Investigation of Methylation Profiles of TP53, Caspase 9, Caspase 8, Caspase 3 Genes Treated with DNA Methyl Transferase Inhibitor (DNMTi) Zebularine (ZEB) and Caffeic Acid Phenethyl Ester (CAPE) on MCF-7 and MDA-MB-231 Breast Cancer Cell Lines

Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined application of TP53, caspase-9, caspase 8 and caspase-3 genes as a result of the use of single and combined drug methylation profiles are aimed to be evaluated by specific PCR method. Material-Metods: In the MCF-7 and MDA-MB-231 breast cancer cell lines, MTT test and survival analysis were performed as a result of single and combined application of CAPE and Zebularine and Methylation Specific PCR was performed to examine the methylation of caspase-3, caspase-8, caspase-9 and TP53 genes. Results: According to the results of 24-hour drug administration, the IC50 for the MCF-7 cell line was determined as 200 μM, for CAPE 40 μM and for the combined values of 50 μM ZEB + 5 μM CAPE. The effects of caspase-3, caspase-8, caspase-9 and TP53 genes on the methylation level of ZEB, CAPE and ZEB + CAPE drug combination were determined by using bisulfite modified DNAs in MCF-7 and MDA-MB-231 cell lines. Discussion: In the MCF-7 cell line, the 120 μM ZEB viability rate was 51%, and the viability of 80 μM ZEB MDA-MB-231 breast cancer cells decreased by 59.7%. After 20 μM CAPE, viability in MCF-7 cells decreased by 31% in 120 μM CAPE and MDA-MB-231 cells decreased by 41%. The viability with 40 μM CAPE decreased by 19% in MDA-MB-231 cells. It was found that 20 μM CAPE concentration was associated with TP53 methylation in MCF-7 cell lines. The 80 μM ZEB concentration was found to be closely related to the unmethylated status of the TP53 gene. These results obtained with 50 μM ZEB + 5 μM CAPE application were found to be related to the methylated-unmetylated status of the TP53 gene in half (50%). For the caspase-9 gene of MDA-MB-231 cells, 80 μM ZEB concentration was found to be associated with unmetylated status. The effective use of drugs with low concentrations of the drug dose provides a more appropriate approach in terms of treatment.

Methanolic extract of Abrus precatorius promotes breast cancer MDA-MB-231 cell death by inducing cell cycle arrest at G0/G1 and upregulating Bax

Objective:To determine the anti-proliferative activity of Abrus precatorius(A.precatorius)leaf extracts and their effect on cell death.Methods:A.precatorius leaves were extracted successively with hexane,ethyl acetate and methanol by Soxhlet extraction.Aqueous extract was prepared by decoction at 50 ℃.Extracts of A.precatorius leaves were used to treat selected cancer and normal cell lines for72 h.Furthermore,3-(4,5-dimethyl thiazol-2-yl)2,5-diphenyl tetrazolium bromide assay was performed to determine cell viability.Analysis of cell cycle arrest,apoptosis assay and apoptosis protein expressions were determined by flow cytometry.Results:Methanolic extract of A.precatorius leaves showed the lowest IC50 on MDA-MB-231 cells at(26.40±5.40)μg/mL.Flow cytometry analysis revealed that cell arrest occurred at G0/G1 phase and the apoptosis assay showed the occurrence of early apoptosis at 48 h in MDAMB-231 cells treated with methanolic extract of A.precatorius leaves.Methanolic extract of A.precatorius leaves induced apoptosis by upregulation of Bax,p53 and caspase-3 and downregulation of Bcl-2.Conclusions:Methanolic extract of A precatorius leaves promotes MDA-MB-231 cell death by inducing cell cycle arrest and apoptosis possibly via the mitochondrial-related pathway.

Anticancer property of sediment actinomycetes against MCF-7 and MDA-MB-231 cell lines

ObjectiveTo investigate the anticancer property of marine sediment actinomycetes against two different breast cancer cell lines.MethodsIn vitro anticancer activity was carried out against breast (MCF-7 and MDA-MB-231) cancer cell lines. Partial sequences of the 16s rRNA gene, phylogenetic tree construction, multiple sequence analysis and secondary structure analysis were also carried out with the actinomycetes isolates.ResultsOf the selected five actinomycete isolates, ACT01 and ACT02 showed the IC50 value with (10.13±0.92) and (22.34±5.82) μg/mL concentrations, respectively for MCF-7 cell line at 48 h, but ACT01 showed the minimum (18.54±2.49 μg/mL) level of IC50 value with MDA-MB-231 cell line. Further, the 16s rRNA partial sequences of ACT01, ACT02, ACT03, ACT04 and ACT05 isolates were also deposited in NCBI data bank with the accession numbers of GQ478246, GQ478247, GQ478248, GQ478249 and GQ478250, respectively. The phylogenetic tree analysis showed that, the isolates of ACT02 and ACT03 were represented in group I and III, respectively, but ACT01 and ACT02 were represented in group II. The multiple sequence alignment of the actinomycete isolates showed that, the maximum identical conserved regions were identified with the nucleotide regions of 125 to 221st base pairs, 65 to 119th base pairs and 55, 48 and 31st base pairs. Secondary structure prediction of the 16s rRNA showed that, the maximum free energy was consumed with ACT03 isolate (-45.4 kkal/mol) and the minimum free energy was consumed with ACT04 isolate (?7.6 kkal/mol).ConclusionsThe actinomycete isolates of ACT01 and ACT02 (GQ478246 and GQ478247) which are isolated from sediment sample can be further used as anticancer agents against breast cancer cell lines.

Down-Regulation of Notchl and NF-κB by Curcumin in Breast Cancer Cells MDA-MB-231

Objective： To test whether the down-regulation of Notchl gene expression by curcumin could inhibit cell growth and induce apoptosis, which may be associated mechanistically with the down-regulation of NF-κB in breast cancer cells. Methods： Breast cancer cell lines MDA-MB-231 were cultured in vitro and treated with different dosages of curcumin （0, 1.25, 5.0, 20.0μmol/L） for dose-dependent assay and different time （0, 24, 48, 72 h） at the dosage of 5.0μmol/L for time course assay. The changes of the mRNA and protein expression of Notchl and NF-κB were measured by RT-PCR and Western Blot, and MTT assay was used to measure the change of proliferation. Results： The mRNA and protein levels of Notchl and NF-κB were decreased significantly in human breast cancer cell line with the increase of dosage of curcumin（P〈0.05）, and with the extension of time course（P〈0.05）. These changes suggested a dose- and time-dependent manner. The proliferation rate of cells also was significantly inhibited（P〈0.05）. Conclusion： The current results show that the Notch-1 signaling pathway is associated mechanistically with NF-κB activity during curcumin-induced cell growth inhibition and apoptosis of breast cancer cells. These results suggest that the down-regulation of Notch signaling by curcumin may be a novel strategy for the treatment of patients with breast cancer.

Inhibition of γ-synuclein (SNCG) expression in breast cancer MDA-MB231 cell line

Objective:The aim of the study was to evaluate the inhibition of different chemotherapy drugs on γ-synuclein (SNCG) positive expression of breast cancer cell line MDA-MB231,and the effects on cell cycle and apoptosis,and to explore the related mechanism as well.Methods:We treated the breast cancer cell line MDA-MB231 for the inhibition of SNCG with chemotherapy drugs such as irinotecan,nedaplatin and 5-fluorouracil using RT-PCR and immunohistochemistry,and adopted flow cytometry to detect cell cycle distribution and apoptosis.Results:At the transcription and translation levels,the SNCG expression level in nedaplatin group and 5-fluorouracil group was lower than that of other groups and there was statistically significance (P < 0.01) compared with the control group,while there was not statistically significant between irinotecan group and the control group.After drugs action,cell cycle and distribution in each experiment group changed obviously,where the cells in G0G1 phase increased,especially the cells in the nedaplatin group and 5-fluorouracil group changed most significantly,as well as the obvious change in the cells of nedaplatin group and 5-fluorouracil group in the apoptosis period.Conclusion:There was a stronger inhibition of SNCG expression in nedaplatin and 5-fluorouracil groups,and can cause significant cell cycle and apoptosis changes.It may also be concluded that nedaplatin and 5-fluorouracil could make effects by the mechanisms of inhibiting cancer cell proliferation and inducing cell apoptosis.

不同浓度吗啡复合罗哌卡因对乳腺癌MDA-MB-231细胞增殖、迁移、侵袭和细胞周期的作用影响

目的 观察不同浓度的吗啡复合罗哌卡因对乳腺癌MDA-MB-231细胞增殖、转移侵袭和细胞周期的影响。方法 人乳腺癌细胞MDA-MB-231细胞接种于培养板24h,随机分为8组:对照组(C组)、罗哌卡因400μg/ml组(R组)、吗啡3μg/ml组(LM组)、吗啡30μg/ml组(MM组)、吗啡300μg/ml组(HM组)、罗哌卡因400μg/ml组+吗啡3μg/ml组(R+LM组)、罗哌卡因400μg/ml+吗啡30μg/ml组(R+MM组)和罗哌卡因400μg/ml+吗啡300μg/ml组(R+HM组)。处理乳腺癌细胞MDA-MB-231细胞24h后,检测其增殖能力、迁移能力、侵袭能力和细胞周期。结果 人乳腺癌细胞MDA-MB-231细胞的增殖抑制情况:单独应用吗啡时,LM组、MM组、HM组均对人乳腺癌细胞MDA-MB-231细胞的增殖具有抑制作用(P<0.05),并且抑制率随着吗啡浓度的升高而依次增加。单独应用罗哌卡因时对人乳腺癌细胞MDA-MB-231细胞的增殖具有抑制作用(P<0.05)。吗啡与罗哌卡因联合应用时,高浓度吗啡组与罗哌卡因具有协同作用。人乳腺癌细胞MDA-MB-231细胞的迁移情况:单独应用吗啡时,LM组、MM组、HM组均能够抑制细胞迁移率(P<0.05),迁移率随着吗啡浓度的升高而依次降低。单独应用罗哌卡因能够抑制细胞迁移率(P<0.05)。吗啡与罗哌卡因联合应用时,低浓度和中浓度吗啡组与罗哌卡因具有协同作用(P<0.05)。人乳腺癌细胞MDA-MB-231细胞的侵袭情况:单独应用吗啡时,MM组、HM组均能够抑制细胞侵袭能力(P<0.05),并且侵袭能力随着吗啡浓度的升高而依次降低,单独应用罗哌卡因时能够抑制细胞的侵袭能力(P<0.05)。吗啡与罗哌卡因联合应用时,中、高浓度组吗啡和罗哌卡因具有协同作用。人乳腺癌细胞MDA-MB-231细胞的细胞周期情况:单独应用高浓度吗啡,能够抑制细胞进入G2/M期(P<0.05)。单独应用罗哌卡因,能够抑制细胞进入G2/M期(P<0.05),低浓度吗啡与罗哌卡因联合应用对于将细胞抑制在G0/G1期和S期具有协同作用(P<0.05)。结论 吗啡复合罗哌卡因能够抑制乳腺癌MDA-MB-231细胞的增殖、迁移和侵袭,具有联合作用并呈剂量依赖性。

Exploring the molecular mechanism of Epimedium brevicornu Maxim.in treating breast cancer via network pharmacology and in vitro experiments Author links open overlay panel

Objective To evaluate the therapeutic effects of Epimedium brevicornu Maxim.(EBM,Yin Yang Huo)on breast cancer using network pharmacology and in vitro validation.It also aimed to explore the novel targets and mechanisms of EBM in the treatment of breast cancer to facilitate the discovery of new drugs and their clinical application.Methods Network pharmacology was used to identify and screen the components and targets of EBM for breast cancer treatment.Molecular docking was further screened the effective components and targets of EBM.Wound-healing assays and flow cytometry analysis were used to detect the ability of two compounds to intervene in the migration and apoptosis of MDA-MB-231 cells,and their mechanism of action was further explored using western blotting experiments.Results EBM contained 19 active components.Among them wereβ-anhydroicaritin(Anhy)and isoliquiritigenin(Iso),which were selected for in vitro experiments.Treatment resulted in a dose-dependent suppression of MDA-MB-231 cell viability,with an IC50 of 23.73μmol/L for Iso and 21.28μmol/L for Anhy.In the wound healing assay,cells in Anhy and Iso groups exhibited considerable inhibition of migration at 48 h.In flow cytometry analysis,treatment with Iso(20μmol/L)for 96 h resulted in significantly higher levels of both early and late apoptosis in the Iso group than that in the control group(P=.004 and P=.014,respectively).Additionally,both Iso(20μmol/L)and Anhy(10 and 20μmol/L)induced cell necrosis at 96 h.Western blotting revealed that Anhy and Iso increased the expression of Bax and TBK1/NAK.Conclusion These findings suggested that Anhy and Iso,the two components of EBM,inhibit MDA-MB-231 cell proliferation and migration of and induce their apoptosis,providing substantial support for future studies on breast cancer.

基于ERK/NF-κB通路探讨苦杏仁苷对乳腺癌MDA-MB-231细胞增殖、侵袭、迁移的影响

目的探讨苦杏仁苷对乳腺癌MDA-MB-231细胞增殖、侵袭、迁移及细胞外信号调节激酶(ERK)/核因子(NF)-κB通路的影响。方法将乳腺癌MDA-MB-231细胞随机分成对照组、紫杉醇组(10μg/ml)、苦杏仁苷低剂量组(20μg/ml)、苦杏仁苷高剂量组(40μg/ml);以上各组每孔设6个平行样,培养72 h。实验结束后,噻唑蓝(MTT)法测定乳腺癌MDA-MB-231细胞增殖水平,Transwell小室测定乳腺癌MDA-MB-231细胞侵袭迁移水平,实时荧光定量聚合酶链反应(PCR)及Western印迹法测定乳腺癌MDA-MB-231细胞ERK、NF-κB mRNA和蛋白表达水平。结果与对照组比较,紫杉醇组、苦杏仁苷低、高剂量组光密度(OD)值、细胞存活率、穿膜数、ERK、NF-κB mRNA和蛋白表达水平显著降低(P<0.05);与紫杉醇组比较,苦杏仁苷低剂量组上述指标显著升高(P<0.05),苦杏仁苷高剂量组上述指标无显著差异(P>0.05);与苦杏仁苷低剂量组比较,苦杏仁苷高剂量组上述指标显著降低(P<0.05)。结论苦杏仁苷能明显抑制乳腺癌MDA-MB-231细胞增殖、侵袭、迁移,其机制可能与苦杏仁苷抑制ERK、NF-κB mRNA和蛋白表达进而抑制ERK/NF-κB通路的激活有关。

Cytotoxicity study of ethanol extract of the stem bark of asam kandis (Garcinia cowa Roxb.) on T47D breast cancer cell line

Objective:To investigate the cytotoxic effect of ethanol extract of the stem bark of asam kandis[Garcinia cowa Roxb.(G.cowa)]on T47 D breast cancer cell line.Methods:The cytotoxicity of ethanol extract was carried out against human breast cancer cell line(T47D) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay.The extract was added at various concentrations(0.1.1,10 and 100 μg/mL).The level of cytotoxicity was determined by calculating the level of IC_(50),that was based on the percentage of the cell death after 24 h treatment with the extract.Cell morphological changes were observed by using inverted microscope.Results:The 3-(4.5-dimelhylthiazol-2-yl)-2.5-diphenyltelrazolium bromide assay showed that ethanol extract of G.cowa exhibited significant cytotoxic effect on T47 D with IC_(50) value of(5.10+1.68) μg/mL.Morphological alteration of the cell lines after exposure to ethanol extract of G.cowa was observed under phase contrast microscope in a dosc-dependent manner.ConclusionsThe results suggest the possible use of ethanol extract of asam kandis for preparing herbal medicine for cancer-related ailments.

Effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells

Objective： To elucidate the effects of amlodipine on the proliferation and apoptosis of human breast carcinoma MDA-MB-231 cells. Methods： Light microscopy was used to determine the effects of amlodipine on cell morphology; Flow cytometry was used to quantitate cells undergoing apoptosis; the expression of a cell cycle-related protein, proliferating cell nuclear antigen （PCNA） and an antiapoptosis protein, Bcl-2 were assessed by immunocytochemistry. Results： Amlodipine concentration of 8.25umol/L （1/2 of ICs0） affected the morphology, decreased the expression of PCNA and Bcl-2 and induced apoptosis of human breast carcinoma MDA-MB-231 cells. Conclusion： The effect of amlodipine on the antiproliferation of human breast carcinoma MDA-MB-231 cells is related to inducement of apoptosis, and the decrease of the expression of Bcl-2 and PCNA may be the possible mechanism for proliferation inhibitory and inducement of apoptosis.

Cytotoxic Effect of Chitosan Based Nanocomposite Synthesized by Radiation： In Vitro Liver and Breast Cancer Cell Line

A silver nanoparticle （AgNP） is likely to provide an attractive object for combining a variety of biochemical properties with great therapeutic potential by using radiation. The present study explores the ICs0 value of chitosan-poly （vinyl alcohol） hydrogel （Cs/PVA） and Ag-doped chitosan-poly （vinyl alcohol） （Cs/PVA/Ag） nanocomposite in view of their anticancer application. The aim was to develop （Cs/PVA） based hydrogel synthesized by gamma radiation which could behave both as a nanoreactor for Ag nanoparticle with promising anticancer applications. The （Cs/PVA/Ag） nanocomposite was confirmed by FTIR （Fourier transform infrared） spectroscopy, XRD （X-ray diffraction） and EDX （energy dispersive X-ray） analysis. The anti-cancer activity of the prepared nanocomposites was demonstrated in human liver cancer cell line （HEPG2） and breast cancer cell lines （MCF7）. It has significant effects against human liver cancer cell line HEPG2 compared to breast cancer cell line MCF7. Further quantitative analysis on the molecular and protein levels is still required to confirm the impact of chitosan on genotoxic effect before reaching a final conclusion and starting its biomedical application.

Inhibitory effect of gold nanoparticles conjugated with interferon gamma and methionine on breast cancer cell line

Objective: To develop a gold nanoparticles complex conjugated with interferon-gamma(IFN-g) and methionine along with application of hyperthermia using near-infrared laser beams for the treatment of cancer cells.Methods: Gold nanorods(10 nm) were conjugated with IFN-g and methionine using carbodiimide family and characterized after purification by dialysis bags. Breast cancer cells were cultured and incubated with gold nanorods at different concentrations followed by irradiation with near-infrared laser beam. Samples were then evaluated for their viability in order to determine the effect of treatment and variables by MTT assy.Results: Zetasizer results confirmed the conjugation of gold nanorods with methionine and IFN-g. The median percentage of cell viability in 0.30 mg/m L concentration of gold nanorods was 82%. The cell viability reached to 85% at the same concentration of gold nanorods, which existed in the assayed complex. The results of MTT assay showed that the 0.60 mg/m L concentration of gold nanoparticles complex was toxic on tumor cells(P < 0.05). After exposure to hyperthermia, the viability of cells at 6 min decreased to77% in 0.30 mg/m L concentration of gold nanorods complex.Conclusions: The size and concentration of gold nanorods was not cytotoxic. However,their presence during irradiation near-infrared laser increased the number of dead cells during the treatment of cells.

Investigating the effects of Pentoxifylline on human breast cancer cells using Raman spectroscopy

Breast cancer is one of the leading causes of cancer-related deaths in a global scenario.In the present study,biochemical changes exerted upon Pentoxifylline(PTX)treatment had been ap-praised in human breast cancer cells using Raman spectrosecopy.There are no clinically approved methods to monitor such therapeutic responses available.The spectral profiling is suggestive of changes in DNA,protein and lipid contents showing a linear relationship with drug dosage.Further,multivariate analysis using principal component based linear-discriminant-analysis(PC-LDA)was employed for dlassifying the control and the PTX treated groups.These findings support the feasibility of Raman spectroscopy as an alternate/adjunct label-free,objective method for monitoring drug-induced modifications against breast cancer cells.

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.

THE EFFECTS OF ESTRADIOL ON BREAST CANCER CELL LINES

The estradiol effects on breast cancer cell lines including estrogen receptors (ER) positive and negative were studied with flow cytometry analysis, scanning and transmission electron microscopy (SEM and TEM), immunogold and immunofluorescence staining techniques. The results showed that estradiol markedly stimulated the division and proliferation of the ER( + ) MCF-7 cells at 10 nM, but had no marked effect on the cell cycle of the ER(-) H466B cells at the same concentration, and that tamo-xifen inhibited the stimulation of estradiol on MCF-7 cells. Estradiol obviously influenced the ultrastruc-ture of MCF-7 cells. Immunocytochemical localization of epidermal growth factor receptor (EGFR) on the MCF-7 cell membrane surface indicated that one of the mechanisms involving the growth of MCF-7 breast cancer cell and the stimulating effect on MCF-7 cells growth by estradiol is autocrine secretion.

REVERSION OF MULTIDRUG RESISTANCE IN THE P-GLYCOPROTEIN POSITIVE BREAST CANCER CELL LINE(MCF-7/ADR) BY INTRODUCTION OF HAMMERHEAD RIBOZYME

A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

咪达唑仑诱导乳腺癌细胞MDA-MB-231凋亡并抑制细胞恶性增殖、干样特性及AKT磷酸化

目的 探讨咪达唑仑对乳腺癌细胞MDA-MB-231增殖、凋亡、干样特性及蛋白激酶B(AKT)的影响。方法 CCK8法检测不用浓度咪达唑仑对乳腺癌细胞MDA-MB-231活力的影响,筛选合适的剂量;克隆形成检测细胞增殖;流式检测细胞凋亡;Western印迹检测凋亡相关蛋白的表达[半胱天冬酶(Caspase)-3、Caspase-9];显微观察干细胞成球;Western印迹检测干细胞标志物八聚体结合转录因子(OCT)4、性别决定区γ框蛋白(SOX)2和AKT的磷酸化;免疫荧光检测AKT的核转位。结果 与对照组相比,75μmol/L咪达唑仑组对MDA-MB-231细胞活力的影响无明显差异,随浓度的增加,细胞活力明显减少。CCK8实验结果,筛选4个剂量组(0、75、150、300μmol/L),进行后续实验;与对照组相比,除75μmol/L剂量组差异无统计学意义外,150、300μmol/L剂量组,细胞凋亡率、凋亡蛋白表达水平明显升高(P<0.05);克隆细胞形成率、干细胞成球数量及直径、OCT4及SOX2表达量、AKT的磷酸化表达均明显降低。结论 咪达唑仑诱导乳腺癌细胞MDA-MB-231凋亡并抑制肿瘤细胞恶性增殖、干样特性,其机制可能是通过下调SOX2、OCT4表达,减少癌细胞内AKT信号通路的调节,从而抑制AKT磷酸化水平及AKT核转位。缓解乳腺癌的发生发展。

In Vivo Animal Model Evaluation of a Powerful Oral Nanomedicine for Treating Breast Cancer in BALB/c Mice Using 4T1 Cell Lines without Chemotherapy

Nanopharmaceuticals containing quantum dot nanoparticles (Q-Dot NPs) for treating serious cancers such as breast cancer have made fantastic proposals. In this study, ZnO quantum dot NPs are formulated via ZnO@PVP nanopolymer as co-assistants coordinating with efficacious suitable wetting agents, PEG-binding compound, and W/O emulsifier for producing eco-friendly water-based nanodrug. Several characterization techniques containing SEM, TEM, FTIR, photoluminescence, zeta potential, and UV-Vis absorption were employed for ZnO Q-Dot NPs in nanodrug. This work aims to investigate the anti-tumor effects of such nanomedicine on the 4T1 breast cancer cell line in BALB/c mice, being elaborated through intraperitoneal, injection (IVP) and oral therapy. The impressive findings showed that ZnO nanodrug caused changes in blood factors, having the most effectiveness at 40 μg/ml concentration after two weeks of oral treatments. The significant increase in white blood cells (WBC) neutrophils and meaningful decreases in lymphocytes and especially cholesterol were powerful simultaneous impacts, successfully treating malignant breast cancer masses. In this significant animal model research for breast cancer, the sick mice recovered entirely and even had a safe space to mate. Histopathological results showed no evidence of breast tumor formation or metastasis in the group treated with nanodrug and their children. This nanomedicine has a therapeutic effect, and is ready to be applied for treating volunteer breast cancer patients. However, its prevention (inhibitory) effect can also be analyzed and added to current data in future studies.

Studies on mechanism of cis9,trans11-CLA and trans10,cis12-CLA inducing apoptosis of human breast cancer cell line MCF-7

Objective： The aim of the study was to explore the activities of cis9, trans11-CLA （C9, t11-CLA） and transl0, cis12-CLA （t10, c12-CLA） inhibiting tumor, and investigate their relationships with PPARy and apoptotic proteins, and mechanism of anti-cancer. Methods： The inhibitory rate, cell growth curve and apoptotic morphological observation of MCF-7 cells were obtained by MTT assay, trypan blue staining and Hoechst33342 fluorescence staining. The apoptotic rate and cell cycle were detected with flow cytometry. Transcriptional level of genes was detected with RT-PCR semi-quantitative method, and Western blot was performed to detect proteins levels. Results： The two CLA isomers could reduce cell proliferation （P 〈 0.05）, increase apoptotic rate （P 〈 0.05）, and increase obviously the transcriptional and protein levels of PPARy （P 〈 0.01）. The synchronism and correlation between the effects of CLA to PPARy and apoptotic proteins Bax, Bcl-2, Caspase 3 changes were found with the dose- and time-dependent manners. There was cooperative relation between the levels of PPARy and the rates of Bax/Bcl-2, Caspase 3 （small fragment） by experiments of PPARy inhibitor GW9662 and ligand Rosiglitazone. Conclusion： The apoptotic pathway of PPARy-Bcl-2-Caspase 3 signaling was found. The C9, t11-CLA and tl0, c12-CLA could inhibit MCF-7 cell proliferation and promote apoptosis via activating PPARy-Bcl-2-Caspase 3 pathway. CLA may be a kind of activator of PPARv.

过表达lncRNA LINC00886靶向调控miR-451a影响乳腺癌MDA-MB-231细胞生物学行为

目的:探究过表达长链非编码RNA(lncRNA)LINC00886通过调控微小RNA-451a(miR-451a)对乳腺癌(BC)MDA-MB-231细胞生物学行为的影响。方法:荧光定量PCR(qRT-PCR)检测人乳腺上皮细胞系(MCF-12A)和4种BC细胞系(HCC1937、SUM159、SK-BR-3和MDA-MB-231)中lncRNA LINC00886、miR-451a表达。将MDA-MB-231细胞分为对照组、LINC00886-NC组、LINC00886组、LINC00886+miR-NC组、LINC00886+miR-451a组。qRT-PCR法检测MDA-MB-231细胞中lncRNA LINC00886、miR-451a表达水平;克隆形成实验和EdU染色检测MDA-MB-231细胞增殖能力;流式细胞术、Transwell小室实验、划痕愈合实验分别用来检测MDA-MB-231细胞凋亡、侵袭、迁移;蛋白印迹法检测MDA-MB-231细胞中增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、活化的半胱氨酸天冬氨酸蛋白酶3(Cleaved caspase-3)、基质金属蛋白酶(MMP)-2、MMP-9蛋白表达;双荧光素酶报告基因检测lncRNA LINC00886与miR-451a的靶向关系。结果:与人乳腺上皮细胞MCF-12A相比,BC细胞系中lncRNA LINC00886表达水平降低,miR-451a表达水平升高(P<0.05);过表达lncRNA LINC00886可升高MDA-MB-231细胞凋亡率、Bax和Cleaved caspase-3蛋白表达,降低miR-451a表达、集落形成数、EdU阳性细胞百分比、侵袭细胞数、迁移率以及PCNA、Bcl-2、MMP-2、MMP-9蛋白表达(P<0.05);上调miR-451a表达可减弱lncRNA LINC00886过表达对MDA-MB-231细胞的影响(P<0.05);lncRNA LINC00886可靶向调控miR-451a表达。结论:过表达lncRNA LINC00886可通过靶向抑制miR-451a表达促进MDA-MB-231细胞凋亡并抑制MDA-MB-231细胞增殖、侵袭和迁移。