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.

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.

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.

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.

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

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 IC_(50) 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。

Piperine suppresses growth and migration of human breast cancer cells through attenuation of Rac1 expression

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.

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.

Meclizine Chloridrate and Methyl-β-Cyclodextrin Associated with Monophosphoester Synthetic Phosphoethanolamine Modulating Proliferative Potential in Triple-Negative Breast Cancer Cells

Synthetic phosphoethanolamine(Pho-s)is a monophosphoester ester with anti-inflammatory and pro-apoptotic properties.Meclizine chloridrate(MC)is a histamine H1 receptor blocker that is also able to inhibit cellular respiration.However,MC does not inhibit cellular respiration in isolated mitochondria such as antimycin and rotenone.Methyl-β-cyclodextrin(MβCD)belongs to theβ-cyclodextrin family,which is capable of removing cholesterol from the plasma membrane.The aim of this study was to evaluate the proliferative effects of meclizine chloridrate and methyl-β-cyclodextrin compounds associated with synthetic phosphoethanolamine in a triple-negative human breast tumor line,MDA-MB-231 Cell viability of the tumor line and normal cells FN1 was evaluated by MTT colorimetric test;the production of free radicals was determined by lipoperoxidation(LPO)test;and the percentage of cell cycle phases and proliferative index was evaluated by flow cytometry.Cell viability demonstrated a significant decrease with the treatments of MβCD,MC and Pho-s associated with MC.The production of free radicals decreases significantly in all treatments.In addition,a significant increase of DNA fragment and decrease in G0/G1 cell cycle phase were observed in cellular percentage with concentrations of 20 and 30 mM of Pho-s in association with MC and MβCD,respectively.

Effect of Guizhi Fuling capsule and its extracts on human breast cancer cells proliferation

OBJECTIVE To evaluate the effect of Guizhi Fuling Capsule active pharmaceutical ingredient(API)and its fractions on human breast cancer cells proliferation by high-throughput screening assay.METHODS The crude fractions were obtained from the extraction and elution of the API of Guizhi Fuling Capsule,and 929 standard fractions were obtained by the optimal separation conditions.Sulforhodamine B(SRB)method was used to evaluate the effects of the Guizhi Fuling capsule API and929 kinds of fractions on the proliferation of human breast cancer cells MCF-7 and MDA-MB-231.RESULTS The Guizhi Fuling capsule API had a strong ability to inhibit the proliferation of MCF-7 cells at high concentration and the ability to inhibit MDA-MB-231 cells' proliferate at low concentration following 72 h treatment;some samples of 929 fractions(5μg·mL^(-1))was found to have a breast cancer cell growth inhibition rate above 50%,without toxicity on HUVECs proliferation.CONCLUSION The API of Guizhi Fuling capsule had significant cytotoxicity effects on these two human breast cancer cells,with significant concentration-and time-dependent manner.

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.

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.

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。

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。

去甲斑蝥素通过诱导自噬体聚集促使乳腺癌MDA-MB-231细胞凋亡

探究去甲斑蝥素(norcantharidin,NCTD)对三阴性乳腺癌MDA-MB-231细胞增殖和凋亡的影响。采用Western blot实验检测NCTD对MDA-MB-231细胞中凋亡相关蛋白Bax/Bcl-2、cleaved-PARP/PARP、cleaved-caspase-9、cleaved-caspase-3和MCL-1表达水平的影响;采用Western blot实验检测NCTD对MDA-MB-231细胞中自噬相关蛋白LC3-II/LC3-I,Parkin和PINK1表达水平的影响;采用流式细胞术检测NCTD对MDA-MB-231细胞线粒体膜电位及线粒体活性氧(reactive oxygen species,ROS)变化情况的影响;采用共聚焦显微镜检测NCTD对表达mCherry-EGFP-LC3的MDA-MB-231细胞自噬流的影响;采用流式细胞术检测NCTD联合使用氯喹(chloroquine,CQ)或3-甲基腺嘌呤(3-methyladenine,3-MA)后,对MDA-MB-231细胞凋亡情况的影响。实验结果显示,NCTD可显著上调Bax/Bcl-2、cleaved-PARP/PARP、cleaved-caspase-9、cleavedcaspase-3以及LC3-II/LC3-I蛋白的表达水平,促进Parkin的线粒体易位,阻断自噬流。此外,NCTD联合使用CQ加剧了细胞凋亡,而NCTD联合使用3-MA则减少了细胞凋亡。研究结果表明,NCTD可以诱导自噬体积累并导致MDA-MB-231细胞凋亡。

TRAIL联合顺铂诱导三阴乳腺癌MDA-MB-231细胞过度性自噬的分子机制

流行病学报道,乳腺癌成为目前女性发病率和死亡率最高的癌症[1]。而铂类药物也是临床上最常用的周期非特异性抗肿瘤药物,在临床治疗实体肿瘤中显示了良好的疗效[2]。其中顺铂作为一线临床的传统用药,其靶点选择范围窄、疗效差、易耐药等问题,尚未得到解决。肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)属于肿瘤坏死因子(tumor necrosis factor,TNF)超家族。

复方山慈菇对人乳腺癌MDA-MB-231细胞裸鼠移植瘤生长的抑制作用及网络药理学分析

目的探讨复方山慈菇对人乳腺癌MDA-MB-231细胞裸鼠移植瘤生长的抑制作用及其潜在的作用机制。方法将人乳腺癌MDA-MB-231细胞裸鼠移植瘤模型随机分为模型组、阳性对照多柔比星(doxorubicin,DOX)组和复方山慈菇醇提物(compound cremastra appendiculata ethanol extraction,CCAEE)高剂量组(1.8 g/kg)以及低剂量组(0.9 g/kg),每组5只。按照分组连续灌胃给药21 d后,处死裸鼠并计算各组裸鼠移植瘤体积、重量和抑瘤率。采用TCMSP 2.3数据库选出复方山慈菇有效成分及其对应的作用靶点,OMIM、GeneCards 5.14、DrugBank 5.1.10、TTD数据库筛选关键靶点并利用STRING11.5数据库构建蛋白质相互作用(PPI)网络,然后筛选核心靶点,以构建“药物-成分-靶点”网络并筛选出核心有效成分。利用Metascape 3.5平台进行GO和KEGG富集分析。结果各组荷瘤裸鼠分别给药处理21 d后,与模型组比较,DOX组和CCAEE高、低剂量组裸鼠移植瘤体积、重量均明显减小(均P<0.05),抑瘤率分别为89.67%、67.39%和43.48%。通过网络药理学分析获得复方山慈菇有效成分27个,抗乳腺癌关键靶点108个,其中核心靶点23个,主要包括TP53、MAPK3、MAPK1、RELA、AKT1、FOS、ESR1、TNF、IL6、MAPK14等;核心有效成分5个,分别为槲皮素、山柰酚、β-谷甾醇、2-甲氧基-9,10-二氢菲-4,5-二醇、去氧鬼臼毒素。GO和KEGG富集分析显示复方山慈菇主要通过激素调节等生物过程以及PI3K-AKT、MAPK等信号通路发挥抗乳腺癌作用。结论复方山慈菇能抑制人乳腺癌移植瘤生长,其可能通过槲皮素、山柰酚等核心有效成分作用于TP53等核心靶点及PI3K-AKT、MAPK等信号通路而发挥抗乳腺癌作用。

蚊母草提取物对乳腺癌MDA-MB-231细胞体外运动能力的影响

目的研究蚊母草提取物(Extracts from Veronica peregrina L.,EVP)对乳腺癌MDA-MB-231细胞体外运动能力的影响。方法采用MTT法检测EVP 40、80、160、320、640 g·L^(-1)生药浓度(相当于提取物浓度0.168、0.336、0.672、1.344、2.688 mg·mL^(-1))对乳腺癌MDA-MB-231细胞体外增殖的影响,计算各EVP处理组的细胞相对存活率。将MDA-MB-231细胞分为空白组、阳性对照组(BB-94组,终浓度为20 nmol·L^(-1))、EVP不同浓度组(终质量浓度为生药40、80、160 g·L^(-1)的EVP)。采用AP48 Chamber system和OrisTM细胞侵袭试剂盒分别检测EVP对乳腺癌MDA-MB-231细胞体外迁移和侵袭能力的影响;鬼笔环肽(Phalloidin)染色法观察MDA-MB-231细胞骨架F-actin排列情况;Western Blot法检测细胞Runx2、磷酸化Runx2(p-Runx2)及基质金属蛋白酶9(MMP-9)蛋白表达;采用SensoLyte^(■)570 Generic MMP检测试剂盒分析MDA-MB-231细胞的MMPs活性。结果EVP的终浓度分别为生药40、80、160、320、640 g·L^(-1)时,MDA-MB-231细胞相对存活率分别为(105.63±4.62)%、(92.81±8.34)%、(103.32±17.47)%、(69.08±4.32)%、(41.82±12.63)%,低浓度EVP对MDA-MB-231细胞的生存无明显影响,后续研究中采用生药40、80、160 g·L^(-1)作为各EVP处理组的终浓度。与空白组相比,BB-94组及EVP生药40、80、160 g·L^(-1)浓度组的迁移细胞数显著减少(P<0.01)。空白组的绝大多数细胞可突破基质胶,侵袭至中央空白处,几乎完全覆盖中央圆形无细胞区域;而BB-94组及EVP各浓度组的MDA-MB-231细胞侵袭则明显被抑制,均不能完全侵袭至中央区域,且中央无细胞区域的大小呈现一定的剂量依赖性。空白组MDA-MB-231细胞中,F-actin沿细胞长轴整齐排列,F-actin在细胞周边聚集成应力纤维;而BB-94组及EVP各浓度组的MDA-MB-231细胞内,F-actin均呈无规则的紊乱排列,应力纤维明显减少。与空白组相比,EVP生药40、80、160 g·L^(-1)浓度组MDA-MB-231细胞的Runx2蛋白表达无明显变化(P>0.05),而p-Runx2、MMP-9蛋白表达均显著下调(P<0.01),MMPs活性明显降低(P<0.05,P<0.01)。结论EVP可显著抑制乳腺癌MDA-MB-231细胞的体外运动能力,具有潜在的抗转移作用,其机制可能与抑制Runx2活化,进而干扰其下游MMP-9等靶基因表达有关。

薯蓣丸联合紫杉醇抑制三阴性乳腺癌细胞MDA-MB-231上皮间质转化作用和机制研究

目的:探究薯蓣丸联合紫杉醇抑制人三阴性乳腺癌MDA-MB-231细胞上皮间质转化(EMT)的作用和机制。方法:运用血清药理学方法制备SD大鼠空白血清和薯蓣丸含药血清,体外培养MDA-MB-231细胞。实验将其分组为空白组、紫杉醇组、薯蓣丸联合紫杉醇组三组。运用CCK8实验检测MDA-MB-231细胞的增殖情况;Transwell小室实验法检测细胞的侵袭、迁移能力变化;Western blot检测上皮间质转化相关分子标志物E-cadherin、Vimentin、N-cadherin、MMP2和PI3K/Akt/mTOR信号通路相关蛋白的表达;RT-qPCR检测E-cadherin、Vimentin、N-cadherin、MMP2 mRNA的表达。结果:与空白组相比,紫杉醇组和薯蓣丸联合紫杉醇组可显著抑制细胞的增殖、侵袭和迁移(P<0.01);与紫杉醇组相比,薯蓣丸联合紫杉醇组对细胞增殖、侵袭和迁移的抑制能力增强(P<0.01)。与空白组相比,紫杉醇组和薯蓣丸联合紫杉醇组细胞中E-cadherin蛋白表达上调,PI3K、Akt、mTOR、N-cadherin、Vimentin蛋白表达下调(P<0.05,P<0.01);与紫杉醇组相比,薯蓣丸联合紫杉醇组细胞中E-cadherin蛋白表达上调,Vimentin、MMP2、PI3K、Akt、p-Akt、mTOR、p-mTOR蛋白表达下调(P<0.05,P<0.01)。与空白组比较,紫杉醇组和薯蓣丸联合紫杉醇组均能上调E-cadherin,下调Vimentin、MMP2 mRNA的表达(P<0.01);与紫杉醇组比较,薯蓣丸联合紫杉醇组上调E-cadherin,下调N-cadherin、MMP2 mRNA表达能力增强(P<0.05,P<0.01)。结论:薯蓣丸联合紫杉醇可有效抑制三阴性乳腺癌细胞的侵袭、迁移和EMT进程,且效果优于紫杉醇单用,其机制可能与PI3K/Akt/mTOR信号通路受抑制相关。