Study on regulating mechanisms of oxocrebanine obtained from Stephania hainanensis H.S.Lo et Y.Tsoong on microtubule sites and tubulin in human breast cancer MCF-7 cells

Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.

Fucoidan Induces G_1 Phase Arrest and Apoptosis through Caspases-dependent Pathway and ROS Induction in Human Breast Cancer MCF-7 Cells

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D 1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, cas- pase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS pro- duction was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce GI phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.

Dietary Daidzein Enhances Antiapoptotic Effect of 17β-Estradiol (E_2) on Breast Cancer MCF-7 Cells

Objective： To investigate whether dietary daidzein interact with endogenous 17β-Estradiol （E2） to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods： Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein （1, 5 μmol/L） and E2 （0.1-10 nmol/L） for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha （ERα）, estrogen receptor beta （ERI3） and ERβ-estrogen response element （ERE） dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptotic p53, bax and antiapoptotic bcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot. Results： Daidzein enhanced the antiapoptotic effect in an Ea dose-dependent manner, but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition, daidzein only antagonized E2-upregulated expression of p53 and bax, but had no effect on E2-upregulated expression of bcl-2. Conclusion： Daidzein enhances the antiapoptotic effect of E2 on breast cancer cells by inhibiting E2-mediated p53-bax proapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2 in hormone-dependent breast cancer.

Synthesis, Characterization, and Evaluation of Antitumor Potential in MCF-7 Cells of Ruthenium-Derived Compounds

To synthesize, characterize and evaluate the antitumor potential derived from ruthenium compounds was generated in this study, from the precursor K[RuCl4(bipy)] a route in a simple and reproducible synthesis for a novel compound of coordinating Ru+3 with bipy and L-trip. The spectroscopic characterization in the middle infrared region (FTIR) shows the interactions between Ru-(L-trip), evidenced by the displacement of the carboxylate ion band for higher energies, and also by the displacements of aliphatic amine bands, suggesting that bidentate coordination of the L-trip ligand occurred. Analysis of the results obtained with thermoanalytical techniques showed that the minimum formula of the compound, [RuCl2(bipy)(L-trip)]1/2H2O. Evaluation of the antitumor potential of precursor K[RuCl4(bipy)] showed the toxic effects on MCF-7 cell line, but did not show selectivity and not reached PBMC cells to the same extent. The evaluation of the antitumor potential of the newly synthesized compound, [RuCl2(bipy)(L-trip)], demonstrated that the insertion of an L-tryptophan molecule into the precursor coordination sphere made it selective when compared to PBMC cells, for MCF-7 type tumor cells.

INHIBITION OF PROLIFERATION OF HUMAN BREAST CANCER MCF-7 CELLS BY SMALL INTERFERENCE RNA AGAINST LRP16 GENE

Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.

Plant extracts as natural photosensitizers in photodynamic therapy:in vitro activity against human mammary adenocarcinoma MCF-7 cells

Objective: To examine three plant extracts [Lumnitzera racemosa(Combretaceae)(L.racemosa), Albizia procera(Fabaceae)(A.procera) and Cananga odorata(Annonaceae)] for their potential as source of photosensitizers in photodynamic therapy.Methods: Human mammary adenocarcinoma(MCF-7) cells were treated with the plant extracts, which were irradiated with 5.53 m W and 0.553 mW broadband light.Cell viability was assessed using MTT assay and induction of apoptosis was determined using terminal deoxynucleotidyl transferase-dUTP nick end labeling assay.Results: The crude ethanolic extracts, independently, were nontoxic against cancer and non-cancer cells but when irradiated with 5.53 mW broadband light, L.racemosa and A.procera extracts were cytotoxic against MCF-7 with IC_(50) of 11.63 mg/mL and10.73 mg/mL, respectively.With 0.553 mW broadband light, the IC_(50) values were higher at 17.14 mg/mL and 19.59 mg/mL, respectively.Photoactivated L.racemosa and A.procera extracts were found to be more cytotoxic against MCF-7 than the non-cancer cell line, human dermal fibroblast-neonatal.Moreover, the cytotoxicity of the extracts was mediated by apoptosis.Conclusions: Two of the plant extracts used, L.racemosa and A.procera were toxic and induced apoptosis to mammary cell adenocarcinoma, MCF-7 when photoactivated.These extracts were also more toxic to human cancer than non-cancer cell lines.

The Influence of Concentration of the Antioxidants in Rosemary Extract on MCF-7 Cells

In my research,I studied the antioxidant effect of extracts of various rosemary samples obtained by Soxlet extraction on MCF-7 epithelial cells.I wanted to discover which of the extracts will have the highest content of total phenols determined in vitro with the Folin-Ciocalte reagent.The results show the highest content of total phenols in the rosemary sample from North Macedonia,so I assumed that it would have the biggest effective activity on MCF-7 cells.While doing the free radical inhibition test,I came to the conclusion that the plant with the highest antioxidant content does not necessarily have the best effect on the cells at different concentrations of the extracts.I suppose that it happens because antioxidants are polar and they need to transport through cell membrane conveyors whose number is limited in the membrane,that is,more antioxidants get assembled on the conveyors and cannot go through the membrane.

Study on Cisplatin Aggravating DNA Damage and Causing a High Apoptosis Rate on Breast Cancer MCF-7 Cells

[Objectives] To investigate the mechanism of DNA damage of cisplatin( DDP),a broad spectrum anticancer drug on breast cancer MCF-7 cells,and to study the mechanism of apoptosis induced by DDP.[Methods]MCF-7 cells were treated by DDP( 0 mg/L,2 mg/L,4 mg/L,6 mg/L,6 mg/L,and 10 mg/L) for 48 hours. MTT assay was used to detect the inhibitory effect of DDP on MCF-7 cells and IC50 value was calculated. Western blot was adopted to detect the expression of γ-H2 AX,which was the marker of DNA double stranded breaks( DSBs) and ATM( sensory molecules of DSBs),the apoptotic signal transduction molecule cleaved caspase-3,and the proteins associated with apoptosis calpain.[Results]DDP inhibited MCF-7 cell activity in a concentration-dependent manner and IC50 was 7. 57 mg/L. In contrast to the control group( without DDP treatment),MCF-7 cells with DDP treatment expressed more γ-H2 AX,ATM,cleaved caspase-3 and calpain.[Conclusions] DDP could inhibit the activity of breast cancer MCF-7 cells. Its mechanisms may be associated with inhibition of MCF-7 cell apoptosis,induction of DNA double strand breaking and the expression of pro-apoptotic protein up-regulation.

Time-dependent metabolomics uncover dynamic metabolic adaptions in MCF-7 cells exposed to bisphenol A

The biochemical consequences induced by xenobiotic stress are featured in dose-response and timeresolved landscapes.Understanding the dynamic process of cellular adaptations is crucial in conducting the risk assessment for chemical exposure.As one of the most phenotype-related omics,metabolome in response to environmental stress can vary from seconds to days.Up to now,very few dynamic metabolomics studies have been conducted to provide time-dependent mechanistic interpretations in understanding xenobiotics-induced cellular adaptations.This study aims to explore the time-resolved metabolite dysregulation manner and dynamically perturbed biological functions in MCF-7 cells exposed to bisphenol A(BPA),a well-known endocrine-disrupting chemical.By sampling at 11 time points from several minutes to hours,thirty seven significantly dysregulated metabolites were identified,ranging from amino acids,fatty acids,carboxylic acids and nucleoside phosphate compounds.The metabolites in different pathways basically showed distinct time-resolved changing patterns,while those within the common class or same pathways showed similar and synchronized dysregulation behaviors.The pathway enrichment analysis suggested that purine metabolism,pyrimidine metabolism,aminoacyl-tRNA biosynthesis as well as glutamine/glutamate(GABA)metabolism pathways were heavily disturbed.As exposure event continued,MCF-7 cells went through multiple sequential metabolic adaptations from cell proliferation to energy metabolism,which indicated an enhancing cellular requirement for elevated energy homeostasis,oxidative stress response and ER-αmediated cell growth.We further focused on the time-dependent metabolite dysregulation behavior in purine and pyrimidine metabolism,and identified the impaired glycolysis and oxidative phosphorylation by redox imbalance.Lastly,we established a restricted cubic splinebased model to fit and predict metabolite’s full range dysregulation cartography,with metabolite’sensitivity comparisons retrieved and novel biomarkers suggested.Overall,the results indicated that 8 h BPA exposure leaded to global dynamic metabolome adaptions including amino acid,nucleoside and sugar metabolism disorders,and the dysregulated metabolites with interfered pathways at different stages are of significant temporal distinctions.

雷公藤内酯醇通过调控miR-142-3p/HSP70通路抑制人乳腺癌MCF-7细胞增殖、侵袭和迁移

目的:探究雷公藤内酯醇(TP)通过miR-142-3p/HSP70信号通路对人乳腺癌MCF-7细胞恶性生物学行为的影响。方法:常规培养MCF-7细胞,将其分为6组:对照组、TP组、miR-142-3p inhibitor组、TP+inhibitor组、miR-142-3p mimic组和TP+mimic组,用转染试剂将相应的核酸或质粒转染MCF-7细胞。qPCR法、EdU细胞增殖实验、Transwell小室实验、细胞划痕实验、WB法分别检测转染后各组MCF-7细胞中miR-142-3p和HSP70 mRNA的表达,MCF-7细胞的增殖、侵袭、迁移能力和HSP70蛋白表达水平。结果:TP或miR-142-3p过表达能显著促进MCF-7细胞中miR-142-3p和HSP70的表达,敲减miR-142-3p则可明显抑制MCF-7细胞中miR-142-3p和HSP70的表达,TP可逆转由敲减miR-142-3p对MCF-7细胞中miR-142-3p和HSP70表达的影响;TP、过表达miR-142-3p均可明显抑制MCF-7细胞的增殖、迁移和侵袭能力(均P<0.05),敲减miR-142-3p则均可促进MCF-7细胞的增殖、迁移和侵袭能力(均P<0.05),TP可逆转由敲减miR-142-3p对MCF-7细胞恶性生物学行为的影响(均P<0.05)。结论:TP可通过调控miR-142-3p/HSP70信号通路,进而抑制MCF-7细胞的增殖、侵袭和迁移能力。

DCN通过VEGF因子抑制乳腺癌MCF-7细胞增殖的实验研究

目的探讨核心蛋白聚糖(decorin,DCN)通过下调血管内皮生长因子(Vascular endothelial growth factor,VEGF)表达抑制乳腺癌MCF-7肿瘤细胞增殖的分子机制。方法体外培养MCF-7细胞,质粒转染诱导MCF-7细胞高表达核心蛋白聚糖为DCN组,不转染的MCF-7细胞为正常对照组。MTT法、流式细胞术细胞观察各自细胞增殖情况,RT-PCR、Western blot法检测各组细胞VEGF mRNA和蛋白表达变化。结果与对照组比较,流式细胞术检测DCN转染24、48、72 h后DCN组MCF-7细胞数目明显减少(P<0.05),MTT结果显示DCN组细胞增殖能力受到显著抑制(P<0.05),Western blot检测DCN组细胞VEGF蛋白表达水平有下降趋势。结论核心蛋白聚糖通过降低VEGF mRNA表达水平,使细胞内生成的VEGF蛋白减少,抑制MCF-7细胞增殖。

TNF-ɑ调控LRG1促进乳腺癌MCF-7细胞增殖、侵袭和迁移

目的:研究TNF-α对乳腺癌MCF-7细胞中LRG1蛋白表达的调控及其对乳腺癌MCF-7细胞增殖、侵袭和迁移能力的影响及相关分子机制。方法:MTT实验检测不同浓度TNF-α处理后MCF-7细胞活力;EdU实验、Transwell实验和划痕实验分别检测抑制LRG1表达后细胞增殖、侵袭以及迁移能力;Western blot检测细胞内MAPK信号通路中p-p38蛋白表达。结果:低浓度TNF-α处理乳腺癌MCF-7细胞,细胞活力增强;抑制LRG1表达后细胞增殖能力下降,侵袭细胞数、细胞迁移率以及p-p38蛋白表达均下降。结论:TNF-ɑ通过调控LRG1的表达促进乳腺癌MCF-7细胞增殖、侵袭和迁移,这一过程可能通过激活p38MAPK信号通路来实现。

Forskolin and Phorbol 12-myristate 13-acetate modulates the expression pattern of AP-1 factors and cell cycle regulators in estrogen-responsive MCF-7 cells

Activator protein-1(AP-1)transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus.The phorbol 12-myristate 13-acetate and Forskolin(Fo)are well-known kinase activators/stimulators of Protein Kinase C(PKC)and Protein Kinase A(PKA)respectively.Importantly,these kinases are found to be present in transitional points of many cell signaling pathways,especially those involved in proliferation.The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized.Hence,the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated.Where,cells treated with PMA showed increased cell proliferation,while Fo had no effect,but inhibited the PMA induced proliferation.The RT-PCR results showed the PMA induced c-Jun,c-Fos and Fra-1 expressions compared to control and Fo.However,Fo in combination with PMA,inhibit the PMA induced above mRNA expressions where Fo alone has no effect.Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells.Further,PMA increases the mRNA expression of Cyclin-E1,Cyclin-D1,and CDK-4,whereas Fo decreases their expressions.Thus,mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.

香豆酸诱导MCF-7细胞凋亡的代谢与氧化应激影响

目的 探讨香豆酸诱导乳腺癌细胞MCF-7凋亡的机制及氧化应激与代谢的影响。方法 采用氧化相关蛋白活性研究香豆酸对MCF-7氧化应激的影响;液相质谱仪结合代谢组学探索香豆酸对MCF-7细胞代谢的影响。结果 香豆酸呈浓度依赖式对MCF-7细胞凋亡具有一定的促进作用,并上调细胞的氧化应激性导致MCF-7细胞氧化系统失衡;同时香豆酸影响MCF-7的细胞代谢,主要是体现糖代谢,富集显著通路集糖酵解;并筛选到差异代谢物563个,其中上调463个,下调100个。结论 香豆酸诱导MCF-7细胞凋亡作用机制可能是通过影响细胞的氧化应激性以及调控细胞糖代谢来实现。

毛酸浆内酯通过抑制STAT3诱导人乳腺癌MCF-7细胞凋亡

[目的]旨在探讨信号转导和转录激活因子3(STAT3)在毛酸浆内酯(PPB)诱导人乳腺癌MCF-7细胞凋亡中发挥的作用。[方法]采用荧光染色法分析PPB诱导MCF-7细胞凋亡;使用生物信息学方法预测PPB抗乳腺癌的潜在机制;采用噻唑蓝(MTT)法考察STAT3抑制剂S3I-201以及STAT3小干扰RNA(siRNA)对PPB抑制MCF-7细胞生长的作用;采用蛋白免疫印迹(Western Blot)法考察PPB单独处理或STAT3 siRNA预处理后对MCF-7细胞中STAT3、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶8(Caspase8)、半胱氨酸天冬氨酸蛋白酶9(Caspase9)、细胞色素c(Cytochrome c)以及多聚ADP核糖聚合酶(PARP)蛋白表达的影响。[结果]MCF-7细胞经PPB作用后凋亡形态特征明显,凋亡比例上升;生物信息学结果显示PPB与乳腺癌疾病的共同靶点STAT3在乳腺癌组织中高表达,单基因GSEA结果提示STAT3高表达与凋亡信号通路呈负相关;Western Blot法检测结果显示PPB能够抑制STAT3的磷酸化;S3I-201抑制剂或siRNA敲降STAT3均能进一步促进PPB抑制MCF-7细胞生长;此外,敲降STAT3进一步增加PPB对促凋亡蛋白Bax、Cytochrome c、裂解的Caspase8(Cleaved-Caspase8)、裂解的Caspase9(Cleaved-Caspase9)以及裂解的PARP(Cleaved-PARP)的促进作用,并增加PPB对抗凋亡蛋白Bcl-2的抑制作用。[结论]PPB通过抑制STAT3诱导人乳腺癌MCF-7细胞凋亡。

Synthesis of N-(phenylcarbamothioyl)-benzamide derivatives and their cytotoxic activity against MCF-7 cells

Cancer is one of the leading causes of death both in developing countries and across the globe.In Indonesia,cancer ranks as the fifth primary cause of death following heart disease,stroke,respiratory tract and diarrhea.Therefore,studies on thiourea derivative compounds as anticancer agents have been profoundly conducted but still require further continuous development.In the present study,we aimed to synthesize new anticancer compounds of N-(phenylcarbamothioyl)-benzamide derivatives,namely N-(phenylcarbamothioyl)-4-bromobenzamide and N-(phenylcarbamothioyl)-4-fluorobenzamide compounds and assess their activities against MCF-7 breast cancer cells.The initial step was to predict the drug-receptor activity through docking between the tested compounds using epidermal growth factor receptor(EGFR)(PDB code: 1 M17).The compounds were futher synthesized from the reactions between benzoyl chloride derivatives and N-phenylthiourea.The structures of the new compounds were identified using FTIR,~1 H NMR,13 C NMR and mass spectra.The cytotoxic activities(IC_(50)) to breast cancer cells of MCF-7 N-(phenylcarbamothioyl)-4-bromobenzamide compound and N-(phenylcarbamothioyl)-4-fluorobenzamide were 0.27 mM and 0.31 mM,respectively.These two new compounds had better cytotoxic activities than those of the current hydroxyurea-based anticancer drugs(the reference compound) with an IC_(50) value of 9.76 mM.Furthermore,these two new compounds were not toxic to Vero normal cells.Therefore,they possessed tremendous potentials as the candidates for new drugs against breast cancer.

LMAN2在HR阳性乳腺癌组织中的表达与患者预后的关系及其对MCF-7细胞增殖和迁移的影响

目的:探究甘露糖结合凝集素2(LMAN2)在激素受体(HR)阳性乳腺癌组织中的表达水平与乳腺癌患者预后的关系及其对MCF-7细胞增殖和迁移的影响。方法:通过TCGA、Bc-GenExMiner、GEPIA和Kaplan-Meier Plotter数据库分析LMAN2在乳腺癌组织和正常乳腺组织中的差异性表达及其与患者预后的关系。采用小RNA干扰技术将si-LMAN2#1、si-LMAN2#2及si-NC转染至MCF-7细胞,将过表达LMAN载体(pc-LMAN)及空载体pcDNA3.1阴性对照(pc-NC)转染至MCF-7细胞,实验分为si-LMAN2#1、si-LMAN2#2、si-NC、pc-LMAN2和pc-NC组。通过qPCR和WB实验检测各组细胞中LMAN2 mRNA和蛋白的表达水平,CCK-8、克隆形成、Transwell迁移、WB等实验检测敲低和过表达LMAN 2对MCF-7细胞增殖、克隆形成、迁移及AKT信号通路相关蛋白表达的影响。结果:LMAN2在乳腺癌组织中的表达水平显著高于正常乳腺组织(P<0.001)。HR阳性乳腺癌组织中LMAN2表达水平显著高于HR阴性乳腺癌组织(P<0.001);LMAN2高表达与HR阳性乳腺癌患者不良预后有关联。敲低LMAN2可显著降低MCF-7细胞的增殖和迁移能力(P<0.01或P<0.001),过表达LMAN2可显著提高MCF-7细胞的增殖和迁移能力(均P<0.001)。敲低LMAN2组MCF-7细胞中PTEN和P21蛋白表达水平均显著升高,p-AKT蛋白表达水平显著降低(均P<0.01)。结论:LMAN2在乳腺癌组织和HR阳性乳腺癌组织中高表达,且与不良预后有关联。LMAN2高表达与MCF-7细胞增殖和迁移有关联,其作用机制可能涉及AKT信号通路。

羟基积雪草酸对乳腺癌MCF-7细胞增殖、凋亡和自噬的影响

目的探讨羟基积雪草酸(madecassic acid,MA)对乳腺癌MCF-7细胞增殖、凋亡及自噬的影响。方法将MCF-7细胞分为阴性对照组、不同浓度的MA(80、100、120、140、160μmol/L)组、不同浓度的他莫昔芬(10、20、30、40、50、35μmol/L)组,干预24、36、48 h。初步确定MA发挥抗乳腺癌作用的最佳浓度和最佳时间后,采用MTT法检测细胞活力,计算相应的半抑制浓度(half maximal inhibitory concentration,IC50)值;流式细胞术检测细胞凋亡;JC-1荧光探针检测线粒体膜电位变化;Western blot法检测细胞增殖蛋白细胞核抗原蛋白(proliferating cell nuclear antigen,PCNA)、自噬蛋白苄氯素-1(Beclin-1)、微管相关蛋白1轻链3-Ⅱ/Ⅰ(microtubule-associated protein1 light chain 3Ⅱ/Ι,LC3Ⅱ/Ι)、螯合体1(protein 62,p62)的蛋白相对表达水平。透射电镜检测自噬小体。结果发挥抗肿瘤作用的他莫昔芬最佳浓度为35μmol/L,MA最佳浓度为140μmol/L,最佳时间均为48 h。与阴性对照组相比,MA140μmol/L组和他莫昔芬35μmol/L组的细胞凋亡率,细胞荧光相对强度以及LC3Ⅱ/Ⅰ、Beclin-1蛋白相对表达水平均升高(P<0.05,P<0.01);PCNA、P62蛋白相对表达水平降低(P<0.05,P<0.01)。与MA 140μmol/L组相比,他莫昔芬35μmol/L组细胞凋亡率、细胞荧光相对强度以及Beclin-1蛋白相对表达水平明显升高(P<0.01),PCNA蛋白相对表达水平明显降低(P<0.01)。阴性对照组MCF-7细胞膜完整,核膜清晰,细胞形态良好;MA 140μmol/L组细胞膜、细胞核形态不规则,线粒体基质密度较高、嵴扩张,可见自噬小体;他莫昔芬35μmol/L组细胞膜局部溶解、破损,可见双核仁,线粒体肿胀、基质溶解,可见自噬小体。结论MA可抑制乳腺癌MCF-7细胞的增殖、诱导细胞凋亡和自噬。

菊藻丸含药血清调控PTEN对乳腺癌MCF-7细胞生长和转移的作用研究

目的 研究菊藻丸含药血清调控10号染色体上缺失的磷酸酶和张力蛋白同源物基因(PTEN)对人乳腺癌MCF-7细胞生长和转移的作用。方法 8周龄、体重(200±20)g的SPF级Wistar雌性大鼠12只,采用随机数字表法分为菊藻丸组和对照组,各6只。各组分别以5.4 g/(kg·d)菊藻丸和等量生理盐水灌胃,制备含药血清和空白对照血清。将培养好的人乳腺癌MCF-7细胞分为20%空白血清组、5%含药血清组、10%含药血清组、15%含药血清组和20%含药血清组,分别使用含20%空白对照血清和含5%、10%、15%、20%含药血清的培养基进行培养干预。采用CCK-8检测细胞增殖、集落形成实验观察克隆形成能力、TUNEL染色检测凋亡、流式细胞术检测周期分布、划痕实验和Transwell检测细胞迁移和侵袭能力,q PCR和Western blot检测PTEN m RNA和蛋白表达水平。结果 与20%空白血清组比较,10%、15%、20%含药血清组细胞增殖活性降低、克隆形成率减少、侵袭能力降低(P<0.05);5%、10%、15%、20%含药血清组细胞凋亡水平升高、迁移能力降低、细胞周期阻滞于G1期、PTEN的m RNA和蛋白表达水平均升高(P<0.05)。与5%含药血清组比较,10%、15%、20%含药血清组细胞增殖活性降低、克隆形成率降低、细胞凋亡水平升高、迁移能力降低、侵袭能力降低、细胞周期阻滞于G1期、PTEN m RNA和蛋白表达水平均升高(P<0.05)。结论 菊藻丸含药血清能抑制MCF-7细胞生长和转移,其机制可能与激活PTEN的表达有关。

Acid Water-ground Nano-realgar Is Superior to Crude Realgar in Promoting Apoptosis of MCF-7 Breast Cancer Cells

Objective:Realgar is a traditional mineral Chinese medicine with antitumor effects,but it has high toxicity and low efficacy in its crude form.The purpose of this study was to optimize realgar to increase its efficacy and therapeutic potential.Methods:Crude realgar(CR)was mechanically ground to obtain nano-realgar(NR),and then nano-realgar processed products(NRPPs)were obtained using three different traditional Chinese medicine processing methods:grinding in water,acid water,and alkali water,respectively.Results:By analyzing the size distribution of nanoparticles and the content of arsenic trioxide(As_(2)O_(3);ATO),we found that acid water-ground NRPPs had the characteristics of high purity and low toxicity.The effects of CR,NR,and NRPPs on proliferation,cell cycle,and apoptosis of MCF-7 cells were detected,and the ability of NRPPs to induce apoptosis in MCF-7 cells was analyzed.The results showed that the average particle size of acid water-ground NRPPs was 137.7 nm,and the content of ATO was 2.83 mg/g.Acid water-ground NRPPs showed better effects on inhibiting proliferation,cell cycle,and apoptosis of MCF-7 cells than CR and NR.Western blot assays further confirmed that acid water-ground NRPPs upregulated the protein expression of TP53,Bax,cytochrome c,caspase-9,and caspase-3 in MCF-7 cells(P<0.05)and inhibited the expression of Bcl-2(P<0.05).Conclusion:These results suggest that acid water-ground NRPPs can induce apoptosis of MCF-7 cells through regulating mitochondrial-mediated apoptosis,providing evidence for the clinical application of realgar.