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Objective:To elucidate the potential molecular markers and drug-compound-target mechanism of Epimedii Folium intervention on breast cancer stem cells（BCSCs） through chip analysis combined with network pharmacology and experimental validation. Method:Relevant drug information was retrieved in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） to obtain the active components and potential targets of Epimedii Folium. "Breast Cancer Stem Cells" were searched in Gene Expression Omnibus（GEO）database，and GSE98239 chip data were obtained through analysis and screening. Then GEO2R online analysis tool was used to obtain the differential genes to draw differential gene heat map and volcano map. The differential gene network map of Epimedii Folium intervention for breast cancer stem cells was constructed by Cytoscape 3.8.0，and Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis of drug and disease genes were performed. Human breast cancer MDA-MB-231 cells were divided into 20%，40%，60% Epimedii Folium drug-containing serum group and control group. Cell counting kit-8（CCK-8），and Western blot were used to detect the effect of Epimedii Folium drug-containing serum intervention on cell activity and target protein expression in breast cancer cells. Result:Twenty-three active components including flavones，sterols，alkaloids and sesquiterpenoids were obtained from Epimedii Folium. It was found that Epimedii Folium interacted with B-cell lymphoma-2-like protein 1（BCL2L1），matrix metallopeptidase 2（MMP2），prostaglandin-endoperoxide synthase 2（PTGS2），vascular endothelial growth factor A（VEGFA），transforming growth factor beta receptor 1（TGFBR1） and other pivotal genes in breast cancer stem cells，participated in the induction of new angiogenesis and cell migration，enabled the continuous self-renewal of BCSCs，decreased apoptosis and cell migration，thus promoting the recurrence and metastasis of breast cancer. KEGG results showed that Epimedii Folium intervened in multiple differential expressed genes（DEGs）of transforming growth factor-<italic>β</italic>（TGF-<italic>β</italic>），vascular endothelial growth factor（VEGF），phosphoinositide 3kinase/protein kenase B（PI3K/Akt），mitogen-activated protein kinese（MAPK）and mammalian target of rapamycin（mTOR）subpathways in cancer signaling pathways to exert its efficacy in intervening breast cancer stem cells. Experiments showed that the survival rate of breast cancer cells was significantly reduced and the expression levels of TGFBR1 and Smad2 in breast cancer cells significantly decreased after the intervention of Epimedii Folium drug-containing serum（<italic>P</italic><0.01）. Conclusion:Several components in different concentrations of drug-containing serum of Epimedii Folium can synergistically act on target differentially expressed genes of breast cancer stem cells，and inhibit the proliferation of breast cancer cells by down-regulating the expression levels of TGFBR1，a key molecule in the TGF-<italic>β</italic> pathway，and Smad2，a downstream signal.
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@#Background: It has been widely reported that breast cancer aggressiveness may be driven by breast cancer stem cells (BCSCs). BCSCs display stemness properties that include self-renewal, tumourigenicity and pluripotency. The regulation of gene expression may have important roles in BCSC stemness and aggressiveness. Thus, the aim of this study was to examine the stemness and aggressiveness gene expression profile of BCSCs compared to MCF-7 and MDA-MB-231 breast cancer cells. Methods: Human ALDH1+ BCSCs were grown in serum-free Dulbecco’s Modified Eagle Medium (DMEM)/F12, while MCF-7 and MDA-MB-231 were cultured in DMEM supplemented with 10% foetal bovine serum under standard conditions. Total RNA was extracted using the Tripure Isolation Reagent. The relative mRNA expressions of OCT4, ALDH1A1 and CD44 associated with stemness as well as TGF-β1, TβR1, ERα1 and MnSOD associated with aggressiveness in BCSCs and MCF-7 cells were determined using the quantitative real-time PCR (qRT-PCR). Results: The mRNA expressions of OCT4 (5.19-fold ± 0.338; P = 0.001), ALDH1A1 (3.67- fold ± 0.523; P = 0.006), CD44 (2.65-fold ± 0.307; P = 0.006), TGF-β1 (22.89-fold ± 6.840; P = 0.015), TβR1 (3.74-fold ± 1.446; P = 0.045) and MnSOD (4.6-fold ± 1.096; P = 0.014) were higher in BCSCs than in MCF-7 but were almost similar to MDA-MB-231 cells. In contrast, the ERα1 expression of BCSCs (0.97-fold ± 0.080; P = 0.392) was similar to MCF-7 cells, indicating that BSCSs are oestrogen-dependent breast cancer cells. Conclusion: The oestrogen-dependent BCSCs express stemness and aggressiveness genes at a higher level compared to oestrogen-dependent MCF-7 but are almost similar to oestrogenindependent MDA-MB-231 cells.
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Purpose To investigate the effect of protein tyrosine phosphatase receptor type D (PTPRD) on the characteristics of breast cancer stem cells. Method PTPRD expression in breast cancer cell line MDA-MB231 was down-regulated by small interference RNAs (siRNAs). Self-renewal ability of breast cancer stem cells (BCSCs) was detected by mammosphere formation assay. The holocolony forming ability was detected by colony formation assay. The proportion of CD44+/CD24- BCSCs was detected by flow cytometry. The ability of tumorigenesis of breast cancer cells in mice was sdudied with mouse tumorigenesis test. Separation of CD44+/CD24- stem cell population and non-stem cell population was isolated by immunomagnetic beads. Expression of PTPRD in stem cell and non-stem cell population was detected by Western blot and immunofluorescent. Results Down-regulation of PTPRD promoted the expression of stem cell markers ALDH1 and OCT-4. The expression of PTPRD in breast cancer stem cells was lower than than in non-stem cells (P<0.05). After PTPRD was down-regulated, the number of mammosphere (147±3.51) was significantly higher than that of the control group (106±12.5) (P<0.05), the proportion of holoclone [(35.9±3.4) ％] was significantly higher than that of the control group [(11.2±5.3) ％] (P<0.05), the proportion of CD44+/CD24- cells[(2.88±1.2) ％]was significantly higher than that of the control group [(0.6±0.4) ％], the in vivo tumorigenicity was significantly enhanced in nude mice (P<0.05). Conclusion The expression of PTPRD is lower in BCSCs. PTPRD may inhibit the self-renewal ability of breast cancer stem cells.
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Objective To explore changes of invasion capability of cancer stem cells loading miR30a derived from enlarging and proliferative axillary lymph node of patients with breast cancer in nude mice.Methods MiR30a oligonucleotide fragment loaded by adenovirus vector was transfected into breast cancer stem cells isolated from enlarging and proliferative axillary lymphnodes of patients with breast cancer,MDA-MB-231 cell lines as the control.The cancer cells were injected into axillary subcutaneous fat of the nude mice for several weeks,and then,the expression of Vimentin or N-Cadherin in tumor tissues of each group of the mice was determined by immunohistochemistry and western blot.Results The average transfection rate of cancer stem cells loaded by miR30a was 62.5％,while it was 78.2％ for MDA-MB-231.The tumor volume was larger in adenovirus vector groups or in control groups of nude mice than in experimental groups induced by mir30a.Vimentin or N-cadherin in tumor tissues was significantly downexpressed in experimental groups with mir30a ((13.1±1.7)％,(15.3％±2.1)％)compared with that in adenovirus vector groups or in control groups((21.1±1.4)％,(25.3±1.6)％,P＜0.05),respectively.The difference between adenovirus vector groups and in control groups had no significant difference (P＞0.05).After inoculated for 6 weeks,except the subcutaneous plantations,no distant metastasis in nude mice was found.MDA-MB-231 cell lines groups had similar results.Conclusion The proliferative and invasive capability of cancer stem cells can be inhibited by miR30a,suggesting a new therapy for breast cancer.
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Background and purpose: MORC2 (microrchidia family CW-type zinc finger 2, MORC2) is a newly identified chromatin remodeling protein that plays key roles in DNA-based biological processes including gene transcription and DNA damage repair. However, its functional role in breast cancer development and progression re-mains unknown. ALDH1A3 (aldehyde dehydrogenase 1 family member A3), a member of the aldehyde dehydrogenases (ALDH) superfamily, is a putative breast cancer stem cell marker, but its regulatory mechanism in breast cancer is poor-ly characterized. This study aimed to investigate the effects of knockdown of endogenous MORC2 on the expression levels of ALDH1A3 and the breast cancer stem-like phenotype in MCF-7 cells. Methods: Human breast cancer MCF-7 cells were infected with negative control short hairpin RNAs (shNC) and specific shRNAs targeting human MORC2 (shMORC2), followed by selection with puromycin to generate stable MORC2 gene knockdown cell lines. Western blot and real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) were used to examine the protein and mRNA levels of ALDH1A3 in MCF-7 cells stably expressing shNC and shMORC2. Microsphere formation and fluo-rescence-activated cell sorting (FACS) assays were used to analyze the effects of knockdown of MORC2 on the breast cancer stem-like phenotype. Results: Western blot and RTFQ-PCR analyses revealed that the protein and mRNA levels of ALDH1A3 were significantly down-regulated in shMORC2 expressing cells as compared with shNC -transfected control cells. Moreover, mammosphere formation assay showed that knockdown of endogenous MORC2 in MCF-7 cells significantly reduced the ability of cells to form microspheres. Consistently, FACS assays demonstrated that shMORC2-transfected cells had a lower proportion of ALDH-positive stem cells as compared with shNC expressing cells. In contrast, knockdown of MORC2 did not significantly affect the CD44+CD24- stem cell population. Conclusion:MORC2 promotes a breast cancer stem-like phenotype through, at least in part, regulating ALDH1A3 expression.
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The breast cancer stem cell theory provides a theoretical basis for explaining phenotypic and functional heterogeneity of breast cancer. These breast cancer stem cells(CSCs)promote tumor growth and are closely related to breast cancer intrinsic drug resistance. Therefore,targeted therapy of breast CSCs has become a hot area in basic and clinical research. There is growing evidence that nanoparticles can kill cancer by targeting breast CSCs ,such as targeted tumor stem cell-specific expressed surface markers(AL-DH1,CD44,and CD90),tumor stem cell stemness-related NOTCH,Hedgehog and TGF-βsignaling pathways. In this review,we summarized the characteristics and research status of breast CSCs ,and the application of nanotechnology in the treatment of breast cancer.In addition,we also summarized the research status of epigenetic drugs aimed to restrain the reprogramming of breast cancer cells.
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Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.
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Humanos , Feminino , Neoplasias da Mama/metabolismo , Antígeno CD24/metabolismo , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Apoptose , Proliferação de Células , Sobrevivência Celular , Espaço Extracelular , Regulação Neoplásica da Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The cancer stem cell theory suggests that cancer develops from a subset of tumor cells that possess characteristics of stem cells. Breast cancer stem cells comprise a sub-population, which possesses the capacity of self-renewal and the potential for differentiation and high tumorigenicity. Evidence from bothin vitro andin vivo studies demonstrates breast cancer stem cells are responsible for tumor relapse, invasion and metastasis, chemo- and radio-resistance and epithelial-mesenchymal transition (EMT). Herein, this review highlighted the recent advances in breast cancer stem cells.
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Cancer stem cells (CSCs), a subpopulation of cancer cells with ability of initiating tumorigenesis, exist in many kinds of tumors including breast cancer. Cancer stem cells contribute to treatment resistance and relapse. Conventional treatments only kill differentiated cancer cells, but spare CSCs. Combining conventional treatments with therapeutic drugs targeting to CSCs will eradicate cancer cells more efficiently. Studying the molecular mechanisms of CSCs regulation is essential for developing new therapeutic strategies. Growing evidences showed CSCs are regulated by non-coding RNA (ncRNA) including microRNAs and long non-coding RNAs (lncRNAs), and histone-modifiers, such as let-7, miR-93, miR-100, HOTAIR, Bmi-1 and EZH2. Herein we review the roles of microRNAs, lncRNAs and histone-modifiers especially Polycomb family proteins in regulating breast cancer stem cells (BCSCs).
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Humanos , Neoplasias da Mama , Genética , Metabolismo , Patologia , Histonas , Metabolismo , Células-Tronco Neoplásicas , Metabolismo , RNA não Traduzido , Genética , MetabolismoRESUMO
CD44 is a principal transmembrane hyluronate receptor, which acts as a hook between Extracellular Matrix and the cytoskeleton. CD44 is up regulated in breast cancer, which in turn helps in tumor progression and metastasis. There has been a lot of attention given to CD44 in recent years because of the discovery, CD44+/CD24- lineage marks breast cancer stem cells. Recent clinical and experimental findings show that CD44 is involved in the tumor associated proliferation, invasion, migration, and in many pathways involved in maintaining cancer cells which inturn are correlated with patient's survival. All these findings make CD44 as a potential target for breast cancer treatment. The methods of literature search for this article include Pubmed, BMC cancer and other printed journal article.
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Receptores de Hialuronatos , Neoplasias da Mama/citologia , Humanos , Ácido Hialurônico/análogos & derivadosRESUMO
Breast cancer stem cells (BCSC) are group of cells exhibiting self-renewal and multi-directional differentiation potentials. These cells have an important role in the occurrence, development, metastasis, and recurrence of breast cancer. In normal circumstances, the ability of mammary stem cells to differentiate and undergo self-renewal is governed by related signaling pathways. After this mechanism is destroyed, breast stem cells undergo abnormal differentiation, forming breast cancer stem cells that unlimitedly proliferate to develop into breast cancer. As research on BCSC increasingly deepens, regulation of BCSC by notch signaling and its crosstalk with several signaling pathways have drawn a great deal of attention in this field. This paper reports the signaling pathways of breast cancer stem cells and latest studies on this field to better understand the essential role of notch signaling pathway in the occurrence and development of breast cancer and corresponding clinical targeted therapy.
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microRNAs play a critical role in breast tumorigenesis and progression, in which it might serve as oncogene, tumor suppressor gene, regulators of invasion, apoptosis and therapy resistance. Moreover,there has been a large body of evidences for the involvement of miRNAs in the self-renewal, differentiation and tumor formation of breast cancer stem cells. This review respectively summarizes the relations between microRNAs and breast cancer, breast cancer stem cell as well as the progresses of applying microRNAs to tumor therapy.