MB-231 Human Breast Cancer Cells Research Articles

Myeloid zinc finger-1 regulates expression of cancer-associated fibroblast and cancer stemness profiles in breast cancer

BackgroundOsteopontin acts thru myeloid zinc finger-1 and transforming growth factor-β to drive the adoption of a cancer-associated fibroblast phenotype by local mesenchymal stem cells. Cancer-associated fibroblasts increase cancer cell stemness. MethodsMesenchymal stem cells were exposed to osteopontin or were cocultured with MB231 human breast cancer cells (high osteopontin producer) in the presence or absence of aptamer (inactivates extracellular osteopontin). Myeloid zinc finger-1 phosphorylation sites were identified, and phosphomutants of T134 (SCAN domain) and S453 (zinc finger DNA binding domain) were constructed. Transforming growth factor-β F and cancer-associated fibroblast markers (smooth muscle actin, vimentin, and tenascin-c) were measured in mesenchymal stem cells. In MB231, stemness markers Sox2, Nanog, and Oct4 were measured. ResultsMesenchymal stem cells plus osteopontin increased transforming growth factor-β and cancer-associated fibroblast markers (P < .05 vs mesenchymal stem cells alone); this was abolished by aptamer inactivation of osteopontin. In mesenchymal stem cells transfected with phosphoresistant myeloid zinc finger-1, osteopontin did not increase cancer-associated fibroblast markers or transforming growth factor-β. In contrast, phosphomimetic myeloid zinc finger-1 increased cancer-associated fibroblast markers and transforming growth factor-β (P < .05 vs mesenchymal stem cells alone). In mesenchymal stem cells plus MB231, MB231 stemness markers were increased (P < .05 vs MB231 alone). In MB231 plus mesenchymal stem cells expressing phosphoresistant myeloid zinc finger-1, MB231 stemness markers were not increased in comparison with MB231 plus mesenchymal stem cells. ConclusionMyeloid zinc finger-1 phosphorylation in mesenchymal stem cells drives the osteopontin-mediated cancer-associated fibroblast phenotype, which then increases the cancer cell stemness profile.

Abstract 5069: Breast cancer prevention by triterpenoids from allspice

Abstract Breast cancer ranks second as a lethal cancer in women. Although survival following initial diagnosis is ~ 100% in first five years, cancer progression and mortality is imminent in subsequent years. The slow progression to the lethal form of breast cancer has prompted development of multiple avenues to delay the progression, metastasis and mortality using potent prevention strategies, including the use of nutraceuticals. Oleanolic acid (OA) and ursolic acid (UA) are two triterpenoids found in edible plant parts-fruits and seeds with potent cancer preventive, and selective cytotoxic activities against multiple cancers including breast cancer. We conducted cytotoxic assays of the combination of OA and UA. We found the combination has enhanced efficacy as compared to OA or UA alone. The combination of OA and UA and UA alone caused cell death by increased autophagy but not apoptosis in both MCF7 and MB231 human breast cancer cells. Further analysis revealed increased autopagosomes and autophagic flux, inhibition of either process reduced cytotoxicity, indicating cytotoxic autophagy is the primary mechanism of their action. Therefore, a combination of OA and UA with conventional therapies could enhance their therapeutic efficacy while limiting systemic toxicities of existing therapies. Citation Format: Jie Gao, Kenza Mamouni, Georgios Kallifatidis, Siva Panda, Muthusamy Thangaraju, Bal L. Lokeshwar. Breast cancer prevention by triterpenoids from allspice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5069.

Dietary isoflavone daidzein synergizes centchroman action via induction of apoptosis and inhibition of PI3K/Akt pathway in MCF-7/MDA MB-231 human breast cancer cells

BackgroundDespite advancements in the prognosis and management of breast cancer, it remains a major cause of mortality in women worldwide. Centchroman (CC), an oral contraceptive has been found to exhibit anti-cancer potential against a wide range of cancer including breast cancer. PurposeThe present study is intended to evaluate the ability of soy isoflavone Daidzein (DZ) in enhancing the efficacy of CC in Human Breast Cancer Cells (HBCCs). Methods/Study designSulforhodamine B assay was employed to determine the cytotoxicity induced by 10 µM CC & 50 µM DZ separately and together in MCF-7/MDA MB-231 HBCCs and non-tumorigenic Human Mammary Epithelial Cells (HMECs) MCF-10A as a control. Combination Index (CI) analysis was executed using CompuSyn software. Further, apoptosis was assessed using Annexin V/PI, AO/PI staining and tunel assay. Cell cycle, reactive oxygen species generation and mitochondrial membrane potential alteration was determined using flow cytometry. Western blot analysis was performed to check the expression of respective proteins. ResultsThe results suggest that the combination exerts elevated toxicity as compared to control and each drug per se without affecting HMECs MCF-10A. This therefore implies cancer cell specific action of CC plus DZ administered together. Additionally, combination index analysis suggests synergistic action of CC and DZ combination in HBCCs. Cell cycle analysis, Annexin V/PI staining, tunel assay and western blot analysis confirms the induction of apoptosis by combination in HBCCs. Interestingly, western blot analysis also revealed that the combination down-regulated the expression of proteins involved in cell survival i.e. PI3K, Akt and mTOR, suggesting inhibition of cell survival pathway. ConclusionThe results overall demonstrate that CC plus DZ has higher anticancer efficacy as compared to either drug alone. Hence, the combination of CC plus DZ may offer a novel strategy for the management of breast cancer.

Neem Seed Oil Induces Apoptosis in MCF-7 and MDA MB-231 Human Breast Cancer Cells

Background:In traditional Indian medicine, azadirachta indica (neem) is known for its wide range of medicinal properties. Various parts of neem tree including its fruit, seed, bark, leaves, and root have been shown to possess antiseptic, antiviral, antipyretic, anti-inflammatory, antiulcer, antimalarial, antifungal and anticancer activity.Materials and Methods:MCF-7 and MDA MB-231 cells were exposed to various concentrations of 2% ethanolic solution of NSO (1-30 µl/ml) and further processed for cell viability, cell cycle and apoptosis analysis. In addition, cells were analyzed for alteration in Mitochondrial Membrane Potential (MMP) and generation of Reactive Oxygen Species (ROS) using JC-1 and DCFDA staining respectively.Results:NSO give 50% inhibition at 10 µl/ml and 20 µl/ml concentration in MCF-7 and MDA MB-231 cells respectively and, arrests cells at G0/G1 phase in both the cell types. There was a significant alteration in mitochondrial membrane potential that leads to the generation of ROS and induction of apoptosis in NSO treated MCF-7 and MDA MB-231 cells.Conclusion:The results showed that NSO inhibits the growth of human breast cancer cells via induction of apoptosis and G1 phase arrest. Collectively these results suggest that NSO could potentially be used in the management of breast cancer.

Differentially expressed proteins in ER+ MCF7 and ER- MDA- MB-231 human breast cancer cells by RhoGDI-α silencing and overexpression.

The consequence of Rho GDP dissociation inhibitor alpha (RhoGDIα) activity on migration and invasion of estrogen receptor positive (ER+) and negative (ER-) breast cancer cells has not been studied using the proteomic approach. Changes in expression of RhoGDIα and other proteins interacting directly or indirectly with RhoGDIα in MCF7 and MDA-MB-231, with different metastatic potentials is of particular interest. ER+ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time- of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDIα using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα. The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDIα in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDIα in MCF7, while only one protein was identified in the upregulation of RhoGDIα in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-α activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells. Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDIα with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.

Increased Resistance of Breast, Prostate, and Embryonic Carcinoma Cells against Herpes Simplex Virus in Three-Dimensional Cultures

In previous studies we found that uveal melanoma cells grown in extracellular matrix (ECM)-containing three-dimensional (3D) cultures have increased resistance against herpes simplex virus type 1 (HSV-1)-mediated destruction relative to cells cultured without ECM. Using additional tumor cell types including MB-231 human breast cancer cells, PC-3 human prostate cancer cells, and P19 mouse embryonal carcinoma cells, we show here that tumor cell lines other than melanoma are also more resistant to HSV-1-mediated destruction in 3D cultures than cells grown in 2D. We also demonstrate here that one mechanism responsible for the increased resistance of tumor cells to HSV-1 infection in 3D cultures is an ECM-mediated inhibition of virus replication following virus entry into cells. These findings confirm and extend previous observations related to the role of the ECM in tumor resistance against HSV-1 and may lead to improved strategies of oncolytic virotherapy.

Paclitaxel in combination with AT-101 induces apoptosis via supressing Bcl-2, bcl-XL, mcl-1 proteins in human breast cancer cells.

e13578 Background: Paclitaxel, a microtubule stabilizing agent, has been a standard of care for breast cancer. AT-101, is an -/- enantiomer of gossypol, inhibits the Bcl-2 family proteins which contain BH3 domain. We reported previously that Paclitaxel in combination with AT-101 showed strong synergistic cytotoxic and apoptotic effects in human breast cancer cells. In this study, to elucidate the molecular mechanisms involved in the apoptotic effect of AT-101/Paclitaxel combination treatment in breast cancer cells, we investigated the possible roles of anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins which contain BH3 domain. Involvement of caspase 3 and 7 activation was also investigated. Methods: Human breast cancer cells were treated with increasing concentrations of drugs alone or with the synergistic combination doses of AT-101 and Paclitaxel. Cell Death Detection Elisa Plus Kit (Roche) was used to detect apoptosis. Caspase 3/7 activity was evaluated by Caspase-Glo 3/7 (Promega, Madison, WI) kit. Changes in the mRNA levels of Bcl-2, Bcl-XL and Mcl-1 genes were evaluated by qRT-PCR. Expression levels of these proteins were also investigated by Western blot analysis. Results: Combined treatment was shown to have strong synergistic apoptotic effects in MCF-7 and MDA- MB-231 human breast cancer cells. mRNA levels of Bcl-2, Bcl-XL and Mcl-1 molecules were reduced by the combination treatment in both cell lines. In parallel with mRNA levels, Bcl-2, Bcl-XL and Mcl-1 protein levels were significantly reduced after this novel drug combination. Combined treatment also induced caspase 3/7 activation in breast cancer cells. Conclusions: These preliminary data suggest that anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 may play important role in the underlying mechanistic rationale of apoptotic effect of AT-101/paclitaxel combination, while pro-apoptotic Bcl-2 related genes (Caspase-3 and Caspase-7) also seem to regulate this synergistic interaction.

18F]FDG and [18F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study

Increased 2′-deoxy-2′-[18F]fluoro-D-glucose (FDG) uptake is the most commonly used marker for positron emission tomography in oncology. However, a proliferation tracer such as 3′-deoxy-3′-[18F]fluorothymidine (FLT) might be more specific for cancer. 3′-deoxy-3′-[18F]fluorothymidine uptake is dependent on thymidine kinase 1 (TK) activity, but the effects of chemotherapeutic agents are unknown. The aim of this study was to characterise FDG and FLT uptake mechanisms in vitro before and after exposure to chemotherapeutic agents. The effects of 5-fluorouracil (5-FU), doxorubicin and paclitaxel on FDG and FLT uptake were measured in MDA MB231 human breast cancer cells in relation to cell cycle distribution, expression and enzyme activity of TK-1. At IC50 concentrations, 5-FU resulted in accumulation in the G1 phase, but doxorubicin and paclitaxel induced a G2/M accumulation. Compared with untreated cells, 5-FU and doxorubicin increased TK-1 levels by >300. At 72 h, 5-FU decreased FDG uptake by 50% and FLT uptake by 54%, whereas doxorubicin increased FDG and FLT uptake by 71 and 173%, respectively. Paclitaxel increased FDG uptake with >100% after 48 h, whereas FLT uptake hardly changed. In conclusion, various chemotherapeutic agents, commonly used in the treatment of breast cancer, have different effects on the time course of uptake of both FDG and FLT in vitro. This might have implications for interpretation of clinical findings.

Centchroman induces G 0/G 1 arrest and Caspase-dependent Apoptosis involving Mitochondrial Membrane Depolarization in MCF-7 and MDA MB-231 Human Breast Cancer Cells

Studies with Centchroman (CC) as a candidate anti-breast cancer agent are into phase III multicentric clinical trial in stage III/IV breast cancer. We have previously demonstrated its anti-neoplastic activity in Estrogen Receptor positive (ER+ve) MCF-7 Human Breast Cancer Cells (HBCCs). We now present the basis for anti-neoplastic activity of CC, mediated through apoptosis in both ER+ve/−ve MCF-7 and MDA MB-231 HBCCs respectively, compared to Tamoxifen (TAM) as a positive control. All the experiments were performed with 48 h estrogen-deprived cells exposed to CC/TAM for the subsequent 48 h. Cytotoxic potential of CC was assessed through SRB assay. Cell-cycle analysis, Time-dependent cytotoxicity, Reactive Oxygen Species (ROS) and Mitochondrial Membrane Permeability were investigated by Flow Cytometry. Early-stage apoptosis was detected by Annexin–PI staining. Caspases were assayed colorimetrically whereas nuclear derangements were assessed morphologically through PI staining and finally by DNA fragmentation analysis. Cell viability studies confirmed the IC 50 of CC in MCF-7 and MDA MB-231 cells to be 10 and 20 μM ( P < 0.001) respectively, suggesting enhanced susceptibility of the former cell type to CC. FACS data reveals CC mediated G 0/G 1 arrest ( P < 0.01) along with the presence of prominent sub-G 0/G 1 peak ( P < 0.001) in both the cell types suggesting ongoing apoptosis. Phosphatidylserine externalization, mitochondrial events, caspase evaluation and nuclear morphology changes reveal initiation/progression of caspase-dependent apoptosis even at a dose of 1 μM which eventually leads to DNA fragmentation in both the cell types. Results demonstrate that CC induces caspase-dependent apoptosis in MCF-7 and MDA MB-231 cells irrespective of ER status similar to TAM in terms of anti-neoplastic activity.

Decursin and decursinol angelate inhibit estrogen-stimulated and estrogen-independent growth and survival of breast cancer cells

IntroductionEstrogen and estrogen receptor (ER)-mediated signaling are crucial for the etiology and progression of human breast cancer. Attenuating ER activities by natural products is a promising strategy to decrease breast cancer risk. We recently discovered that the pyranocoumarin compound decursin and its isomer decursinol angelate (DA) have potent novel antiandrogen receptor signaling activities. Because the ER and the androgen receptor belong to the steroid receptor superfamily, we examined whether these compounds affected ER expression and signaling in breast cancer cells.MethodsWe treated estrogen-dependent MCF-7 and estrogen-independent MDA MB-231 human breast cancer cells with decursin and DA, and examined cell growth, apoptosis, and ERα and ERβ expression in both cell lines – and, in particular, estrogen-stimulated signaling in the MCF-7 cells. We compared these compounds with decursinol to determine their structure-activity relationship.ResultsDecursin and DA exerted growth inhibitory effects on MCF-7 cells through G1 arrest and caspase-mediated apoptosis. These compounds decreased ERα in MCF-7 cells at both mRNA and protein levels, and suppressed estrogen-stimulated genes. Decursin and the pure antiestrogen Faslodex™ exerted an additive growth inhibitory effect on MCF-7 cells. In MDA MB-231 cells, these compounds induced cell-cycle arrests in the G1 and G2 phases as well as inducing apoptosis, accompanied by an increased expression of ERβ. In contrast, decursinol, which lacks the side chain of decursin and DA, did not have these cellular and molecular activities at comparable concentrations.ConclusionThe side chain of decursin and DA is crucial for their anti-ER signaling and breast cancer growth inhibitory activities. These data provide mechanistic rationales for validating the chemopreventive and therapeutic efficacy of decursin and its derivatives in preclinical animal models of breast cancer.

Human breast cancer MDA-MB-231 cells fail to express the neurofibromin protein, lack its type I mRNA isoform and show accumulation of P-MAPK and activated Ras

Neurofibromin is a tumor suppressor protein, which is similar in function to the GTPase activating protein (GAP), p120GAP, in that it accelerates inactivation of Ras. Mutations in the NF1 gene cause neurofibromatosis type 1, NF1, an autosomal dominant disease with a diverse spectrum of clinical manifestations, including neurofibromas. Ras activation (GTP binding) is induced by the GTP exchange factor Sos and its inactivation is regulated through the GAPs (p120GAP and neurofibromin). Strikingly, neurofibromin was nearly absent in MB-231 human breast cancer cells and present in the remaining four cell lines studied, with higher levels in BT-474 and MB-453 than in MCF-7 and BT-20 cells, as tested with polyclonal antibodies to both the N-terminal as well as the C-terminal peptides. Coordinated with the near absence of neurofibromin, these cells also presented with much greater levels of P-MAPK and activated Ras. Further, RT-PCR analysis demonstrated the absence of expression of NF1 mRNA type I isoform only in the MB-231 cell lines. This result documents for the first time an altered NF1 expression at the protein and mRNA levels in MDA-MB-231 breast cancer cells.