Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3β at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3β by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3β silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3β activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells.     

SETD8基因表达对乳腺癌细胞周期、活性氧及药物敏感性的影响

目的:构建SETD8重组慢病毒载体,探讨SETD8对人乳腺癌细胞周期、氧化应激的影响,并观察稳定敲低SETD8后对乳腺癌细胞多西他赛敏感性的影响。方法:构建含SETD8基因的重组慢病毒载体,经转染,荧光显微镜观察感染效率;采用RT-qPCR和Western blot检测SETD8 m RNA和蛋白相对表达量;采用流式细胞技术检测细胞周期及活性氧水平,通过CCK-8试剂检测稳定敲低SETD8基因的乳腺癌细胞对多西他赛的敏感性变化。结果:成功包装慢病毒,荧光观察慢病毒感染效率在85%左右,通过PCR和WB验证SETD8过表达及敲低稳转细胞系的效率显著(P0.01)。低表达SETD8的乳腺癌细胞周期停滞在G2/M和S期,细胞内ROS水平高于对照组。不同浓度多西他赛处理的SETD8敲低稳转细胞系的细胞活性较对照组明显降低(P0.01)。结论:慢病毒介导下调SETD8表达,使得乳腺癌细胞周期阻滞在G2/M期,细胞内ROS增多,与多西他赛发生协同作用,从而增加了药物敏感性。     

DNMT3B7 Expression Promotes Tumor Progression to a More Aggressive Phenotype in Breast Cancer Cells

Epigenetic changes, such as DNA methylation, have been shown to promote breast cancer progression. However, the mechanism by which cancer cells acquire and maintain abnormal DNA methylation is not well understood. We have previously identified an aberrant splice form of a DNA methyltransferase, DNMT3B7, expressed in virtually all cancer cell lines but at very low levels in normal cells. Furthermore, aggressive MDA-MB-231 breast cancer cells have been shown to express increased levels of DNMT3B7 compared to poorly invasive MCF-7 cells, indicating that DNMT3B7 may have a role in promoting a more invasive phenotype. Using data gathered from The Cancer Genome Atlas, we show that DNMT3B7 expression is increased in breast cancer patient tissues compared to normal tissue. To determine the mechanism by which DNMT3B7 was functioning in breast cancer cells, two poorly invasive breast cancer cell lines, MCF-7 and T-47D, were stably transfected with a DNMT3B7 expression construct. Expression of DNMT3B7 led to hypermethylation and down-regulation of E-cadherin, altered localization of β-catenin, as well as increased adhesion turnover, cell proliferation, and anchorage-independent growth. The novel results presented in this study suggest a role for DNMT3B7 in the progression of breast cancer to a more aggressive state and the potential for future development of novel therapeutics.     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 μM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.     

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.     

Design,synthesis and biological evaluation of new carbazole derivatives as anti-cancer and anti-migratory agents

Based on the efficacy of EHop-016 as an inhibitor of migration and Rac1 activation, a new series of carbazole derivatives has been synthesized. Cytotoxic and anti-migratory effects of these compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. Preliminary investigations of their anticancer activity demonstrated that several compounds have moderate antiproliferative effects on cancer cell lines with GI₅₀ values in the range of 13–50?µM. Furthermore, compounds 3b and 11b inhibit migration activity of metastatic cell line MDA-MB-231 by 32% and 34%, respectively. Compound 11b was shown to inhibit activation of the Rho GTPase Rac1 by 55% at 250?nM in both MDA-MB-231 and MDA-MB-435 cell lines. Compared with the IC₅₀ of Rac1 inhibition by lead compound EHop-016 of 1.1?µM, compound 11b demonstrates 4X improved in vitro efficacy.     

SOX7基因启动子甲基化水平对乳腺癌MDA-MB-231细胞株体外迁移和侵袭的影响

目的:探讨乳腺癌MDA-MB-231细胞中,Y性别决定区基因7(SOX7)基因启动子甲基化水平对细胞的体外迁移和侵袭的影响。方法:脂质体转染pcDNA3.0-DNA甲基转移酶3a(DNMT3a)质粒至MDA-MB-231细胞中,并于24h、48h及72h后,采用蛋白质免疫印迹实验(WB)检测细胞内DNMT3a蛋白表达水平;甲基化特异性定量PCR(Q-MSP)检测DNMT3a处理组、5-aza-C处理组及对照(Control)组MDA-MB-231细胞中的SOX7基因启动子DNA甲基化水平;实时荧光定量PCR(qRT-PCR)及WB实验检测各组MDA-MB-231细胞中的SOX7 m RNA和蛋白表达水平;细胞划痕实验及细胞侵袭实验检测各组MDA-MB-231细胞的迁移和侵袭能力。结果:pcDNA3.0-DNMT3a质粒转染MDA-MB-231细胞24h时,细胞内的DNMT3a蛋白表达水平最高。DNMT3a能够显著提高SOX7基因启动子DNA甲基化水平,而5-aza-C则抑制了SOX7基因启动子DNA甲基化水平(P0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞中,SOX7的m RNA及蛋白表达水平均明显下降,而5-aza-C处理组SOX7的m RNA及蛋白表达水平均明显增加(P0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞的迁移和侵袭能力均显著增强(P0.05),而5-aza-C处理组的MDA-MB-231细胞的迁移和侵袭能力变化不大(P0.05)。结论:在恶性肿瘤中,SOX7低表达表受其基因启动子高甲基化调节,且乳腺癌MDA-MB-231细胞中低表达的SOX7能够影响细胞的外迁移和侵袭能力。     

Synthesis and anti breast cancer activity of biphenyl based chalcones

A series of (2E,2′E)-1,1′-(3-hydroxy-5-methylbiphenyl-2,6-diyl)-bis(3-pheylprop-2-ene-1-ones (5–33) were prepared by the reaction of 1,3-diacetyl biphenyls (1–4) with different aldehydes in presence of catalytic amount of solid KOH in ethanol in excellent yields. The compounds were evaluated for anticancer activity against human breast cancer MCF-7 (estrogen responsive proliferative breast cancer model) and MDA-MB-231 (estrogen independent aggressive breast cancer model) cell lines, HeLa (cervical cancer) cell line, and human embryonic kidney (HEK-293) cells. Most of the compounds preferentially inhibited the growth of the aggressive human breast cancer cell lines, MDA-MB-231 in the range of 4.4–30 μM. The two compounds 9 and 29 proved to be better anticancer agents than the standard drug tamoxifen against the MDA-MB-231 cell lines. Mode of action of these compounds was established to be apoptosis, cell cycle arrest and loss of mitochondrial membrane potential.     

Embryonic Morphogen Nodal Promotes Breast Cancer Growth and Progression

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.     

BMP4 enhances anoikis resistance and chemoresistance of breast cancer cells through canonical BMP signaling

Bone morphogenetic proteins (BMPs) regulate cell fate during development and mediate cancer progression. In this study, we investigated the role of BMP4 in proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells. We utilized breast cancer cell lines and clinical samples representing different subtypes to understand the functional effect of BMP4 on breast cancer. The BMP pathway was inhibited with the small molecule inhibitor LDN193189 hydrochloride (LDN). BMP4 signaling enhanced the expression of stem cell genes CD44, ALDH1A3, anti-apoptotic gene BCL2 and promoted anoikis resistance in MDA-MB-231 breast cancer cells. BMP4 enhanced self-renewal and chemoresistance in MDA-MB-231 by upregulating Notch signaling while LDN treatment abrogated anoikis resistance and proliferation of anoikis resistant breast cancer cells in the osteogenic microenvironment. Conversely, BMP4 downregulated proliferation, colony-forming ability, and suppressed anoikis resistance in MCF7 and SkBR3 cells, while LDN treatment promoted tumor spheroid formation and growth. These findings indicate that BMP4 has a context-dependent role in breast cancer. Further, our data with MDA-MB-231 cells representing triple-negative breast cancer suggest that BMP inhibition might impair its metastatic spread and colonization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00649-9.     

Folic acid enforces DNA methylation-mediated transcriptional silencing of PTEN,APC and RARbeta2 tumour suppressor genes in breast cancer

Folate, one of the most studied dietary compounds, has recently become the main topic of debates on food fortification. Although low folate levels may be associated with increased risk of cancer development, simultaneously several reports indicate a detrimental effects mediated by high folate concentrations. Using the methylation sensitive restriction analysis (MSRA) and real-time RT-PCR we tested the effect of folic acid on DNA promoter methylation and expression of PTEN, APC and RARbeta2 tumour suppressor genes in MCF-7 and MDA-MB-231 breast cancer cell lines with different invasive capacity. The tested genes encode proteins involved in regulation of oncogenic intracellular signaling pathways. The results show that the increasing concentrations of folic acid lead to a dose-dependent down-regulation of tumour suppressor genes which may be linked to the increased DNA methylation detected within their promoter regions. The effects were more remarkable in non-invasive MCF-7 cells where we also observed 30% up-regulation of DNMT1 expression at the highest folate concentration used. Our findings show that caution need to be used when introducing folic acid supplementation since it may lead to cancer progression.     

Cellular effect of styrene substituted biscoumarin caused cellular apoptosis and cell cycle arrest in human breast cancer cells

BackgroundCoumarins occurs naturally across plant kingdoms exhibits significant pharmacological properties and pharmacokinetic activity. The conventional, therapeutic agents are often associated with poor stability, absorption and increased side effects. Therefore, identification of a drug that has little or no-side effect on humans is consequential. Here, we investigated the antiproliferative activity of styrene substituted biscoumarin against various human breast cancer cell lines, such as MCF-7, (ER-) MDA-MB-231 and (AR+) MDA-MB-453. Styrene substituted biscoumarin induced cell death by apoptosis in MDA-MB-231 cell line was analyzed.MethodsAntiproliferative activity of Styrene substituted biscoumarin was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Styrene substituted biscoumarin induced apoptosis was assessed by Hoechst staining, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining and flow cytometric analysis. Migratory and proliferating characteristic of breast cancer cell line MDA-MB-231 was also analyzed by wound healing and colony formation assay. Furthermore, mRNA expression of BAX and BCL-2 were quantified using qRT-PCR and protein expression level analyzed by Western blot.ResultsThe inhibition concentration (IC₅₀) of styrene substituted biscoumarin was assayed against three breast cancer cell lines. The inhibition concentration (IC₅₀) value of styrene substituted biscoumarin toward MDA-MB-231, MDA-MB-453 and MCF-7 cell lines was 5.63, 7.30 and 10.84 μg/ml respectively. Styrene substituted biscoumarin induced apoptosis was detected by Hoechst staining, DAPI/PI analysis and flow-cytometric analysis. The migration and proliferative efficiency of MDA-MB-231 cells were completely arrested upon styrene substituted biscoumarin treatment. Also, mRNA gene expression and protein expression of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) genes were analyzed by qRT-PCR and western blot analysis upon styrene substituted biscoumarin treatment to MDA-MB-231 cells. Our results showed that styrene substituted biscoumarin downregulated BCL-2 gene expression and upregulated BAX gene expression to trigger apoptotic process.ConclusionStyrene substituted biscoumarin could induce apoptosis through intrinsic mitochondrial pathway in breast cancer cell lines, particularly in MDA-MB-231. Our data suggest that styrene substituted biscoumarin may act as a potential chemotherapeutic agent against breast cancer.