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The effects of oenothein B(OEB) on the proliferation, apoptosis, invasion, and migration of breast cancer MCF-7 and MDA-MB-231 cells were investigated by cell culture in vitro, network pharmacology, and molecular docking. In vitro cell experiments revealed that OEB inhibited the proliferation and colony formation ability, and promoted the apoptosis and formation of apoptotic bodies in breast cancer cells, as well as inhibited the invasion and migration of breast cancer cells. The targets of OEB were obtained using SwissTargetPrediction database and breast cancer targets were obtained from GeneCards. The targets of OEB and breast cancer were entered separately in Venny 2.1 software to obtain the Venn diagram of common targets of OEB and breast cancer. The common targets of OEB and breast cancer were input into STRING database to construct a protein-protein interaction(PPI) network, which was entered into Cytoscape 3.7.2 software for network topology analysis. Key targets were screened according to protein association strength, and analyzed for KEGG pathway enrichment. Molecular docking of OEB to key targets using AutoDock software revealed that OEB stably bound to the active pocket of P53, while OEB promoted the expression of P53 protein. MCF-7 and MDA-MB-231 cell viability and migration ability increased after silencing P53, and this change was reversed after treatment with OEB. Therefore, this study showed that OEB inhibited the proliferation, migration, and invasion of breast cancer MCF-7 and MDA-MB-231 cells, and promoted the apoptosis of breast cancer MCF-7 and MDA-MB-231 cells, which may be related to the targeted regulation of P53.
Subject(s)
Humans , Female , Cell Proliferation , Breast Neoplasms/drug therapy , Tumor Suppressor Protein p53/genetics , Molecular Docking SimulationABSTRACT
@#Objective To investigate the effects of live attenuated measles vaccine Hu191 strain(MV-Hu191)on epithelial mesenchymal transition(EMT),proliferation and migration of 4T1 breast cancer cells.MethodsCCK-8 and clone formation assay were used to analyze the effect of MV-Hu191 on the proliferation of 4T1 cells;The effect of MV-Hu191 on 4T1cell migration was analyzed by cell scratch test;The expression of EMT pathway proteins(MMP-2,MMP-9,E-cadherin)in4T1 cells was detected by Western blot;4T1 tumor-bearing mouse model was established in female BALB/c mice. The model mice were divided into control group(PBS),MV-Hu191(1 × 106TCID50)group and paclitaxel group(15 mg/kg),with 10 mice in each group,and injected into tumor at the dosage of 100 μL every 2 d for 5 times. At 28 d after administration,the effects of MV-Hu191 on survival time,tumorigenicity and metastasis in vivo were observed;The pathological characteristics of lung tissue and tumor tissue were observed by HE staining under microscope;The expression of EMT pathway proteins(MMP-2,MMP-9 and E-cadherin)in tumor tissue was detected by immunohistochemical staining.Results The results of in vitro experiment showed that,compared with the control group,MV-Hu191 inhibited the proliferation and migration of 4T1 cells(F = 2. 811 and 13. 535,P = 0. 001 and 0. 002,respectively),down regulated the expression of MMP-2 and MMP-9(F = 45. 433 and 9. 744,P = 0. 011 and 0. 038,respectively),and up regulated the expression of Ecadherin(F = 7. 001,P = 0. 032);The results of in vivo experiment showed that MV-Hu191 significantly prolonged the survival time of tumor-bearing mice,and decreased the tumor quality(F = 8. 301,P = 0. 003)and the number of pulmonary nodules metastasis compared with the control group(F = 33. 792,P = 0. 000);MV-Hu191 treated tumor tissue gap was small,the cells were round,and the alveolar contour was clearly visible;The expression of MMP-2 and MMP-9 in MVHu191 treated tumor tissue decreased significantly(F = 6. 705 and 9. 047,P = 0. 028 and 0. 023,respectively),while the expression of E-cadherin increased significantly(F = 3. 468,P = 0. 039).ConclusionMV-Hu191 signi-ficantly inhibits the proliferation and migration of 4T1 breast cancer cells,antagonizes the tumorigenicity and lung meta-stasis of 4T1 tumorbearing mice,and prolongs the survival time of mice. The possible mechanism of MV-Hu191 against breast cancer is closely related to the regulation of EMT pathway protein expression.
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Objective: To investigate the effect of piperine on human breast cancer cells. Methods: The effect of piperine on proliferation and migration of human breast cancer cells, MCF-7 and MDA-MB-231, was investigated using colony formation assays, wound healing assays, Matrigel migration assays, flow cytometry, RT-qPCR, and Western blotting assays. Results: Piperine inhibited the growth of MCF-7 and MDA-MB-231 cells and suppressed colony formation. Cell reduction at the G 0 / G 1 phase and cell arrest at the G 2 /M phase were observed in breast cancer cells. However, the significant effect was only demonstrated in MDA-MB-231 cells. Moreover, cancer cell migration was suppressed by piperine at low concentration. RT-qPCR and Western blotting assays showed that piperine downregulated Rac1 gene and protein expression. Conclusions: Piperine could inhibit growth and migration of breast cancer cells by reducing Rac1 gene and protein expression.
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Abstract Objective: This study aimed to investigate the effect of Zinc Finger E-box Binding Homeobox 1 (ZEB1) regulation by Micro Ribonucleic acid (miR)-448 on Breast Cancer (BC) cells and their sensitivity to chemotherapy. Methods: miR-448 and ZEB1 mRNA levels in BC and normal tissues were detected by qPCR, and ZEB1 protein was detected by Western Blotting (WB). The correlation between miR-448 and tumor metastasis, clinical staging, and ZEB1 expression was analyzed. MCF-7 cells were transfected or co-transfected with the miR-448 mimic, oe-ZEB1, or their negative controls. Changes in miR-448 and ZEB1 expression were detected by qPCR and WB. Cell proliferation was determined by CCK-8 assays, invasion changes were analyzed by Transwell assays, and apoptosis was detected by flow cytometry. Results: miR-448 expression in BC tissues was lower than that in normal tissues, while ZEB1 expression was increased in the former. ZEB1 expression was lower in BC patients with lymph node metastasis than in those without. In patients with clinical stage I-III BC, miR-448 expression decreased with an increase in tumor stage, which was negatively correlated with ZEB1 expression. Upregulation of miR-448 expression can suppress MCF-7 cell proliferation and invasion and promote apoptosis. Upregulation of ZEB1 expression in cells overexpressing miR-448 can partially reverse the inhibition of BC cell growth induced by miR-448. miR-448 can enhance the sensitivity of cells toward paclitaxel and 5-fluorouracil. Conclusions: miR-448 suppresses cell proliferation and invasion and promotes apoptosis by targeting ZEB1. Moreover, it can increase the sensitivity of cells toward paclitaxel and 5-fluorouracil.
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Breast cancer is one of the most malignant tumors and is associated with high mortality rates among women. Lycium barbarum polysaccharide (LBP) is an extract from the fruits of the traditional Chinese herb, L. barbarum. LBP is a promising anticancer drug, due to its high activity and low toxicity. Although it has anticancer properties, its mechanisms of action have not been fully established. Ferroptosis, which is a novel anticancer strategy, is a cell death mechanism that relies on iron-dependent lipid reactive oxygen species (ROS) accumulation. In this study, human breast cancer cells (Michigan Cancer Foundation-7 (MCF-7) and MD Anderson-Metastatic Breast-231 (MDA-MB-231)) were treated with LBP. LBP inhibited their viability and proliferation in association with high levels of ferroptosis. Therefore, we aimed to ascertain whether LBP reduced cell viability through ferroptosis. We found that the structure and function of mitochondria, lipid peroxidation, and expression of solute carrier family 7 member 11 (SLC7A11, also known as xCT, the light-chain subunit of cystine/glutamate antiporter system Xc-) and glutathione peroxidase 4 (GPX4) were altered by LBP. Moreover, the ferroptosis inhibitor, Ferrostatin-1 (Fer-1), rescued LBP-induced ferroptosis-associated events including reduced cell viability and glutathione (GSH) production, accumulation of intracellular free divalent iron ions and malondialdehyde (MDA), and down-regulation of the expression of xCT and GPX4. Erastin (xCT inhibitor) and RSL3 (GPX4 inhibitor) inhibited the expression of xCT and GPX4, respectively, which was lower after the co-treatment of LBP with Erastin and RSL3. These results suggest that LBP effectively prevents breast cancer cell proliferation and promotes ferroptosis via the xCT/GPX4 pathway. Therefore, LBP exhibits novel anticancer properties by triggering ferroptosis, and may be a potential therapeutic option for breast cancer.
Subject(s)
Female , Humans , Breast Neoplasms/drug therapy , Drugs, Chinese Herbal/pharmacology , Ferroptosis , Glutathione/metabolism , Iron/metabolismABSTRACT
OBJECTIVE To study the mechanism of curcumol inhibiting the pro liferation of breast cancer cells T 47D. METHODS MTT assay was used to detect the inhibitory effects of different doses of curcumol （0，6.25，12.5，25，50，100 μg/mL）on the proliferation of T 47D cells. After treated with curcumol （12.5，25，50，100 μg/mL），the morphology of T 47D cells was observed by inverted phase contrast microscope. The cell cycle and the levels of reactive oxygen species （ROS）were detected by flow cytometry. Quantitative real-time PCR （qRT-PCR）was used to detect the expressions of proliferating cell nuclear antigen （PCNA），cell cycle regular p 21 and cyclin-dependent kinase 2（CDK2）mRNA. Western blot assay was used to detect the protein expression of CDK 2，CDK6，Cyclin D ，PCNA，nucler transcription factor E 2-related factor （Nrf2）and Kelch-like ECH associated protein 1（Keap1）. Breast cancer cells T 47D were divided into 2 groups，one group was given different doses of curcumol ，and another group was given curcumol 33 μg/mL for 6，12，24，48 h. After the optimal oxidation time and administration concentration were determined according to the results of the above two groups ，the blank control group ，N-acetylcysteine（NAC）group（ROS antioxidant NAC alone ），curcumol group （curcumol alone ），curcumol combined with NAC group （pretreatment with ROS antioxidant NAC ，and then adding into curcumol ）. Cell cycle and fluorescence intensity of ROS were detected. RESULTS Curcumol could significantly increase the inhibitory rate of the proliferation of T 47D cells （P＜0.05 or P＜0.01），and showed a certain dose and time dependent trend. Curcumol blocked the ， cycle in the G 1 phase and significantly increased the level of ROS （P＜0.05 or P＜0.01）；ROS antioxidant NAC could significantly reverse above inductive effect of curcumol （P＜ 0.01）. qRT-PCR showed that curcumol down-regulated the com expression of PCNA and CDK 2 mRNA and up-regulated the expression of p 21 mRNA（P＜0.05 or P＜0.01）. Western blot assay showed that curcumol significantly down-regulated the edu.cn protein expression of Keap 1，Nrf2，CDK2，CDK6 and Cyclin D（P＜0.05，P＜0.01）；ROS antioxidant NAC could reverse the down-regulation effects of curcumol on the expression of these proteins（P＜0.05 or P＜0.01）. CONCLUSIONS Curcumol may induce oxidative stress and cell arrest in G 1 phase to inhibit the proliferation of T 47D cells.
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Objective To investigate the effect of LncRNA WT1-AS on the invasion and migration of triple-negative breast cancer MDA-MB-231 cells. Methods qRT-PCR was used to detect the expression of WT1-AS and the efficiency of gene silencing in MDA-MB-231 cells. Transwell invasion test and scratch test were used to detect the invasion and migration of MDA-MB-231 cells; Western blot was used to detect the expression of E-cadherin, N-cadherin, Vimentin and Snail in MDA-MB-231 cells. Results Compared with normal human mammary epithelial cells, the expression of WT1-AS in MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells were significantly up-regulated. Knockdown of WT1-AS significantly reduced the invasion and migration abilities of MDA-MB-231 cells, the expression of E-cadherin was increased but N-cadherin, Vimentin and Snail expression were decreased. Conclusion WT1-AS could promote the invasion and migration of triple-negative breast cancer cells MDA-MB-231 via regulating epithelial-mesenchymal transition.
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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.
Subject(s)
Humans , Plant Extracts/pharmacology , Apoptosis , Ocimum basilicum/chemistry , Flowers/chemistry , Mitochondrial Dynamics , Breast Neoplasms , Cold Temperature , MCF-7 CellsABSTRACT
Objective To study the effect of three kinds of commonly used liquid culture media for in vitro cell experiments on elastic modulus of breast cancer cells, so as to provide references for developing novel diagnosis and treatment approach of tumour based on mechanics principles. Methods The elastic modulus and adhesion force of breast cancer cells MCF7 to atomic force microscopy (AFM) probes in phosphate buffered solution(PBS), Dulbecco’s modified eagle media (DMEM) and DMEM+10% fetal bovine serum (FBS) were measured using AFM technology. Results The elastic moduli of breast cancer cells in PBS, DMEM and DMEM+10% FBS were 2.59, 2.11 and 1.59 kPa, respectively. The cell adhesion forces in the above three kinds of liquid medium environment were 63.81, 66.09 and 121.97 pN, respectively. Cell adhesion force in DMEM+10%FBS was significantly different from that of the other two kinds of liquid media. Conclusions There are significant differences in elastic modulus of breast cancer cells in three kinds of liquid media. The difference between DMEM and DMEM+10%FBS might be caused by the different adhesion force caused by serum proteins in the media, and the difference between DMEM and PBS might be attributed to the difference in pH of the media.
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Objective: To investigate the effect of citronellol (citronellol, CT) on the proliferation of HEp-2 and MCF-7 cells, and prepare CT self-emulsifying drug delivery system (CT-SMs). Its antitumor activity and cell uptake ability of HEp-2 cells in vitro was evaluated. Methods: The effect of CT on the cell proliferation of HEp-2 and MCF-7 were investigated by MTT assay. The pseudo- ternary phase diagram method was used to optimize the formulation of CT-SMs, and the appearance morphology, mean particle size, and Zeta potential were characterized. The effect of CT-SMs on the proliferation of HEp-2 cells was detected by MTT assay and cellular uptake was determined by fluorescence inversion microscopy and flow cytometry. Results: After a certain concentration of CT treatment, MCF-7 cells proliferation was not affected, and the difference was not statistically significant (P > 0.05 compared with the control group), while the proliferative capacity of HEp-2 cells was significantly inhibited (P < 0.05 compared with the control group) in a dose-time dependent manner. The best prescription for CT-SMs was as following: Km (emulsifier:co-emulsifier) was Kolliphor® HS 15:absolute ethanol = 7:3, CT:Km = 3:7, the mean particle size was (354.0 ± 9.5) nm, the appearance was round and spherical with uniform distribution, and the Zeta potential was (-13.4 ± 0.3) mV. The results of cellular uptake experiments showed that the intake of CT-SMs (545.70 ± 11.56) was higher than that of CT (230.00 ± 17.76) in HEp-2 cells treating the same concentration of CT-SMs and CT. Conclusion: CT-SMs could significantly inhibit the proliferation of HEp-2 cells. In this study, CT-SMs were successfully prepared by dropping water method and the quality of CT-SMs was stable and controllable.
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Objective:To explore the potential mechanisms of Panax Notoginseng Saponins （PNS） on growth inhibition of breast cancer cell line 4T1 in tumor-bearing mice by investigating the mitogen-activated protein kinase kinase kinase 1 （MEKK1）/stress activated protein kinase （SAPK）/extracellular regulated protein kinases （Erk） Kinase （SEK1）/c-Jun N-terminal kinase 1 （JNK1）/activator protein-1 （AP-1） signaling pathways. Method:The 4T1 breast cancer mice model was established. Forty-eight mice with successful modeled and randomly divided into the low， medium and high-dose PNS groups （10， 20， 40 mg·kg-1） and the model control group （12 mice in each group）. The PNS groups received intraperitoneal injection with dosage of 10 mL·kg-1， while the controlled group was given the same dosage of saline. After administration with PNS for 28 days， tumor tissues were isolated， weighed， sliced and homogenized. Tumor cell apoptosis was detected by TdT mediated-dUTP nick end labeling （TUNEL） staining. The mRNA expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissue were detected by Real-time polymerase chain reaction（Real-time PCR）. The protein expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissue were detected by immunofluorescence staining and Western blot. Result:Compared with model group， the tumor weights of medium-dose and high-dose PNS groups were decreased significantly （P<0.05）. TUNEL staining showed that the number of apoptotic tumor cells increased with the rise of dosage of PNS （P<0.05）. The medium-dose and high-dose PNS groups showed a significant increase in the mRNA expressions of MEKK1， SEK1， JNK1 and AP-1 as well as the protein expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissues （P<0.05）， with statistically significant differences （P<0.05）. Conclusion:PNS could inhibit the tumor growth of breast cancer cell line 4T1 in tumor-bearing mice， which may be related to the activation of MEKK1/SEK1/JNK1/AP-1 signaling pathways.
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Orthogonal experiments were used to optimize the process parameters of curcumin TPP-PEG-PCL nanomicelles; the particle size, electric potential and morphology under the electron microscope were systematically detected for the curcumin TPP-PEG-PCL nanomicelles; and the stability and in vitro release of the curcumin TPP-PEG-PCL nanomicelles were investigated. With DID fluorescent dye as the fluorescent probe, flow cytometry was used to study the uptake of nanomicelles by breast cancer cells, and laser confocal microscopy was used to study the mitochondrial targeting and lysosomal escape functions of nanomicelles. Under the same dosage conditions, the effect of curcumin TPP-PEG-PCL nanomicelles on promoting the apoptosis of breast cancer cells was evaluated. The optimal particle size of curcumin TPP-PEG-PCL nanomicelle was(17.3±0.3) nm, and the Zeta potential was(14.6±2.6) mV in orthogonal test. Under such conditions, the micelle appeared as regular spheres under the transmission electron microscope. Fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote drug uptake by tumor cells, escape from lysosomal phagocytosis, and target the mitochondria. The cell survival rate and Hoechst staining positive test results showed that curcumin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of breast cancer cells. The curcumin TPP-PEG-PCL micelles can significantly reduce the mitochondrial membrane potential of breast cancer cells, increase the release of cytochrome C, significantly increase the expression of pro-apoptotic protein Bcl-2 and reduce the expression of anti-apoptotic Bax protein. These test results were significantly better than those of curcumin PEG-PCL nanomicelles and curcumin, with statistically significant differences. The results revealed that curcumin TPP-PEG-PCL nanomicelles can well target breast cancer cell mitochondria and escape from the lysosomal capture, thereby enhancing the drug's role in promoting tumor cell apoptosis.
Subject(s)
Humans , Apoptosis , Breast Neoplasms/drug therapy , Cell Line, Tumor , Curcumin/pharmacology , Lysosomes , Micelles , Mitochondria , Phosphatidylethanolamines , Polyethylene GlycolsABSTRACT
OBJECTIVE To study the effect of tumor suppressor gene p53 on valproic acid (VPA)-caused radiosensitivity of breast cancer cells MCF7 and its mechanism of homologous recombination (HR) repair. METHODS By infecting breast cancer cells MCF7 with PLKO.1 and p53 shRNA lentiviral particle solution, the isogenic pairing MCF7 cells with down-regulated p53 expression, including MCF7/wild-type p53 (MCF7/wtp53) and MCF7/defective p53 (MCF7/dp53) cells, were established. By infecting MCF7/pDR-GFP cells, the MCF7/pDR-GFP/wfp53 and MCF7/pDR-GFP/cfp53 cells were established. MCF7/wtp53 and MCF7/dp53 cells were divided into control group, VPA group (VPA 0.5mmol«L-1 treatment for 24 h), ionizing radiation (IR, 8 Gy) group and VPA+IR group (VPA 0.5 mmol • L-1 pretreatment for 24 h combined with IR). The expression level of protein P53 was detected by Western blotting, while the nucleus tail length and percentage of cells containing phosphorylated histone (yH2AX) focus formation were detected by comet assay and immunofluorescence assay. The clone formation rate was detected by cell clone formation assay. The HR repair efficiency was detected by flow cytometry, and the percentage of cells containing breast cancer susceptibility gene 1 (BRCA1) and recombinase 51 (Rad51) focus formation was detected by immunofluorescence assay. RESULTS Western blotting results showed that the expression of P53 protein was observed in MCF7/wtp53 cells, however, it was decreased in bACF7/dp53 cells significantly (P<0.05). Comet assay and immunofluorescence assay results showed that in WiCF7/wtp53 cells, the nucleus tail length and percentage of cells containing yH2AX focus formation in the VPA+IR group increased compared with the IR group (P<0.05). In MCF7/dp53 cells, the nucleus tail length and percentage of cells containing yH2AX focus formation in VPA + IR group increased compared with the IR group (P<0.05), but still lower than those of the VPA + IR group in MCF7lwtp53 cells (P<0.05). Cell clone formation assay showed that in MCF7/w?p53 cells, the cell viability of the VPA+IR group was lower than that of the IR group (P<0.05). In MCF7Idp53 cells, the cell viability of the VPA+IR group was lower than that of the IR group, but still higher than that of the VPA+IR group in MCF7/wtp53 cells (P<0.05). Flow cytometry results showed that in MCF7/pDR-GFPIwtp53 cells, compared with cell control group, the HR efficiency of VPA group decreased (P<0.05). In MCF7/pDR-GFP/dp53 cells, the HR efficiency of the VPA group was lower than that of the cell control group, but higher than the VPA group in MCF7/pDR-GFP/wtp53 cells (P<0.05). Immunofluorescence assay results showed that in MCF7/wtp53 cells, the percentage of cells containing BRCA1 and Rad51 focus formation in the VPA+IR group was lower than in the IR group respectively (P<0.05). In MCF7/dp53 cells, the percentage of cells containing BRCA1 and Rad51 focus formation in VPA+IR group was lower than in the IR group respectively (P<0.05), but higher than that of VPA + IR group in MCF7/wtp53 cells (P< 0.05). CONCLUSION VPA can enhance the sensitivity of breast cancer cells to IR and is capable of radio sensitization. The inhibition of p53 expression can down-regulate the radiosensitization of VPA, which is associated with the BRCA1-Rad51-mediated over-enhancement of the HR mechanism.
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Objective: To identify the cytotoxic natural products from the deep-sea derived Ochrobactrum sp. OUCMDZ-2164. Methods: The isolations and purifications of compounds were performed by means of column chromatography over silica gel and Sephadex LH-20 as well as HPLC. Their structures were elucidated through the analysis of UV, IR, MS, NMR and ECD spectra. The cytotoxicities against MCF-7, A549 and K562 cells were evaluated by MTT and CCK-8 methods. Results: From the fermentation broth of Ochrobactrum sp. OUCMDZ-2164, we isolated and identified four compounds (1-4). Compound 1 was identified as a new ansamycin and named trienomycin J, and the structures of 1-4 were identified as 3-O-demethyltrienomycinol, flazin, flazin-3- carboxylic acid and thymine, respectively. Compound 1 showed cytotoxic effect on the MCF-7 cells with 61.5% inhibition rate at 10 μmol/L. Conclusion: Compound 1 was a new ansamycin named trienomycin J, with cytotoxic activity against human breast cancer cells (MCF-7).
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@#To investigate whether lncRNA MALAT1 affects the migration and proliferation of breast cancer cells through the regulation with histone methyltransferase SMYD3, the endogenous MALAT1 in the MCF-7 and MDA-MB-231 breast cancer cells were knocked down by siRNA, and then the migration and proliferation of cells were detected by wound healing migration and MTT assay. The effects of si-MALAT1 on the mRNA and protein levels of miRNA-124, SMYD3 and its downstream genes were detected by Real time PCR and Western blot. The results showed that siRNA-targeted knockdown of MALAT1 reduced the migration and proliferation of breast cancer cells, and inhibited the transcriptional expression of SMYD3 and its downstream genes, including N-cadherin, MYL9, MMP9 and CYR61, and up-regulated miR-124. Overexpression of miR-124 reduced the expression of SMYD3 in breast cancer cells, and knockdown of MALAT1 attenuated the promotion of SMYD3 protein expression by miR-124 inhibitors. In addition, SMYD3 overexpression activated MALAT1 transcription, whereas siRNA interference with SMYD3 downregulated MALAT1. These results indicate that LncRNA MALAT1 acted as a competing endogenous RNA(ceRNA)of miR-124 to regulate expression of SMYD3 in breast cancer cells, and SMYD3 can activate the transcription of MALAT1, which will affect the proliferation and migration of breast cancer cells.
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p90 ribosomal S6 kinase (p90RSK), one of the downstream effectors in ERK1/2 pathways, shows high expression in human breast cancer tissues. However, its role in breast cancer development and drug resistance is not fully understood. Here, we demonstrate that Cis-DDP treatment failed to increase cytotoxicity in MDA-MB-231 cells compared to MCF-7 cells and p90RSK activation was involved in Cis-DDP-resistance to MDA-MB-231 cells. In the study, we found that inhibition of p90RSK expression or activation using a small interfering RNA (siRNA) or dominant-negative kinase mutant (DN-p90RSK) plasmid overexpression increased Cis-DDP-induced cytotoxicity of MDA-MB-231 cells, respectively. Mechanistically, we found that Cis-DDP resistance was associated with up-regulation of epithelial growth factor (EGF) expression and EGF treatment induced cancer survival signaling pathway including activation of ERK1/2, p90RSK, and Akt. We also examined the expression of epithelial-mesenchymal transition (EMT)-associated proteins using a reverse transition-quantitative PCR analysis. Cis-DDP treatment induced EMT by increasing the expression levels of N-cadherin, Snail, and Twist, while decreasing the expression levels of E-cadherin. Furthermore, we examined the epithelial marker, Zonula occludens-1 (ZO-1) using immunofluorescence analysis and found that Cis-DDP-inhibited ZO-1 expression was recovered by p90RSK deactivated condition. Therefore, we conclude that Cis-DDP resistance is involved in EMT via regulating the EGF-mediated p90RSK signaling pathway in MDA-MB-231 cells.
Subject(s)
Humans , Breast Neoplasms , Cadherins , Cisplatin , Drug Resistance , Epidermal Growth Factor , Epithelial-Mesenchymal Transition , Fluorescent Antibody Technique , MCF-7 Cells , Phosphotransferases , Plasmids , Polymerase Chain Reaction , Ribosomal Protein S6 Kinases, 90-kDa , RNA, Small Interfering , Snails , Triple Negative Breast Neoplasms , Up-RegulationABSTRACT
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
Aim: To explore the effect of chloroxoquinoline (Chl) on cell proliferation, apoptosis and cell cycle in breast cancer cells and its potential mechanism. Methods: Breast cancer cells of Bcap37, MDA-MB-231 and MDA-MB-453 were treated with Chl at different doses. Cell proliferation was detected by MTT assay. Cell apoptosis and cell cycle were assayed by Annexin V-/PI kit using flow cytometry. Protein expression related to apoptosis and cell cycle were determined by Western blot. Results: Chl over the dose of 50 mg · L-1 inhibited cell proliferation in all three cell lines in a dose-dependent manner, and Ch; at the dose of 200 mg · L-1 exhibited similar efficiency as 40 mg · L-1 Paclitaxel did. When the cells were treated with 100 or 200 mg · L-1 Chl for 48 h, the apoptotic ratio was dose-dependently up-regulated in all three cell lines (P < 0.05), especially in the MDA-MB-231 cells. It was further proved by the protein expression of cleaved-PARP and cleaved-caspase related to apoptosis. On the contrary, Chl at the dose of 100mg · L-1 significantly increased the cell number of G2/M stage in Bcap37 and MDA-MB453 cells (P < 0. 05). Correspondingly, Western blot analysis demonstrated that Chl dose-dependently down-regulated p21 protein and up-regulated CDC2 protein, especially in Bcap37 and MDA-MB453 cells. Conclusions: Chl significantly inhibits cell proliferation in breast cancer cells of Bcap37, MDA-MB-231 and MDA-MB453, which may mainly result from its induction of cell apoptosis in MDA-MB-231 cells and cell cycle arrest in Bcap37 and MDA-MB-453 cells. These data provide new idea of chemotherapy using Chl for breast cancer treatment.
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@#Objective: To explore the difference in the proliferation inhibition of doxorubicin and dual specific oncolytic adenoviruses (Ad-VT, Ad-T, Ad-VP3 and d-Mock) on breast cancer cells and normal mammary cells. Methods: The proliferation inhibition rates of doxorubicin and recombinant adenovirus(Ad-VT, Ad-T, Ad-VP3and Mock) on breast cancer cells were detected through WST-1 experiment, and the effects of two drugs on the inhibitory rates of normal mammary epithelial cells were also detected. Moreover, the apoptosis rates of doxorubicin and oncolytic adenoviruses on breast cancer cells and normal mammary epithelial cells were evaluated by Annexin V flow cytometry, Hoechst and JC-1 staining, and the difference in the apoptosis rates were also compared. Results: All the recombinant adenovirus could effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), the inhibition effects followed the order ofAd-VT>Ad-T>Ad-VP3>Ad-MOCK, and the inhibition effect was positively correlated with time. Doxorubicin could also effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), and the inhibition effect was markedly enhanced with the increases in does and time. However, doxorubicin also showed strong inhibition effect on the normal mammary epithelial cells, and the inhibition rate achieved 80% under 72 h and 5 ug/ml doxorubicin, while that of oncolytic adenovirus Ad-VT on MCF-10A was 20% at 72 h. The apoptosis effects of oncolytic adenoviruses-induced breast cancer cellwere increased with time, and the apoptosis rate efficiency followed the order of Ad-VT>Ad-T>Ad-VP3>Ad-MOCK, but they displayed low ability to induce normal mammary cell apoptosis. The apoptosis effects of doxorubicin-induced breast cancer cell were similar to that of the normal mammary epithelial cell (P <0.05 or P<0.01), which followed the dose of 0.05<0.5<5 μg/ml. Conclusion: Dual specific oncolytic adenoviruses can effectively suppress the proliferation of breast cancer cells, but they have low inhibition on normal mammary cells, which have displayed superior safety and provide a new method for the biotherapy of tumor.
ABSTRACT
Objective To investigate whether miR-210 plays a crucial role in the process of inducing the transfor-mation from co-cultured adipose derived(AD)-MSCs into CAFs. Methods Co-cultured adipose derived (AD)-MSCs with breast cancer cells (BCCs) lines in vitro and detected the expression of miR-210 using RT-qPCR. The expression of CAFs characteristic markers including α-SMA and tenascin-c were measured by RT-qPCR and the ex-pression of α-SMA and FAPA were assessed by Western blot.Co-injected MCF-7 cells transfected with miR-210 in-hibitor or miR-NC with MSC at 1 : 1 ratio into the immunodeficient nude mice subcutaneously and observed tumor growth in vivo constantly. Results miR-210 was all up-regulated when a variety of breast cancer cells were co-cul-tured with MSCs (P<0.05). Inhibition of miR-210 in tumor cell could inhibit the transformation from MSCs into CAFs(P<0.05). Animal experiments further confirmed that inhibition of miR-210 in tumor cell could reduce tumor growth (P<0.05). Conclusions As an important information molecule, miR-210 mediates the transforma-tion from MSCs to CAFs in the tumor microenvironment.