Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand–mediated triple negative breast cancer cell death

Nearly 40 000 women die annually from breast cancer in the United States. Clinically available targeted breast cancer therapy is largely ineffective in triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). TNBC is associated with a poor prognosis. Previous reports show that aryl hydrocarbon receptor (AhR) partial agonist 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) selectively inhibits the growth of breast cancer cells, including those of the TNBC subtype. We previously demonstrated that 5F 203 induced the expression of putative tumor suppressor gene cytoglobin (CYGB) in breast cancer cells. In the current study, we determined that 5F 203 induces apoptosis and caspase-3 activation in MDA-MB-468 TNBC cells and in T47D ER⁺ PR ⁺ Her2 ⁻ breast cancer cells. We also show that caspases and CYGB promote 5F 203–mediated apoptosis in MDA-MB-468 cells. 5F 203 induced lysosomal membrane permeabilization (LMP) and cathepsin B release in MDA-MB-468 and T47D cells. In addition, silencing CYGB attenuated the ability of 5F 203 to induce caspase-3/-7 activation, proapoptotic gene expression, LMP, and cathepsin B release in MDA-MB-468 cells. Moreover, 5F 203 induced CYGB protein expression, proapoptotic protein expression, and caspase-3 cleavage in MDA-MB-468 cells and in MDA-MB-468 xenograft tumors grown orthotopically in athymic mice. These data provide a basis for the development of AhR ligands with the potential to restore CYGB expression as a novel strategy to treat TNBC.     

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca²⁺ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.     

C-藻蓝蛋白对乳腺癌细胞的抗癌作用研究

C-藻蓝蛋白具有抗癌、抗氧化、抗炎活性等多种功能,然而其对乳腺癌细胞的抗癌作用及机制尚不明确。本研究应用不同浓度(0~500μg/mL)的C-藻蓝蛋白处理人乳腺癌细胞系MDA-MB-468。研究显示,C-藻蓝蛋白以剂量依赖性方式抑制MDA-MB-468细胞的增殖并降低细胞的菌落形成能力。C-藻蓝蛋白通过上调了Fas和cleaved-caspase 3的表达并下调Bcl-2的表达来诱导细胞凋亡。C-藻蓝蛋白处理以剂量依赖性方式显著降低COX-2的表达并抑制细胞迁移能力。C-藻环蛋白以剂量依赖性方式促进p38 MAPK和JNK的磷酸化,p38 MAPK和JNK抑制剂处理可显著抑制C-藻红蛋白诱导的细胞死亡。本研究表明C-藻蓝蛋白能够抑制三阴性乳腺癌MDA-MB-468细胞的增殖,促进细胞凋亡,抑制细胞迁移能力。C-藻蓝蛋白的抗癌机制可能与激活p38 MAPK和JNK信号传导有关。     

Growth hormone-releasing hormone induced transactivation of epidermal growth factor receptor in human triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a subset of breast cancers which is negative for expression of estrogen and progesterone receptors and human epidermal growth factor receptor-2 (HER2). Chemotherapy is currently the only form of treatment for women with TNBC. Growth hormone-releasing hormone (GHRH) and epidermal growth factor (EGF) are autocrine/paracrine growth factors in breast cancer and a substantial proportion of TNBC expresses receptors for GHRH and EGF. The aim of this study was to evaluate the interrelationship between both these signaling pathways in MDA-MB-468 human TNBC cells. We evaluated by Western blot assays the effect of GHRH on transactivation of EGF receptor (EGFR) as well as the elements implicated. We assessed the effect of GHRH on migration capability of MDA-MB-468 cells as well as the involvement of EGFR in this process by means of wound-healing assays. Our findings demonstrate that in MDA-MB-468 cells the stimulatory activity of GHRH on tyrosine phosphorylation of EGFR is exerted by two different molecular mechanisms: i) through GHRH receptors, GHRH stimulates a ligand-independent activation of EGFR involving at least cAMP/PKA and Src family signaling pathways; ii) GHRH also stimulates a ligand-dependent activation of EGFR implicating an extracellular pathway with an important role for metalloproteinases. The cross-talk between EGFR and GHRHR may be impeded by combining drugs acting upon GHRH receptors and EGFR family members. This combination of GHRH receptors antagonists with inhibitors of EGFR signalling could enhance the efficacy of both types of agents as well as reduce their doses increasing therapeutic benefits in management of human breast cancer.     

Isochromanoindolenines suppress triple-negative breast cancer cell proliferation partially via inhibiting Akt activation

As the most malignant subtype of breast cancers, triple-negative breast cancer (TNBC) lacks effective targeted therapeutics clinically to date. In this study, one lead compound FZU-0025-065 with isochromanoindolenine scaffold was identified by a cell-based screening. Among nine breast cancer cell lines tested, TNBC are the most sensitive cell lines to FZU-0025-065. FZU-0025-065 inhibits TNBC cell growth in a time- and dosage-dependent manner. FZU-0025-065 suppresses the expression of cell cycle dependent kinase 4 (CDK4), Cyclin D1 and Cyclin B1; meanwhile, elevates the expression of cell cycle dependent kinase inhibitor p21 and p27. Importantly, we found that FZU-0025-065 suppresses AKT activation in a time- and dosage-dependent manner. Over-expression of constitutive active AKT partially rescues FZU-0025-065 induced cell growth inhibition in MDA-MB-468 cells, indicating FZU-0025-065 suppresses TNBC cell growth partially via inhibiting AKT activation. Finally, FZU-0025-065 suppresses TNBC cell growth in a xenograft mouse model. Taken together, our findings suggested that isochromanoindolenine derivative FZU-0025-065 inhibits TNBC via suppressing the AKT signaling and that FZU-0025-065 may be useful for TNBC treatment.     

Glucose promotes breast cancer aggression and reduces metformin efficacy

Metformin treatment has been associated with a decrease in breast cancer risk and improved survival. Metformin induces complex cellular changes, resulting in decreased tumor cell proliferation, reduction of stem cells, and apoptosis. Using a carcinogen-induced rodent model of mammary tumorigenesis, we recently demonstrated that overfeeding in obese animals is associated with a 50% increase in tumor glucose uptake, increased proliferation, and tumor cell reprogramming to an “aggressive” metabolic state. Metformin significantly inhibited these pro-tumorigenic effects. We hypothesized that a dynamic relationship exists between chronic energy excess (glucose by dose) and metformin efficacy/action.

Media glucose concentrations above 5 mmol/L was associated with significant increase in breast cancer cell proliferation, clonogenicity, motility, upregulation/activation of pro-oncogenic signaling, and reduction in apoptosis. These effects were most significant in triple-negative breast cancer (TNBC) cell lines. High-glucose conditions (10 mmol/L or above) significantly abrogated the effects of metformin. Mechanisms of metformin action at normal vs. high glucose overlapped but were not identical; for example, metformin reduced IGF-1R expression in both the HER2+ SK-BR-3 and TNBC MDA-MB-468 cell lines more significantly at 5, as compared with 10 mmol/L glucose. Significant changes in gene profiles related to apoptosis, cellular processes, metabolic processes, and cell proliferation occurred with metformin treatment in cells grown at 5 mmol/L glucose, whereas under high-glucose conditions, metformin did not significantly increase apoptotic/cellular death genes. These data indicate that failure to maintain glucose homeostasis may promote a more aggressive breast cancer phenotype and alter metformin efficacy and mechanisms of action.     

Lamin B2 promotes the progression of triple negative breast cancer via mediating cell proliferation and apoptosis

Triple negative breast cancer (TNBC) is a more common type of breast cancer with high distant metastasis and poor prognosis. The potential role of lamins in cancer progression has been widely revealed. However, the function of lamin B2 (LMNB2) in TNBC progression is still unclear. The present study aimed to investigate the role of LMNB2 in TNBC. The cancer genome atlas (TCGA) database analysis and immunohistochemistry (IHC) were performed to examine LMNB2 expression levels. LMNB2 short hairpin RNA plasmid or lentivirus was used to deplete the expression of LMNB2 in human TNBC cell lines including MDA-MB-468 and MDA-MB-231. Alterations in cell proliferation and apoptosis in vitro and the nude mouse tumorigenicity assay in vivo were subsequently analyzed. The human TNBC tissues shown high expression of LMNB2 according to the bioinformation analysis and IHC assays. LMNB2 expression was correlated with the clinical pathological features of TNBC patients, including pTNM stage and lymph node metastasis. Through in vitro and in vivo assays, we confirmed LMNB2 depletion suppressed the proliferation and induced the apoptosis of TNBC cells, and inhibited tumor growth of TNBC cells in mice, with the decrease in Ki67 expression or the increase in caspase-3 expression. In conclusion, LMNB2 may promote TNBC progression and could serve as a potential therapeutic target for TNBC treatment.     

Is miR-144 an effective inhibitor of PTEN mRNA: a controversy in breast cancer

Breast cancer is the first common cancer among women worldwide. One of the major signaling pathways playing a role in the onset and progression of this disease is PI3K/Akt/mTOR, which can be inhibited by PTEN. miRNAs are small non-coding molecules that regulate the expression of their targets by inhibition or suppression, and thus, their dysregulated expression results in the development of cancer. Using various software applications predicting miRNAs and evaluating GEO microarray data, miR-144 was selected as an inhibitor of PTEN. The expression of miR-144 and PTEN was evaluated in 18 triple negative breast cancer (TNBC) clinical samples and cell lines including 4T1, MDA-MB-231, MDA-MB-468, SK-BR-3, and MCF-7 in comparison with normal cells. PTEN and miR-144 expression analysis revealed their elevated expression in MCF-7 cells. MDA-MB-468, SK-BR-3, and MDA-MB-231 cells showed decreased levels of PTEN and increased levels of miR-144. In contrast, 4T1 cells had an increased expression of PTEN and decreased expression of miR-144. In clinical samples, miR-144 was up-regulated in 22% of the cases and PTEN was down-regulated in 78% of the cases. The results showed that the expression of PTEN and miR-144 was inversely correlated in metastatic breast cancer cell lines. However, in TNBC clinical samples, there was no correlation between the expression of miR-144 and PTEN. Literature shows that there are other influencing factors affecting the expression of miRNAs. Therefore, care should be taken in interpreting the results of gene expression studies and its relation with cancer diagnosis/prognosis.     

Cyclic GMP induced apoptosis via protein kinase G in oestrogen receptor-positive and -negative breast cancer cell lines

The activation of protein kinase G (PKG) by cyclic guanosine 3,5-monophosphate (cGMP) has become of considerable interest as a novel molecular approach for the induction of apoptosis in cancer cells. The present study was designed to examine the effects of cGMP and PKG on cell growth and apoptosis in the human breast cancer cell lines, MCF-7 and MDA-MB-468. To achieve this, 1-benzyl-3-(5P-hydroxymethyl-2P-furyl) indazole (YC-1), a soluble guanylyl cyclase activator, and 8-bromo-cGMP (8-br-cGMP), a membrane-permeant and phosphodiesterase-resistant analogue of cGMP, were employed in MCF-7 and MDA-MB-468 cells. Then, the role of PKG in the induction of apoptosis was evaluated using KT5823 and Rp-8-pCPT-cGMP as specific inhibitors of PKG. The expression of PKG isoforms in these cell lines was also investigated. KT5823 and Rp-8-pCPT-cGMP significantly attenuated the loss of cell viability caused by YC-1 and 8-br-cGMP in these cells. This study provides direct evidence that the activation of PKG by cGMP induces growth inhibition and apoptosis in MCF-7 and MDA-MB-468 breast cancer cell lines.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

Suberoylanilide Hydroxamic Acid,an Inhibitor of Histone Deacetylase,Enhances Radiosensitivity and Suppresses Lung Metastasis in Breast Cancer In Vitro and In Vivo

Triple-negative breast cancer (TNBC), defined by the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, is associated with an early recurrence of disease and poor outcome. Furthermore, the majority of deaths in breast cancer patients are from metastases instead of from primary tumors. In this study, MCF-7 (an estrogen receptor-positive human breast cancer cell line), MDA-MB-231 (a human TNBC cell line) and 4T1 (a mouse TNBC cell line) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with suberoylanilide hydroxamic acid (SAHA, an inhibitor of histone deacetylase (HDAC)) and to determine the underlying mechanisms of these effects in vitro and in vivo. We also evaluated the ability of SAHA to inhibit the metastasis of 4T1 cells. We found that IR combined with SAHA showed increased therapeutic efficacy when compared with either treatment alone in MCF-7, MDA-MB-231 and 4T1 cells. Moreover, the combined treatment enhanced DNA damage through the inhibition of DNA repair proteins. The combined treatment was induced primarily through autophagy and ER stress. In an orthotopic breast cancer mouse model, the combination treatment showed a greater inhibition of tumor growth. In addition, SAHA inhibited the migration and invasion abilities of 4T1 cells and inhibited breast cancer cell migration by inhibiting the activity of MMP-9. In an in vivo experimental metastasis mouse model, SAHA significantly inhibited lung metastasis. SAHA not only enhances radiosensitivity but also suppresses lung metastasis in breast cancer. These novel findings suggest that SAHA alone or combined with IR could serve as a potential therapeutic strategy for breast cancer.     

Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation,Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

Treatments for triple-negative breast cancer (TNBC) are limited; intermediate-conductance calcium-activated potassium (SK4) channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC) and western blotting (WB), increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05). Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05). Further investigation revealed that treatment with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT) and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.     

Progesterone signals through membrane progesterone receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear progesterone receptor

The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.     

Non-stimulated, agonist-stimulated and store-operated Ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry

In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca(2+) reserves, molecular components of the store-operated Ca(2+) entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca(2+) influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca(2+) influx. The potential roles for specific Ca(2+) channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca(2+) influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca(2+) influx in a manner dependent on Ca(2+) influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca(2+) release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (time to peak [Ca(2+)](CYT) = 188.7 ± 34.6 s (TRPC1 siRNA) versus 124.0 ± 9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca(2+) influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca(2+) influx in non-stimulated cells.     

LIN9 confers paclitaxel resistance in triple negative breast cancer cells by upregulating CCSAP

LIN9 functions to regulate cell mitotic process.Dysregulation of LIN9 expression is associated with development of human cancers.In this study we assessed the association of LIN9 expression with paclitaxel resistance and clarified the underlying mechanisms for the first time.LIN9 expression in breast cancer tissues was retrieved from publicly available online databases and statistically analyzed.Human TNBC cell lines MDA-MB-231 and MDA-MB-468 and their corresponding paclitaxelresistant sublines 231PTX and 468PTX were used to assess the expression of LIN9 by qRT-PCR and Western blot,cell growth by cell counting,cell viability by MTS assay,and cell apoptosis by flow cytometry.The data showed that high LIN9 expression in breast cancer patients receiving chemotherapy was related to poor overall survival (OS).LIN9 expression was upregulated in paclitaxel-resistant TNBC cells compared to their parental cells.Knockdown of LIN9 or treatment of paclitaxel-resistant TNBC cells with a bromo-and extra-terminal domain inhibitor (BETi) JQ1 which also decreased LIN9 expression enhanced the sensitivity of paclitaxel-resistant TNBC cells to paclitaxel.Mechanistically,decreased LIN9 in resistant cell lines reduced tumor cell viability,promoted multinucleated cells formation and induced tumor cell apoptosis,potentially by directly regulating microtubule-binding protein CCSAP.In conclusion,high LIN9 expression contributed to poor clinical outcomes and paclitaxel resistance in TNBC and BETi,targeting LIN9 expression,could be a reversible drug for PTX-resistant TNBC patients.     

ANXA2 could act as a moderator of EGFR-directed therapy resistance in triple negative breast cancer

Triple negative breast cancer (TNBC) patients cannot benefit from EGFR-targeted therapy even though the EGFR is highly expressed, because patients exhibit resistance to these drugs. Unfortunately, the molecular mechanisms remain relatively unknown. ANXA2, highly expressed in invasive breast cancer cells, is closely related with poor prognosis, and acts as a molecular switch to EGFR activation. In this study, MDA-MB-231 cells and MCF7 cells were used. Our results showed that ANXA2 expression is inversely correlated with cell sensitivity to gefitinib. Knockdown of ANXA2 expression in MDA-MB-231 cells increased the gefitinib induced cell death. When ANXA2 was overexpressed in MCF7 cells, the gefitinib induced cell death was decreased. Furthermore, we demonstrated that phosphorylation of ANXA2 at Tyr23 is negatively correlated with the sensitivity of TNBC to gefitinib. Altogether, our results suggest a new role of ANXA2 in regulating sensitivity of TNBC MDA-MB-231 cells to the EGFR inhibitor gefitinib.     

Induced expression of miR-1250-5p exerts tumor suppressive role in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it has a prevalence rate of 15%–20% among all breast cancer cases in younger women. Still, the underlying molecular mechanisms of its pathogenesis are not entirely understood. In the previous study, we identified that microRNA (miR)-1250-5p is significantly down-expressed in TNBC cells. Thus, in the present study, we explore the functional anticancer role of miR‑1250‑5p in the transient mimic transfected TNBC cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to examine the effect of miR-1250-5p on cell viability of TNBC (MDA-MB-231 and MDA-MB-453) cells. The confocal microscopy, quantitative real-time polymerase chain reaction, and western blot analysis techniques were used to assess the effect of miR-1250-5p on cancer hallmarks in test cells. Induced miR‑1250-5p expression in MDA-MB-231 and MDA-MB-453 cells decreased cell viability in a time-dependent manner. Increased miR‑1250-5p expression levels significantly decreased cell cycle G1/S phase transition markers (Cyclin D1 and CDK4) at messenger RNA (mRNA) and protein levels in TNBC cells compared to scrambled sequence transfected cells. Transient transfection of TNBC cells with miR-1250-5p mimic increased apoptosis in TNBC cells by increasing the level of active caspase (Caspase 8 and Caspase 3) of the intrinsic pathway. Apoptosis-related morphological changes were also observed in the test cells. Further, the induced expression of miR-1250-5p significantly decreased epithelial-mesenchymal transition (EMT) by altering the mRNA and protein levels of E-cadherin and Vimentin. Moreover, results of confocal microscopy revealed increased reactive oxygen species generation, and decreased mitochondria membrane potential in miR-1250-5p mimic transient transfected TNBC cells. In conclusion, miR‑1250-5p acts as tumor suppressor in TNBC cells and its induction by therapeutics might be a novel strategy for the disease treatment.     

Chamomile Essential Oil: Chemical Constituents and Antitumor Activity in MDA-MB-231 Cells through PI3K/Akt/mTOR Signaling Pathway

Chamomile essential oil (CEO) is extracted from chamomile and mainly used in aromatherapy. The chemical constituents and its antitumor activity on Triple-negative breast cancer (TNBC) was explored in the present study. Gas chromatography-mass spectrometry (GC/MS) was employed to analyze the chemical constituents of CEO. The cell viability, migration and invasion of TNBC cell MDA-MB-231 were measured using MTT, wound scratch and Transwell assay, respectively. The protein expression of PI3K/Akt/mTOR signaling pathway was determined by Western blot. CEO is rich in terpenoids (63.51 %), among which the identified terpenoids and their derivatives are mainly Caryophyllene (29.57 %), d-Cadinene (12.81 %), Caryophyllene oxide (14.51 %), etc. Three concentration of CEO (1, 1.5, 2 μg/mL) significantly inhibited the proliferation, migration and invasion of MDA-MB-231 cells with a dose dependent manner. Moreover, the phosphorylation of PI3K, Akt and mTOR was inhibited by CEO. The results revealed that there was abundant terpenoids in the CEO which account for 63.51 %. CEO significantly inhibited the proliferation, migration and invasion of MDA-MB-231 cells, exhibiting antitumor effect on TNBC. The antitumor effect of CEO might attribute to its inhibition on PI3K/Akt/mTOR signaling pathway. However, further study should be conducted in more TNBC cell lines and animal models to provide further evidence for TNBC treatment by CEO.     

SAR optimization studies on a novel series of 2-anilinopyrimidines as selective inhibitors against triple-negative breast cancer cell line MDA-MB-468

Triple-negative breast cancers (TNBCs) account for approximately 15% of breast cancer cases and exhibit an aggressive clinical behavior. In this study, we designed and synthesized two series of 2-anilinopyrimidines based on the structure of our previously reported compound 1 that act as a selective inhibitor of the basal-like TNBC cell line MDA-MB-468. Through the fine-tuning of 1, cyclic and acyclic amines at 4-position of the pyrimidine core were turned out to be crucial for the selectivity. An extensive analysis of structure-activity relationships of the analogs revealed that aminoalkyl groups at the end of the propyl chain are amenable to modification. Among the newly synthesized analogs, compound 38, bearing 4-chloropiperidinyl and cyclohexyl groups, was found to be the most potent and selective, and was about three times more potent and selective than 1 was against the TNBC cells.