation pathway.
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BACKGROUND: The preventive and therapeutic medical utilization of this plant is an age-long practice across the globe. This study aimed to validate the impact of dark purple blossoms of basil (Ocimum basilicum L.) aqueous extract at low temperature (0 °C) mediated mitochondrial fission contributed to induced apoptosis in human breast cancer cells. METHODS: Fresh blossoms were extracted at low temperature (0 °C) using a watery solvent. Human MCF7 breast cancer cells were then treated with 3 separate fluctuated concentrations of 0, 50, 150 and 250 µg/mL for 24 and 48 h. RESULTS: The outcomes demonstrated the presence of anthocyanins, anthraquinones, tannins, reducing sugars, glycosides, proteins, amino acids, flavonoids and volatile oils and nonappearance of Terpinoids and alkaloids. Contrastingly, frail presence of steroids in basil blossoms aqueous concentrate was noted. In addition, the results from a phytochemical subjective examination of basil (Ocimum basilicum L.) blossoms aqueous extract demonstrated that most of the credited natural impacts containing more remarkable contents of antioxidants and anticancer compounds in basil blossoms aqueous extract. Moreover, the restraint of glucose take-up was alleviated mediated by a dose-dependent manner in MCF7 cells with basil (Ocimum basilicum L.) blossoms aqueous extract inducted for 24 h, resulting in mitochondrial fission. CONCLUSION: This is the first study that shows the impact of the aqueous extract of basil (Ocimum basilicum L.) blossoms was extracted at low temperature (0°C/6 h) underlined high amounts of flavonoids and phenolic compounds bearing more anticancer and antioxidant activities compared to another aqueous extract (using boiled water solvent) and alcoholic extracts.
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Humans , Plant Extracts/pharmacology , Apoptosis , Ocimum basilicum/chemistry , Flowers/chemistry , Mitochondrial Dynamics , Breast Neoplasms , Cold Temperature , MCF-7 CellsABSTRACT
Objective: To investigate the cell growth inhibitory effect and molecular mechanism of bakuchiol against human breast cancer MCF-7 cells. Methods: The growth inhibitory effect of bakuchiol on MCF-7 cells was tested by MTT assay. Flow cytometry was used to investigate the distribution of cell cycle and ROS generation. Fluorescence microscope was used to observe the change of cell nucleus. Western blotting was used to detect the expression of the protein related to cell cycle and MAPK family. The ROS scavenger and inhibitors of MAPK family were introduced to investigate the effect on the growth inhibitory rate and the levels of cell cycle related protein by bakuchiol. Results: Bakuchiol inhibited the cell growth on the MCF-7 cells in dose- and time-dependent manner, which showed stronger effect than that of 5-fluorouracil. Furthermore, bakuchiol induced S-phase arrest in MCF-7 cells via ROS generation. The production of ROS up-regulated p-p53 and p21 expression, and then decreased CDK2 and CyclinA2. The changes of bakuchiol on these proteins could be reversed by the ROS scavenger Trion, indicating that ROS was associated with bakuchiol-induced S-phase arrest. In addition, pretreatment with p38MAPK inhibitor SB203580 decreased bakuchiol-caused ROS generation, suggesting that the production of ROS was dependent on p38MAPK pathway. Conclusion: The proliferation inhibitory effect of bakuchiol on MCF-7 cells is related with S-phase cell cycle arrest, and ROS plays a role in the bakuchiol-induced S-phase arrest.
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OBJECTIVE：To study the effects of stilbene glucoside （TSG）on the proliferation and estrogen receptor （ER）of human breast cancer T- 47D cells ，and to explore its estrogen-like effect and potential mechanism. METHODS ：Taking ER positive human breast cancer T- 47D cells as subjects ，using β-estradiol（β-E2，1×10－8 mol/L）as positive control ，CCK-8 assay was used to detect the cell proliferation after treated with different concentrations of TSG （1×10－8，1×10－7，1×10－6，1×10－5，1×10－4 mol/L）for 24，48，72 h；the cell proliferation rate was calculated. Western blotting assay and RT-PCR methods were adopted to detect the protein and mRNA expression of ER-α and ER-β in cells after treated with low，medium and high concentrations of TSG （1×10－8， 1×10－6，1×10－4 mol/L）for 48 h. RESULTS ：After treated with different concentrations of TSG for 24，48，72 h，the cell proliferation rate of each administration group at each time point （except for β-E2 group at 48 h）increased significantly ，compared with blank group ；those of TSG groups （1×10－5，1×10－6，1×10－7 mol/L）were significantly higher than β-E2 group（P＜0.05 or P＜0.01）. After treated with low ，medium and high concentrations of TSG for 48 h，protein and mRNA expression of ER-α and ER-β in cells were increased significantly，compared with blank group （P＜0.05 or P＜0.01）；protein expression of ER-β in TSG low concentration group ，mRNA expression of ER-α in TSG groups as well as mRNA expression of ER-β in TSG low and high concentration groups were significantly higher than β-E2 group（P＜0.05 or P＜0.01）. CONCLUSIONS ：TSG can induce the in vitro proliferation of T- 47D cells and exert estrogen-like effects by promoting protein and mRNA expression of ER-α and ER-β， which is stronger than that of β-E2 at a certain concentration.
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OBJECTIVE：To study the effects of costunolide on the proliferation ，migration and apoptosis of breast cancer SK-BR-3 cells and its mechanism. METHODS ：SK-BR-3 cells in logarithmic growth period were collected and cultured with different concentrations （10，20，30，40，50 μmol/L）of costunolide for 24，48，72 h. Inhibitory rate of costunolide on cell proliferation was detected with CCK- 8. The cells were divided into blank control group and costunolide group （10，20，30 μmol/L）. Hoechst 33258 fluorescence was used to observe the morphology and apoptosis of cells ，and apoptotic rate of cells were calculated. Cell scratch test was used to detect the migration ability of cells and calculate the migration rate. Western blotting was used to detect the relative expression level of Bcl- 2，Bax，Caspase-3 and Cleaved Caspase- 3 in cells. RESULTS ：The proliferation of SK-BR-3 cells were significantly inhibited by costunolide （P＜0.05 or P＜0.01），and it shows a trend of concentration and time dependence. In the blank control group ，cells possessed clear contour ，regular shape and good adherence . Compared with blank control group，the number of cells were decreased significantly in 10，20，30 μmol/L costunolide groups，the cell structure was loose，the volume was reduced ，and the gap became larger ，and most of the cell contour disappeared and became round ，the cell adherence was poor ；cell migration rate and Bcl- 2 protein relative expression level were decreased significantly ，while apoptosis rate and the relative expression level of Bax ，Caspase-3 and Cleaved Caspase- 3 protein were significantly increased （P＜0.05 or P＜0.01）. CONCLUSIONS ： Costunolide can inhibit the proliferation and migration ，and induce apoptosis of human breast cancer SK-BR- 3 cells，mechanism of which may be through up-regulating the expression of Bax ，Caspase-3 and Cleaved Caspase- 3 while down-regulating the expression ofBcl-2.
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A popular approach for the study of estrogen receptor α inhibition is to investigate the protein-protein interaction between the estrogen receptor (ER) and the coactivator surface. In our study, we investigated phytochemicals from Rubus coreanus that were able to disrupt ERα and coactivator interaction with an ERα antagonist. The E-screen assay and molecular docking analysis were performed to evaluate the effects of the estrogenic activity of R. coreanus extract and its constituents on the MCF-7 human breast cancer cell line. At 100 µg/mL, R. coreanus extract significantly stimulated cell proliferation (574.57 ± 8.56%). Sanguiin H6, which was isolated from R. coreanus, demonstrated the strongest affinity for the ERα coactivator-binding site in molecular docking analysis, with a binding energy of
Subject(s)
Humans , Breast Neoplasms , Cell Line , Cell Proliferation , Estrogens , Molecular Docking Simulation , Phytochemicals , RubusABSTRACT
OBJECTIVE@#To explore the molecular mechanism underlying the inhibitory effects of aspirin against human breast cancer cell proliferation through bioinformatics analysis.@*METHODS@#Drug Bank 5.1.3 was searched to identify direct protein targets (DPTs) of aspirin, and the protein-protein interaction (PPI) network of the DPTs was constructed online using STRING and the signaling pathways involved were identified. The genetic alterations of 6 DPTs associated with human breast cancer was analyzed and visualized by cBio Portal and OncoPrint, respectively. The transcriptomic data of breast cancer and normal tissues were downloaded from TCGA database, and the overexpressed genes were analyzed by DECenter. The intersection between the genes associated with the DPTs obtained by STRING analysis and the differentially over-expressed genes in TCGA was determined to confirm the candidate DPTs as a potential target of aspirin, and GO functional enrichment analysis was performed using Gene Ontology. The potential targets of aspirin against the proliferation of human breast cancer cells were verified by Western blotting.@*RESULTS@#Eleven DPTs of aspirin were identified. KEGG pathway enrichment indicated that 6 genes (EDNRA, IKBKB, NFKB2, NFKBIA, PTGS2 and TP53) were associated with the occurrence and development of cancer. A total of 10 220 differentially expressed genes were identified from the TCGA database, and among them 4 genes (, , , ) were found to be the potential targets for aspirin. These genes were involved mostly in the regulation of cell cycle and cell division. Western blotting showed that aspirin could down-regulate the expression levels of several pivotal proteins that regulated cell cycle and cell division, including , , and .@*CONCLUSIONS@#, , and may be potential targets for aspirin to inhibit the proliferation of human breast cancer cells, by affecting the progress of cell cycle and cell division.
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SATB1 plays a crucial role in the invasion and metastasis of breast cancer,and inhibition of SATB1 expression can effectively control breast cancer metastasis. In this study,homogeneous polysaccharides were isolated from Poria cocos and their sulfated derivatives were prepared to screen out the polysaccharide compositions with inhibitory effects on SATB1 expression. Smal-molecule components were removed from P. cocos by ethanol extraction,and P. cocos crude polysaccharide PPS was obtained by water extraction and ethanol precipitation. Then PPS was successively separated by DEAE Sepharose fast flow anion-exchange and Superdex-75 gel permeation chromatographic steps to give PPSW-1. The structure of PPSW-1 was identified and its sulfated derivatives were prepared. Then their inhibitory effects on human breast cancer MDA-MB-231 cells were investigated. A kind of polysaccharide,PPSW-1 with inhibitory effect on human breast cancer MDA-MB-231 cells,was obtained from P. cocos,with a relative molecular weight of 3. 06×104,and structure of 1,6-branched 1,3-α-D-galactan. PPSW-1 and its sulfated derivative Sul-W-1 showed good inhibitory effect on cells migration,and the water solubility of Sul-W-1 was better than that of PPSW-1. In addition,it was found that polysaccharide of P. cocos and its sulfated derivative can inhibit expression of SATB1. In this study,a kind of homogeneous polysaccharide with inhibitory effect on human breast cancer MDA-MB-231 cells was isolated from P. cocos,and its sulfated derivative with similar efficacy but better solubility was prepared,laying the foundation for the substance basis study of P. cocos.
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Humans , Breast Neoplasms , Pathology , Cell Line, Tumor , Cell Movement , Matrix Attachment Region Binding Proteins , Metabolism , Phytochemicals , Pharmacology , Polysaccharides , Pharmacology , Sulfates , Wolfiporia , ChemistryABSTRACT
Objective To investigate the effect of safflower polysaccharide on apoptosis of human breast cancer MDA-MB-435 cells by blocking PI3K/Akt/mTOR pathway and explore its mechanism. Methods MDA-MB-435 cells were divided into control group and safflower polysaccharide 0.5 and 1.0 mg/mL groups. MTT assay and flow cytometry were used to detect the effects of different concentrations of safflower polysaccharide on the growth inhibition and apoptosis of MDA-MB-435 cells. RT-PCR and Western blotting were used to detect the effect of different concentrations of safflower polysaccharides on the mRNA and protein expression of PI3K, Akt, and mTOR in MDA-MB-435 cells. Results Compared with the control group, the inhibition rate of MDA-MB-435 cells in safflower polysaccharide 1.0 mg/mL group was (27.73 ± 3.75)%, which was significantly higher than that [(21.52 ± 2.43)%] in safflower polysaccharide 0.5 mg/mL group (P < 0.05). Compared with the control group, safflower polysaccharide could significantly increase the apoptosis rate of MDA-MB-435 cells in a dose-dependent manner (P < 0.01). The results of RT-PCR and Western blotting showed that, compared with the control group, safflower polysaccharide significantly decreased the mRNA and protein expression of PI3K, Akt, and mTOR in MDA-MB-435 cells (P < 0.05, 0.01). Conclusion Safflower polysaccharide can effectively inhibit the growth of MDA-MB-435 cells and promote their apoptosis, which may be achieved by blocking the PI3K/Akt/mTOR pathway.
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OBJECTIVE:To prepare argininate betulinic acid,and to investigate the effect of the proliferation of triple-negative human breast cancer cell MDA-MB-231. METHODS:By using argininate as the solubilization carrier,argininate betulinic acid was prepared by co-grinding equal molar ratio of betulinic acid and argininate. The argininate betulinic acid was characterized with powder X-ray diffractometry,infrared spectroscopy and differential scanning calorimetry. The solubility of betulinic acid and argininate betulinic acid were compared. MTT method was used to assay the effects of 15,30,60,120 μ g/mL betulinic acid, argininate betulinic acid and 5-FU on the proliferation of MDA-MB-231 cell. RESULTS:Prepared argininate betulinic acid was a new phase which was different from the physical mixing of argininate and betulinic acid,among which carboxyl group of betulinic acid and amino group of argininate formed as a salt,and the salt had no obvious melting peak. Betulinic acid was almost insoluble in water. The solubility of betulinic acid in argininate betulinic acid aqueous solution was 50.72 μg/mL. Compared with betulinic acid,the inhibitory rate of argininate betulinic acid on the growth of MDA-MB-231 cell was increased significantly(P<0.05), there was no statistical significance between its effect and 5-FU(P>0.05). CONCLUSIONS:Argininate betulinic acid with good solubility is prepared successfully,and can inhibit the proliferation of MDA-MB-231 cell.
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In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. Methods: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-TricineSDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. Results: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. Conclusion: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.(AU)
Subject(s)
Snake Venoms , Crotalus , Disintegrins , Breast NeoplasmsABSTRACT
OBJECTIVE:To explore the effects and mechanism of extracts,active constituents and constituent combination of Sinopodophylli Fructus on cell proliferation of human breast cancer. METHODS:Acid phosphatase method was conducted to deter-mine the effects of 4 extracts [ethanol extract (Xc),petroleum ether extract from ethanol extract (Xp),ethyl acetate extract from ethanol extract (Xe),n-butanol extract from ethanol extract (Xz)],5 active constituents [podophyllotoxin (S1),deoxypodophyllo-toxin (S2),4-desmethyl deoxypodophyllotoxin (S3),8-isopentenyl kaempferol (S4),8,2′-diisoprenyl quercetin-3-methyl ether (S5)] and 3 active constituent combination [combination 1,S1-S2-S3-S4-S5 (2:4:1:4:32),Z1;combination 2,S2-S4 (1:1),Z2;combination 3,S3-S4(1:4),Z3] on the MDA-MB-231,MCF-7 cell proliferation;flow cytometry was adopted to detect the effects of above-mentioned samples on MDA-MB-231,MCF-7(T47D)cell cycle and mitochondrial membrane potential. RESULTS:The active constituent combination Z1 showed significant inhibitory effects on MDA-MB-231,MCF-7 cells,the half inhibitory concen-trations(IC50)were(0.27±0.2),(0.11±0.1)μg/mL;extracts Xc,Xp,Xe,active constituents S2,S4 and active constituent combi-nation Z2,Z3 showed relatively strong inhibitory effects on MDA-MB-231,MCF-7 (T47D) cell proliferation (IC50<15 μg/mL). Both extracts and active constituents can block MDA-MB-231,MCF-7 cell cycle in G2/M phase;all active constituents can block MDA-MB-231,T47D cell cycle in G0/G1 phase,and can reduce MDA-MB-231,T47D cell mitochondrial membrane potential. CONCLUSIONS:The active constituents and constituent combination of Sinopodophylli Fructus can inhibit cell proliferation of breast cancer by affecting cell cycle and mitochondrial mem-brane potential.
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Objective:To investigate the effects and mechanisms of anti-cancer by bacailein combined with U0126 on human breast cancer in vitro. Methods: The human breast cancer cell line MCF-7 was treated by baicalein,U0126 and baicalein combined with U0126 respectively. CCK8 assay measured cell proliferation of MCF-7;flow cytometry tested the cell cycle and apoptosis of MCF-7;microscopy observed the amount;TUNEL assay evaluated the apoptosis of MCF-7;Western blot detected the protein level of proliferation and apoptosis related protein;scratch assay measured the ability of migration. Results: Human breast cancer cell line MCF-7 was treated by baicalein or U0126 at different concentration for 24 h, CCK8 assay suggested that both of them can dramatically inhibit MCF-7 proliferation in a dose-dependent way (P<0. 05). Compared to the blank and DMSO groups,the human breast cancer cell line MCF-7 was treated with baicalein for 24 h,the cellular rate at G0-G1 phase increased a lot (91%) (P<0. 05),while the cellular rate at S phase reduced dramatically (P<0. 05),cell apoptosis increased dramatically by microscopy and TUNEL assay(P<0. 05),the level of ERK1/2,CyclinD1 and JNK reduced quickly (P<0. 05). Compared to the baicalein group,MCF-7 was treated by baicalein combined with U0126,the cellular rate at S phase decreased remarkably (P<0. 05),apoptosis was much obvious (P<0. 05),the phosphorylation level of ERK1/2 and JNK reduced a lot (P<0. 05),and the proliferation accelerator CyclinD1 highly decreased (P<0. 05);the scratch assay demonstrated that cell migration was dramatically inhibited when MCF-7 was treated by 20 μmol/L baicalein ( P<0. 05 ) . Conclusion:Both of baicalein and U0126 can inhibit the proliferation and migration,induce the apoptosis of human breast cancer cell line MCF-7 through decreasing the level of ERK, JNK and CyclinD1. Baicalein and U0126 can provide some novel avenues to treat breast cancer in clinic.
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Aim Toinvestigatetheinducementeffect of isatin on apoptosis of breast cancer cell line MCF-7 , andexploreitsdetailedmechanism.Methods MCF-7 cell lines were exposed to isatin at different concentra-tions(0,50,100,200 μmol·L-1 )for 48 h.Apop-totic features were demonstrated by nuclei staining with Hoechst 33258.Bcl-2,Bax and p53 mRNA were ana-lyzed by RT-PCR.Caspase-9 activation and mitochon-drial depolarization were assayed by flow cytometry. Bcl-2,Bax,p53 and cytochrome c proteins were ana-lyzedbyWesternblot.Results Isatininducesapopto-sis of MCF-7 cells.Furthermore,Bcl-2 expression was decreased and the ratio of Bcl-2 to Bax was significant-ly decreased by isatin.While,p53 expression relative-ly decreased.The mitochondrial transmembrane poten-tial was markedly reduced and the release of cyto-chrome c into the cytosol was increased after treatment with isatin.Simultaneously,caspase-9 was activated. Conclusions Isatinsignificantlyinducedtheapopto-sis of MCF-7 cells in vitro.These results strongly sug-gest that the p53 dependent mitochondrial pathway is involved in apoptosis.
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Objective:To screen fatty acid synthase (FAS) inhibitors from natural products and study their inhibitory effects on the proliferation of MCF-7 breast cancer cells.Methods: CCK-8 method was used to detect the inhibitory effects of Fructus Amomi, Polygonum cuspidatum Sieb, Cinnamomi Ramulus and their main compounds, such as polydatin, resveratrol and cinnamic acid on the proliferation of MCF-7 breast cancer cells for 24 h.Results: The results showed that the IC50 values of the 60% ethanol extracts of Fructus Amomi, Polygonum cuspidatum Sieb, and Cinnamomi Ramulus were 24.86μg/ml, 153.67 μg/ml and 178 μg/ml respectively. The IC50 value of Resveratrol was 61.75 μg/ml. The inhibitory effect of Resveratrol was better than that of Polygonum cuspidatum Sieb. Cinnamic acid, the main component of Cinnamomi Ramulus had better inhibitory activity at lower concentration.Conclusion: The 60% ethanol extracts of Fructus Amomi, Polygonum cuspidatum Sieb, and Cinnamomi Ramulus all showed inhibitory effects on the proliferation of MCF-7 breast cancer cells in a dose-dependent manner. Among them, Fructus Amomi had the best inhibitory activity.
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Objective:To explore the expressions of Disabled-1 (Dab1 )in human breast epithelial cells and breast cancer cells,and to clarify its role in cell cycle.Methods:Real-time PCR was used to analyze the Dab1 mRNA expressions in breast epithelial cells MCF-10A and breast cancer cells MCF-7,BT-549,and MDA-MB-231. The Dab1 protein expressions in those cells were tested by Western blotting method. The BT-549 cells at logarithmic growth phase were divided into control,pKH3,and pKH3-Dab1 groups;the cell cycle was investigated by flow cytometry.Results:The Real-time PCR results showed that the Dab1 mRNA expression levels in MCF-7 cells (0.504 ± 0.037),BT-549 cells (0.302 ± 0.027),and MDA-MB-231 cells (0.330 ± 0.031 )were reduced compared with MCF-10A cells (0.998±0.020)(P <0.05).The Western blotting results showed that the Dab1 protein expression levels in breast cancer cells MCF-7 (0.134±0.014),BT-549 (0.076±0.01),and MDA-MB-231 (0.074±0.005)were reduced compared with MCF-10A cells (0.227±0.021)(P <0.05).Compared with control group and pKH3 group,the cell cycle in pKH3-Dab1 group was inhibited at G1 phase detected by flow cytometry analysis. Conclusion:The expression of Dab1 is down-regulated in breast cancer cells,and the over-expression of Dab1 can inhibit the cell cycle at G1 phase.
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<p><b>OBJECTIVE</b>To explore the viral etiology of human breast cancer to determine whether there are novel molecular targets for gene therapy of breast cancer and provide evidence for the research of gene therapy and vaccine development for breast cancer.</p><p><b>METHODS</b>PCR was used to screen HPV16 and HPV18 oncogenes E6 and E7 in the SKBR3 cell line and in 76 paraffin embedded breast cancer tissue samples. RNA interference was used to knock down the expression of HPV18 E6 and E7 in SKBR3 cells, then the changes in the expression of cell-cycle related proteins, cell viability, colony formation, metastasis, and cell cycle progression were determined.</p><p><b>RESULTS</b>HPV18 oncogenes E6 and E7 were amplified and sequenced from the SKBR3 cells. Of the patient samples, 6.58% and 23.68% were tested to be positive for HPV18 E6 and HPV18 E7. In the cell culture models, the knockdown of HPV18 E6 and E7 inhibited the proliferation, metastasis, and cell cycle progression of SKBR3 cell. The knockdown also clearly affected the expression levels of cell cycle related proteins.</p><p><b>CONCLUSION</b>HPV was a contributor to virus caused human breast cancer, suggesting that the oncogenes in HPV were potential targets for gene therapy of breast cancer.</p>
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Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Base Sequence , Breast Neoplasms , Genetics , Therapeutics , Genetic Therapy , Methods , Oncogene Proteins, Viral , Genetics , Metabolism , Papillomaviridae , Physiology , Papillomavirus Infections , Genetics , Therapeutics , Sequence AlignmentABSTRACT
Objective To study the nuclear protein association of high-mobility group box-1 (HMGB1) and histone deacetylase 1 (HDAC1),and the effect of interaction on radiosensitivity in human breast cancer cells.Methods The protein-protein interaction was determined by immunoprecipitationWestern blot and glutathione-S-transferase capture assays.Cell growth was examined by MTT (methyl thiazolyl tetrazolium)assay and clonogenic assay.Histone deacetylase activity was analyzed by histone deacetylase assay.Results A significant increase of HMGB1 protein and radiosensitivity was observed in MDA-MB-231 and MDA-MB-468 cells transfected with a pCMV-Tag2B expression vector carrying with a full-length of HMGB1 cDNA.HMGB1 binding to HDAC1 was demonstrated as GST (glutathione Stransferase)-pull down and immunoprecipitation Western blot assay,and the association was elevated by irradiation.An LXCXE motif was required for the HMGB1-HADC1 interaction and HMGB1 radiosensitization.A significant difference of IC50 value was observed,for example,1.8 and 2.2 Gy (wtHMGB1 transfectants,P ＜ 0.05),3.6 and 3.8 Gy (HMGB1/C103F transfectants,P ＞ 0.05),both compared with 3.9 and 4.1 Gy (pCMV-Tag2B transfectants) in MDA-MB-231 and MDA-MB-468 cells,respectively.A specific HDAC1 inhibitor trichostatin A markedly reduced the HMGB1-mediated radiosensitivity,0.5 Gy in the presence of trichostatin A versus 1.8 Gy in absence of trichostatin A in MDA-MB-231 transfectants,1.2 Gy (with trichostatin A) versus 2.2 Gy (without trichostatin A) in MDA-MB-468 transfectants,P ＜ 0.05.Histone deacetylase activity was also detected in immunoprecipitates prepared from these cells with antibodies to HMGB1,and this activity was abolished by the histone trichostatin A.Conclusions These results suggest a previous unanticipated role for HDAC1 in modification of HMGB1-mediated radiosensitivity by its direct interaction with HMGB1.
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Objective To study the effect of knockdown A20 expression on the proliferation, apoptosis and migra-tion of MCF-7 cells and to evaluate the potential value of the A20 gene as the therapeutic target of breast cancer. Methods Synthesized siRNA targeted to A20 gene or negative control siRNA were transfected into MCF-7 cells by using lipofectamine 2000. CCK8 assay, Annexin V and 7-AAD double staining cytometry, Transwell assay were performed to investigate the effect of knockdown A20 mRNA expression on the proliferation, apoptosis, migration of MCF-7 cells, respectively. Results It can inhibit the proliferation and migration as well as promote the apoptosis in MCF-7 cells by knockdown A20 mRNA expression. Conclusion A20 gene plays an important role in the prolif-eration, apoptosis and migration of MCF-7 cells and it could be a potential therapeutic target of breast cancer.
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OBJECTIVE: To study the effect of selective COX (cyclooxygenase)-2 inhibitor valdecoxib on the growth of human breast cancer MCF-7/ADR(MCF-7/adriamycin) cells. METHODS: MTT assay was used to observe the effect of drugs on the growth of cells. Flow cytometry and Hoechst 33258 dye were used to detect apoptosis of MCF-7/ADR cells. The levels of GSH and GSSG were detected by kit; Laser confocal microscopy was used to detect the levels of ROS. RESULTS: Valdecoxib significantly inhibited the growth of human breast cancer MCF-7/ADR cells and induced apoptosis of the cells. Caspase 3 inhibitor Ac-DEVD-CHO and caspase inhibitor Z-VAD-FMK antagonized the inhibitory effect of valdecoxib on MCF-7/ADR cell growth. Valdecoxib significantly decreased GSH/GSSG ratio and increased the level of ROS, and antioxidant V-acetylcysteine antagonized the inhibitory effect of valdecoxib on MCF-7/ADR cell growth. CONCLUSION: The apoptosis of human breast cancer MCF-7/ADR cells induced by valdecoxib is associated with increase of ROS.