MDA-MB231 Cells Research Articles

Abstract 967: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT)

Abstract Triple-negative breast cancers (TNBC) are a subset of breast cancer that is highly aggressive and has a poor prognosis. The TNBC subtype has no effective molecular targeted therapies. Gene expression studies have identified a subset of TNBC that is enriched with androgen receptors (AR) and androgen receptor signaling. AR levels are most abundant in the luminal AR (LAR) molecular subtype of TNBC, but other non-LAR molecular TNBC subtypes also display high levels of AR expression and activity. Interference with androgen signaling in TNBC with AR-inhibiting drugs have shown a reduction in epithelial-to-mesenchymal transition (EMT), which is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal-like stem cells. Approximately one-third of TNBC expressed AR, and evaluation of AR-positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. Previous studies have shown that AR inhibition or AR knockdown significantly reduces migration and invasion and EMT in different TNBC cell lines. γ-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer effects and have little or no effect on normal cell viability. Studies show that treatment with 0-7 µM γ-tocotrienol reduced AR levels, proliferation, migration and invasion in a dose-responsive manner in TNBC MDA-MB-231 and MDA MB-453 cell lines. Results also show that treatment with 5 µM (MDA-MB-231) and 7 µM (MDA-MB-453) γ-tocotrienol induced a reversal in EMT cell biomarkers, as well as reversal in EMT morphological and behavioral characteristics in these TNBC cell lines. Western blot analysis show that similar treatment with γ-tocotrienol significantly decreased DHT-induced N-cadherin (mesenchymal cell biomarker) and increased expression in cytokeratin 18 and E-cadherin (epithelial cell biomarkers) in these cells. AR signaling is known to play an important role in stimulating DHT-induced EMT by stimulating the STAT3/Snail signaling pathway in MDA-MB231. However, results in the present study shows that γ-tocotrienol treatment significantly inhibited activation of STAT3 and Snail proteins. Additional immunocytochemistry experiments showed that γ-tocotrienol treatment significantly inhibited DHT-induced cytoskeleton changes in TNBC MDA-MB-231 and MDA MB-453 cell lines. In conclusion, these results demonstrated that γ-tocotrienol treatment inhibits AR expression and DHT-dependent EMT and cytoskeleton changes in TNBC cells. These findings suggest that AR may be a potential therapeutic target for treating both LAR and non-LAR TNBC subtypes. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Nayef Aldabaan, Tasmin A. Sultana, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 967.

Abstract 1334: MerTK tumorigenesis and immune evasion mechanisms in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) cells lack estrogen, progesterone, and HER2 receptors. TNBC tends to be more aggressive than most types of breast cancer and is more likely to recur even after surgery and treatment. Recently, a few targeted therapies were approved for TNBC, but additional molecular targeting agents are still needed. Previous studies have found that activated MerTK is associated with many types of human cancers, including breast cancer. In this study, we focused on MerTK expression and its tumorigenesis and immunoregulatory functions in TNBC. We first examined MerTK expression levels in human TNBC tissue samples by analyzing tissue microarrays (TMA) using immunohistochemistry (IHC). IHC revealed that ~40% of TNBC samples expressed high levels of MerTK. We further examined MerTK expression levels in ten TNBC cell lines by immunoblot analysis. Three cell lines (BT549, MDAMB231 and MDAMB436) showed strong MerTK expression levels. Next, to evaluate MerTK’s effect on cell proliferation, we treated MDAMB231 cells with MerTK siRNA, which inhibited cell proliferation by 20% compared to siNon-target (siNT). These data indicate that TNBC cells remain dependent on MerTK-activated signaling pathways for proliferation and survival. To further investigate the impact of MerTK expression in TNBC cells, we stably overexpressed MerTK in the TNBC cell line SUM102, which naturally has very low MerTK levels. The results indicated that overexpression of MerTK in SUM102 cells led to: 1) increased proliferation in vitro 2) robust tumor growth, 3) marked migration potential and 4) increased metastasis in mice. Collectively, these results demonstrate that activation of a MerTK-driven pathway could be involved in tumorigenesis in TNBC. To understand the role of MerTK in cell signaling and pro-tumor immunity, we utilized a cytokine array to examine which molecules were impacted in MerTK-overexpressing SUM102 cells. The results showed that production of CXCL4, CD74, and IL-1α was increased, and production of CXCL10 was decreased in MerTK-overexpressing SUM102 cells as compared to control cells. Interestingly, PD-L1 was upregulated and MHC class I was downregulated in MerTK-overexpressing SUM102 cells as compared to control cells. These preliminary findings suggested that MerTK-overexpressing TNBC could be creating a favorable immune microenvironment for proliferation of tumor cells. To further explore the cytokine, PD-L1, and MHC class I results, we overexpressed MerTK in 4T1 murine TNBC-like cells. Immunoblot analysis showed that PD-L1 was upregulated in MerTK-overexpressing 4T1 cells. We will utilize this cell line to evaluate tumor growth and immunosuppression in the tumor microenvironment in BALB/C mice. Citation Format: Mari Iida, Kourtney Kostecki, Carlene A. Kranjac, Christine Glitchev, David T. Yang, Deric L. Wheeler. MerTK tumorigenesis and immune evasion mechanisms in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1334.

Green synthesized Ag-nanoparticles using Scutellaria multicaulis stem extract and their selective cytotoxicity against breast cancer

Scutellaria multicaulis is a medicinal herb which has been extensively prescribed in Iranian traditional medicine for treatment of infections, constipation, wounds, and also as medication for anxiety. To evaluate biological activities of biosynthesized silver nanoparticles (Sm-AgNPs), Sm-AgNPswere synthesized using S. multicaulis stem extract as capping and reducing agent. Characterization studies using UV–vis, XRD, DLS, Zeta potential, FESEM, EDX, TEM, FTIR, RAMAN and TGA assays indicated that Sm-AgNPs had optimal shape, size (∼60 nm) and stability (−46.4 mV) to be used as a drug. Findings also demonstrated that Sm-AgNPs display higher Total Phenolics and Total Flavonoids Content and possesses higher antioxidant activity. HPLC-MSn analysis of constituents in the S. multicaulis stem extract and Sm-AgNPs-free supernatant in negative ion mode showed that flavonoids, mainly jaceidin, skullcapflavon II, wogonin, oroxylin A and dihydroxy, trimethoxyflavone from extract have contributed to the synthesis of Sm-AgNPs. Additionally, Sm-AgNPs demonstrated effective anticarcinogenic properties on MDA-MB231 cells proliferation with IC50 value of 81.2 μg/mL at 48 h by inducing cell apoptosis. Overall, results confirmed the potential role of S. multicaulis stem extract as a potential raw material for synthesis of biologically active Sm-AgNPs, for development of new antitumor agents which could be utilized to combat breast cancer.

Brain infiltration of breast cancer stem cells is facilitated by paracrine signaling by inhibitor of differentiation 3 to nuclear respiratory factor 1.

Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood-brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCsID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCsID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCsID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCsID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCsID3 help guide BCSCsNRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.

DNA cleavage, cytotoxic and antioxidant properties of Cistus laurifolius L. extracts

In this study, extracts of the root, branch and leaf parts of Cistus laurifolius L. were prepared using hexane, methylene chloride and ethanol solvents. DNA cleavage, cytotoxic and antioxidant properties of the prepared extracts were then investigated. The cytotoxic properties of the extracts were investigated in A549, DU-145, PNT-1A, MDA-MB231, CRL-4010 and HCT-116 cell lines. Among all prepared extracts, T4 (seed–methylene chloride) extract showed cytotoxic effects on more cancer cell lines compared to other extracts. Antioxidant tests were performed by DPPH (2,2-diphenyl-1-picrylhydrazil) radical scavenging and Folin-Ciocalteu methods. In both tests, it was determined that T7 (seed–ethanol) extract has a very strong antioxidant effect (IC50 = 0,000015 μg/ml for the DPPH method). In DNA cleavage studies, the results obtained confirm the protection of DNA from hydroxyl radical damage in the presence of methanolic plant extracts. Consequently, we determined that some of the Cistus laurifolius L. extracts have significant biological properties on the studied cell lines.

Lipoic acid blocks autophagic flux and impairs cellular bioenergetics in breast cancer and reduces stemness

Autophagy inhibition is currently considered a novel therapeutic strategy for cancer treatment. Lipoic acid (LA), a naturally occurring compound found in all prokaryotic and eukaryotic cells, inhibits breast cancer cell growth; however, the effect of LA on autophagy-mediated breast cancer cell death remains unknown. Our study identified that LA blocks autophagic flux by inhibiting autophagosome-lysosome fusion and lysosome activity which increases the accumulation of autophagosomes in MCF-7 and MDA-MB231 cells, leading to cell death of breast cancer cells. Interestingly, autophagic flux blockade limits the recycling of cellular fuels, resulting in insufficient substrates for cellular bioenergetics. Therefore, LA impairs cellular bioenergetics by the inhibition of mitochondrial function and glycolysis. We show that LA-induced ROS generation is responsible for the blockade of autophagic flux and cellular bioenergetics in breast cancer cells. Moreover, LA-mediated blockade of autophagic flux and ROS generation may interfere with the regulation of the BCSCs/progenitor phenotype. Here, we demonstrate that LA inhibits mammosphere formation and subpopulation of BCSCs. Together, these results implicate that LA acts as a prooxidant, potent autophagic flux inhibitor, and causes energetic impairment, which may lead to cell death in breast cancer cells/BCSCs.

Design, synthesis, biological evaluation, and in silico studies of 2-aminobenzothiazole derivatives as potent PI3Kα inhibitors.

A new series of 2-aminobenzothiazole derivatives was designed, synthesized and evaluated for their anticancer activity against the MCF7, MDAMB-231, and HepG2 cancer cell lines. All synthesized derivatives (8a-8n) demonstrated moderate to high anticancer activity against the tested cell lines. As the most potent compound in the series, compound 8i displayed excellent inhibitory potency with an IC50 value of 6.34 μM and compound 8m displayed an IC50 value of 8.30 µM against the MCF7 cell line compared to the standard drug HS-173 (IC50 = 10.25 μM). PI3K enzyme activity assays demonstrated that compound 8i is highly selective against PI3Kα, with an IC50 value of 1.03 nM. Wound healing assays and cell cycle analysis of compounds 8i and 8m revealed that both compounds suppressed the migration of MCF7 cells and induce cell cycle arrest in the S phase. In the cell death assay, compound 8i was revealed to induce apoptosis in a dose-dependent pattern; further Western blot assays revealed that compound 8i obviously decreases the levels of the antiapoptotic proteins Bcl-xL and Mcl-1. Downregulation of the expression of p-Akt confirmed the PI3K inhibitory activity of compound 8i. The molecular docking and molecular dynamicssimulation studies performed were found in agreement with the PI3Kα inhibitory activity assessments performed experimentally.

Overexpression of pigment epithelium-derived factor in breast cancer cell-derived exosomes induces M1 polarization in macrophages

M2 macrophages, the major component of tumor microenvironment, are recognized as important player in tumor progression. M2 macrophages mediate this effect by promoting tumor angiogenesis, tumor metastasis, and suppression of tumor immunity. Reprogramming of M2 macrophages can serve as a promising strategy in cancer immunotherapy. In this study, we constructed pigment epithelium-derived factor (PEDF) expressing vector and transfected MDA-MB-231 cells with this construct. Then, exosomes were isolated from transfected cells and M2 macrophages were incubated with isolated exosomes from transfected cell. The effect of isolated exosomes on macrophage polarization was examined by real-time PCR and ELISA. The results demonstrated reprogramming of M2 macrophages after incubation with isolated exosomes from PEDF transfected cells. M2-to-M1 repolarization of macrophages was confirmed by upregulation of CD80, IRF5, MCP1, and IL-1β and repression of CD206, Arg, TGM2, and TGF-β. Therefore, these findings revealed that introducing PEDF into exosomes by genetic manipulation of parent cells may be a potential approach for reprogramming of M2 macrophages in cancer.

MiR-150 expression in breast cancer attracts and activates immune cells, and is associated with better patient outcome.

570 Background: MicroRNA (miRNA) epigenetically regulate a large number of cancer-related genes and is known as a key player in cancer biology. MiR-150 promote cancer cell proliferation, migration, and invasion. However, there has been no study that investigated the role of miR-150 in the tumor microenvironment (TME) of breast cancer patients. Methods: Total of 1,961 breast cancer patients from multiple independent large cohorts were analyzed. These observational results were validated by in vitro experiments. Lymphocyte attraction was assessed using Transwell system. Results: miR-150 expression was the highest in triple negative breast cancer (TNBC) among subtypes and correlated with Nottingham histological grade (all p<0.001). A high miR-150 was significantly associated with better overall survival (OS) in both METABRIC and TCGA cohorts regardless of subtypes (all p<0.05 except for TNBC in TCGA). MiR-150 expression was strongly correlated with Hallmark immune-related gene sets (Allograft rejection, IL6 signaling, IFN-γ response, Inflammatory response, IL2 signaling, and apoptosis), with cytolytic activity, with infiltration of CD8+ T cells, CD4+ memory T cells, and dendritic cells, as well as with gene expressions of major immune checkpoint molecules (PD-1, CTLA4, IDO1, TIGIT, BTLA, and LAG3), consistently in both METABRIC and TCGA cohorts (all r>0.65, p<0.01). Unexpectedly, overexpression of miR-150 by mimic did not increase growth, migration, nor invasion of neither MDA-MB231 or BT-549 breast cancer cell lines. On the other hand, overexpression of miR-150 in either MDA-MB231 or BT-549 significantly increased attraction of lymphocyte cell line (Jurkart cells), which was abolished by addition of miR-150 inhibitor. By comparing the gene expression profile between miR-150 overexpressed and control cells, we found that many inflammation related gene sets including NF-kB signaling that enhance cell migration was enriched to miR-150 overexpressed cells, implicating that is how miR-150 high cells attract lymphocytes to TME. Conclusions: miR-150 expression is associated with immune cell infiltration and immune response, as well as with better survival in breast cancer patients. Overexpression of miR-150 in breast cancer cells did not promote cancer cell proliferation, migration, nor invasion; however, increased attraction of lymphocytes. Multiple inflammation-related genes were expressed higher in miR150 overexpressed MDA-MB231.

Lamotrigine derivatives‐synthesis, anti‐cancer, and anti‐MDR‐bacterial activities

A new series of quaternary ammonium salts 3–21 of lamotrigine (6-(2, 3-dichlorophenyl)-1, 2, 4-triazine-3, 5-diamine) was synthesized via N-alkylation of lamotrigine using different benzyl bromides. The new analogues were characterized by using FT-IR, NMR, and mass spectrometric techniques. Single-crystal X-ray diffraction analysis of compound 10 was also carried out to confirm the position of substitution and salt formation. All the compounds 3-21 were tested for various biological activities, including anti-bacterial, and anti-cancer properties. All compounds, except 20, exhibited a potent growth inhibition of Staphylococcus aureus strains as compared to the ofloxacin, the standard drug. Compounds 4, 7, 10, and 13 showed a significant toxicity towards HeLa cell line (derived from cervical carcinoma), whereas compounds 3, and 5 showed a significant activity towards HeLa, MCF-7 (estrogen and progesterone positive breast cancer cell line), and MDA-MB cell lines (epithelial, human breast cancer cell line), as compared to the standard drug doxorubicin. To the best of our knowledge all analogues of 6-(2, 3-dichlorophenyl)-1, 2, 4-triazine-3, 5-diamine, and their activity against MDR, and anti-cancer is reported here for the first time.

DMT1‐mediated endosome‐mitochondria interactions regulates iron homeostasis and mitochondrial metabolism

Mitochondria‐early endosome (EE) interactions have been shown to facilitate the translocation of iron into mitochondria. Here we show that Divalent Metal Transporter 1 (DMT1) modulates iron exit from endosomes and transport into mitochondria via regulation of EE‐mitochondria interactions. In cancer cells, mitochondria are the ultimate cellular iron sink, where iron can be either stored or used for example to shift cellular metabolism towards glycolysis (Warburg effect), a key adaptive mechanism of cancer cells. Moreover, a gene signature associated with reduced intracellular iron content, including low transferrin receptor (TfR) (anti‐import) and high ferroportin (FPN) (pro‐export) expression levels, has been related to favorable breast cancer prognosis. Similarly, reduced DMT1 expression associates with improved breast cancer patient survival. We evaluated the role of DMT1 in two distinct breast cancer cell lines: estrogen receptor positive T47D and triple‐negative MDAMB231. In both cell lines, we demonstrate colocalization between EE, DMT1 and mitochondria. Interestingly, DMT1 is localized to the surface contact area between endosomes and mitochondria. To demonstrate that DMT1 plays a role in endosome‐mitochondria interactions and Mitochondrial Iron Translocation (MIT), we have generated MDAMB231 as well as T47D CRISPR/Cas9 based DMT1 knockout (KO) stable cell lines. Several lines of evidence show that DMT1 regulates MIT and labile iron pool (LIP) levels via modulation of EE‐mitochondria interactions in MDAMB231 cells. MIT decrease via DMT1 silencing was partially rescued by re‐expression of DMT1 in MDAMB231, but not in T47D cells. MDAMB231 DMT1 KO cells showed increased Ferro‐Orange staining, indicating higher LIP levels, as well as decreased TfR and increased FPN protein levels. Importantly, DMT1 silencing significantly reduced EE‐mitochondria interactions and EE speed in MDAMB231 but not in T47D. Thus, DMT1 regulates MIT and LIP levels via EE‐mitochondria interactions in MDAMB231. These results are in agreement with previous results showing that MDAMB231 display a delay in iron release in comparison to T47D, making them more sensitive to disruptions in MIT. Since mitophagy has been shown to act as a tumor suppressor in breast cancer, we tested whether it could be modulated by DMT1‐mediated MIT. We found that DMT1 silencing increases mitochondrial ferritin, global autophagy marker LC3B and PINK1/Parkin‐dependent mitophagy markers in MDAMB231; levels of all proteins evaluated were rescued to basal levels upon re‐expression of DMT1 in DMT KO cells. Moreover, DMT1 silencing decreases Tom 20 (outer mitochondrial membrane marker) with PMPCB, a known DMT1 interactor that is required for PINK1 turnover. Concurring with the role of DMT1 in mitophagy and iron metabolism, both mitochondrial metabolism and invasive cell migration are significantly impaired by DMT1 silencing and are partially rescued by re‐expression of DMT1. Overall, our results implicate DMT1 in the regulation of EE dynamics and EE‐mitochondria interactions to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cell migration. Thus, we propose DMT1 as a key player associated with aggressive phenotypes in breast cancer

Effect of Knock‐out of the a4 Isoform of Vacuolar H+‐ATPase following CRISPR/Cas9 on Activation of Cathepsins in MDA‐MB231 Breast Cancer Cell

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases creates a low pH in a localized environment which promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells cleave extracellular matrix proteins and activate other proteases, facilitating tumor cell invasion. Our lab is investigating a connection between the isoforms of the a subunit for V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but not cathepsin L. However, although there was a trend, differences in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data supports the conclusion that there is a significant difference in the effects of V‐ATPase inhibition on secreted cathepsins. Inhibiting V‐ATPases does not appear to affect the activity of secreted cathepsin L, however it reduces the activity of secreted cathepsins B and D. To further investigate the role of V‐ATPases in the activation of cathepsins, we are using CRISPR/Cas9 to knockout the genes of the four V‐ATPase “a” subunit isoforms, a1, a2, a3 and a4. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. The a1 and a2 isoforms are found in intracellular compartments. Thus far we have made the most progress with knocking out the a4 isoform. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsins B and D while not effecting activation of cathepsin L.

Simulation and fabrication of an integrating well-aligned silicon nanowires substrate for trapping circulating tumor cells labeled with Fe3O4 nanoparticles in a microfluidic device.

Introduction: Circulating tumor cells (CTCs) are the transformed tumor cells that can penetrate into the bloodstream and are available at concentrations as low as 1-100 cells per milliliter. To trap CTCs in the blood, one valid and mature technique that has been developed is the magnetophoresis-based separation in a microfluidic channel. Recently, nanostructured platforms have also been developed to trap specific targeted and marker cells in the blood. We aimed to integrate both in one platform to improve trapping. Methods: Here, we developed a numerical scheme and an integrated device that considered the interaction between drag and magnetic forces on paramagnetic labeled cells in the fluid as well as interaction of these two forces with the adhesive force and the surface friction of the nanowires substrate. We aimed on developing a more advanced technique that integrated the magnetophoretic property of some Fe3O4 paramagnetic nanoparticles (PMNPs) with a silicon nanowires (SiNWs) substrate in a microfluidic device to trap MDA-MB231 cell lines as CTCs in the blood. Results: Simulation indicated assuming that the nanoparticles adhere perfectly to the white blood cells (WBCs) and the CTCs, the magnetic moment of the CTCs was almost one order of magnitude larger than that of the WBCs, so its attraction by the magnetic field was much higher. In general with significant statistics, the integrated device can trap almost all of the CTCs on the SiNWs substrate. In the experimental section, we took advantage of the integrated trapping techniques, including micropost barriers, magnetophoresis, and nanowires-based substrate to more effectively isolate the CTCs. Conclusion: The simulation indicated that the proposed device could almost trap all of the CTCs onto the SiNWs substrate, whereas trapping in flat substrates with magnetophoretic force was very low. As a result of the magnetic field gradient, magnetophoretic force was applied to the cells through the nanoparticles, which would efficiently drive down the nanoparticle-tagged cells. For the experimental validation, anti-EpCAM antibodies for specific binding to tumor cells were used. Using this specific targeting method and by statistically counting, it was shown that the proposed technique has excellent performance and results in the trapping efficiency of above 90%.

In vitro effect of PIK3CA/mTOR inhibition in triple-negative breast cancer subtype cell lines.

Agents targeting the PI3K pathway in triple negative breast cancer did not show any significant efficacy so far mostly because of the complex nature of these targeted inhibitors. Targeting the cancer cells with the combination of inhibitors may help in decelerating the regulatory pathways further achieving optimum clinical benefit. In this study, we investigated the effect of PIK3CA and mTOR inhibition in-vitro in triple-negative breast cancer (TNBC) cell lines. Three TNBC cell lines; MDA MB231, MDA MB468, and MDA MB453 were subtyped using immunohistochemistry and were screened for hotspot mutations in PIK3CA and AKT1. All cell lines were treated with different concentrations of inhibitors; PI3K inhibitor (BKM 120), mTOR inhibitor (AZD 8055), and dual PI3K/mTOR inhibitor (BEZ 235), and cell viability was assessed by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide), Trypan blue and Annexin-V/PI Assays. Using immunohistochemistry, TNBC cell lines were subtyped as; mesenchymal subtype-specific cell line (MDA MB231), basal subtype-specific cell line (MDA MD468), and Luminal androgen receptor (LAR) subtype-specific cell line (MDA MB453). PIK3CA hot spot mutation (p.H1047R) in exon 20 was identified in the Luminal androgen receptor subtype (MDA MB453 cells) cell line. Cell viability assays showed that the Mesenchymal subtype-specific cell line (MDA MB231) was the most resistant to all inhibitors and the Luminal Androgen subtype (MDA MB453 cells) cell line was more sensitive to BKM120 (PI3K inhibitor) inhibition compared to other subtypes. This study identified that the Luminal androgen receptor subtype of triple-negative breast cancer with PIK3CA mutation may be targeted with PIK3CA inhibitors with a favorable outcome.

The effects of sertraline on breast cancer (MDAMB-231) cells viability in vitro

Antidepressant drugs have been reported to affect the viability of cancer cells; however, the effects of the antidepressant sertraline on breast cancer cells is still unclear. The present study investigated the cytotoxic effects of sertraline on breast cancer cell viability in vitro. In this experimental-laboratory study, MDAMB-231 breast cancer cells were divided into control (untreated) and groups treated with 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μg / ml of sertraline. Cell viability was measured 24 hours after treatment using MTT assay method. Data were analyzed using one-way analysis of variance. The results showed that treatment with 1.56 μg / ml of sertraline had no significant effect on the viability of MDAMB-231 cells, however, sertraline with concentrations ≥ 3.12 μg / ml significantly reduced the MDAMB-231 cell viability after 24 and 48 hours. In conclusion, the findings of this study indicated that sertraline has cytotoxic effects on breast cancer cells in a dose dependent manner.

Synthesis, Molecular Docking, and 2D-QSAR Modeling of Quinoxaline Derivatives as Potent Anticancer Agents against Triple-negative Breast Cancer.

Breast carcinomas aka triple-negative breast cancers (TNBC) are one of the most complex and aggressive forms of cancers in females. Recently, studies have shown that these carcinomas are resistant to hormone-targeted therapies, which makes it a priority to search for effective and potential anticancer drugs. The present study aimed to synthesize and develop the 2Dquantitative structural activity relationship model (QSAR) of quinoxaline derivatives as a potential anticancer agent. Quinoxaline derivatives were designed and synthesized (8a-8i and 9a-9d) and the 2DQSAR model against TNBC was developed using VLife MDS v4.4. The anticancer activity was investigated against the TNBC MDA-MB-231 cell line using an MTT cytotoxicity assay. Molecular docking studies along with the estimation of ADMET parameters were done using Discovery Studio. The most potent compound was docked against the β-tubulin protein target (PDB: 4O2B), using the Autodock Vina v0.8 program. Eleven derivatives of quinoxaline were designed and synthesized (8a-8i and 9a-9d) and a 2D-QSAR model was developed against the TNBC MDA-MB231 cell line. The regression coefficient values for the training set were (r2) 0.78 and (q2) 0.71. Further, external test set regression (pred_r2) was 0.68. Five molecular descriptors viz., energy dispersive (Epsilon3), protein-coding gene (T_T_C_6), molecular force field (MMFF_6), most hydrophobic hydrophilic distance (XA), and Zcomp Dipole were identified. After ADMET, the best analog 8a showed the best activity against the TNBC cell line. The best-predicted hit '8a' was found to bind within the active site of the β- tubulin protein target. The newly synthesized quinoxaline compounds could serve as potent leads for the development of novel anti-cancer agents against TNBC.

Multi-targeted Drug Repurposing Approach for Breast Cancer via Integrated Functional Network Analysis.

The present study focuses on the interconnected functional network of altered metabolism and EMT (epithelial to mesenchymal transition) signaling in breast cancer. We have interlinked the metabolic and EMT signaling circuits and selected Insulin receptor (IR), Integrin beta 1 (ITGB1), and CD36 as target proteins based on network analysis. Extensive computational approaches discerned the potential drug molecules from the library of 1293 FDA-approved drugs to block all three target proteins. Using molecular docking, molecular dynamics simulation, and MMPBSA binding free energy studies, Capmatinib, Ponatinib, Naldemedine, and Pimozide were identified as potential repurposed drugs to block the function of all three target proteins. Among in silico selected candidate drugs, Pimozide, a known anti-psychotic drug, was further validated using in-vitro studies for its anti-cell proliferative potential on breast cancer cell lines (namely, MCF7, MDAMB231 and MDAMB468). The inhibitory concentration (IC50 ) values of MCF7, MDAMB231 and MDAMB468 was found to be 16.26 μM, 20.82 μM and 13.10 μM, respectively. The effect of Pimozide on EMT-induced MDAMB231 and MDAMB468 cells was evident from their IC50 values of 7.85 μM and 6.83 μM, respectively. The potent anti-cancer property of Pimozide has opened up avenues for drug repurposing towards 'multi-targeted therapy' in EMT dynamics.

Moringa oleifera Methanolic Extract Induces Apoptosis in Human Breast Cancer Cells

Background: Moringa oleifera Lam. (Family: Moringaceae), commonly known as ‘drumstick tree’ is beneficial to obtain sustenance as a food source and applied as a traditional medicine. The therapeutic potential of Moringa oleifera emphasizes on the inhibition of cancer proliferation and its versatile applications in Pharmacology which provide novel by-products and nutritive dietary supplements for human consumption. The objective of the present study is to investigate the efficacy of M. oleifera leaves methanolic extract (MOME) against the invasive breast cancer cell lines such as T-47D and MDA-MB-231. Materials and Methods: The different concentrations of MOME were cultured individually with breast cancer cells, T-47D and MDA-MB-231. The cell viability was determined after 24 hrs of treatment using MTT Assay (-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) to analyse the anti-proliferative effect of MOME on cell lines, T-47D and MDA-MB-231. The cell cycle assay and apoptosis were evaluated by Annexin V- FITC Apoptosis Detection Kit, followed by the analysis through Attune flow cytometer. Results: The IC50 values of MOME in T-47 D and MDA-MB231 were found 45.33 ± 5.2 μg/mL and 24.44 ± 3 μg/mL, respectively. The G2M phase population exhibited an increase of 60.4% in MOME treated T-47D cells and 11.6% in MDA-MB231 cells. The population of early apoptosis cells increased to 37.1% in MOME treated cells. The time and dose dependent treatment with MOME inhibited the growth and induced apoptosis in T-47D and MDA-MB-231 cells. The externalization of phosphatidylserine on membrane of T-47D and MDA-MB-231 cells confirmed the apoptosis. Conclusion: These findings suggest that MOME actively contributes to cell cycle arrest and inhibits the proliferation of breast cancer cells in a dose-dependent manner; thus, evaluated as the first report of apoptosis induced in triple negative breast cancer cells by MOME. The cell cycle arrest at G2/M phase depicted G2/M enrichment to emphasize on the efficacy of MOME, thus representing as an ideal anti-cancer agent against human breast cancer cells. The dose-dependent methanolic extract of leaves of M. oleifera was found as a promising antiproliferating agent against the invasive breast cancer cells.

Glycosphingolipids in human embryonic stem cells and breast cancer stem cells, and potential cancer therapy strategies based on their structures and functions.

Expression profiles of glycosphingolipids (GSLs) in human embryonic stem cell (hESC) lines and their differentiated embryoid body (EB) outgrowth cells, consisting of three germ layers, were surveyed systematically. Several globo- and lacto-series GSLs were identified in undifferentiated hESCs and during differentiation of hESCs to EB outgrowth cells, and core structure switching of these GSLs to gangliosides was observed. Such switching was attributable to altered expression of key glycosyltransferases (GTs) in GSL biosynthetic pathways, reflecting the unique stage-specific transitions and mechanisms characteristic of the differentiation process. Lineage-specific differentiation of hESCs was associated with further GSL alterations. During differentiation of undifferentiated hESCs to neural progenitor cells, core structure switching from globo- and lacto-series to primarily gangliosides (particularly GD3) was again observed. During differentiation to endodermal cells, alterations of GSL profiles were distinct from those in differentiation to EB outgrowth or neural progenitor cells, with high expression of Gb4Cer and low expression of stage-specific embryonic antigen (SSEA)-3, -4, or GD3 in endodermal cells. Again, such profile changes resulted from alterations of key GTs in GSL biosynthetic pathways. Novel glycan structures identified on hESCs and their differentiated counterparts presumably play functional roles in hESCs and related cancer or cancer stem cells, and will be useful as surface biomarkers. We also examined GSL expression profiles in breast cancer stem cells (CSCs), using a model of epithelial-mesenchymal transition (EMT)-induced human breast CSCs. We found that GD2 and GD3, together with their common upstream GTs, GD3 synthase (GD3S) and GD2/GM2 synthase, maintained stem cell phenotype in breast CSCs. Subsequent studies showed that GD3 was associated with epidermal growth factor receptor (EGFR), and activated EGFR signaling in breast CSCs and breast cancer cell lines. GD3S knockdown enhanced cytotoxicity of gefitinib (an EGFR kinase inhibitor) in resistant MDA-MB468 cells, both in vitro and in vivo. Our findings indicate that GD3S contributes to gefitinib resistance in EGFR-positive breast cancer cells, and is a potentially useful therapeutic target in drug-resistant breast cancers.

Role of Neural (N)-Cadherin in Breast Cancer Cell Stemness and Dormancy in the Bone Microenvironment.

Simple SummaryBreast cancer (BrCa) patients experience tumour recurrence 7–25 years after mastectomy. Dormant BrCa cells often home to the bone and, upon reactivation, lead to secondary lesions. We previously demonstrated that, in the bone marrow, dormant BrCa cells are located near the endosteal niche enriched in spindle-shaped N-cadherin-high Osteoblasts (SNOs), show a stem signature like the LT-HSCs and express high levels of Notch2. Here, we observed that Notch2High human BrCa MDA cells are also N-CadherinHigh. When injected in vivo, N-CadherinHigh MDA cells show lower aggressiveness, and higher ability to colonize the endosteal niche and to adhere to SNOs compared to N-CadherinLow MDA cells. These cells have features typical of cancer stem cells, displaying a gene signature similar to the HSCs. In contrast, N-cadherin has a negative role in mouse 4T1 cell stemness and HSC mimicry. In brief, our results identified a role of N-Cadherin in BrCa dormancy and stemness, also highlighting the differences between human and mouse BrCa cell lines.Breast cancer cells that interact with spindle-shaped N-Cadherin+ Osteoblasts (SNOs) are recognised to become dormant through a Notch2-dependent mechanism. We found that Notch2High human BrCa MDA-MB231 (MDA) cells also expressed high level of N-Cadherin. This prompted us to hypothesize that N-Cadherin could have a role in MDA-SNO interaction. Of note, the expression of N-Cadherin in MDA cells reduced tumour incidence and bone osteolysis in BrCa mouse model. Moreover, similarly to Notch2High MDA cells, the N-CadherinHigh MDA cells revealed a high expression of the canonical Haematopoietic Stem cell (HSC) markers, suggesting an HSC mimicry, associated with higher ability to form mammospheres. Interestingly, N-CadherinHigh MDA cells showed greater capacity to adhere to SNOs, while the inhibition of SNO-mediating MDA cell proliferation was unremarkable. To investigate whether these features were shared by mouse BrCa, we used the 4T1 cell line in which N-Cadherin expression was abolished and then rescued. At variance with MDA cells, 4T1 cells expressing N-Cadherin revealed that the latter was associated with a lower expression of the HSC marker, Cxcr4, along with a lower capacity to form mammospheres. Furthermore, the rescue of N-Cadherin expression increased cell-cell adhesion and reduced proliferation of 4T1 cells when they were co-plated with SNOs. In conclusion, we demonstrated that: (i) N-CadherinHigh and Notch2High MDA cells showed similar HSC mimicry and dormancy features; (ii) N-Cadherin mediated BrCa-SNO adhesion; (iii) N-Cadherin had a positive Notch2-dependent role on SNO-induced dormancy and HSC mimicry in MDA cells, and a negative role in 4T1 cell stemness and HSC mimicry.