One-pot three-component synthesis of novel spirooxindoles with potential cytotoxic activity against triple-negative breast cancer MDA-MB-231 cells

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited treatment options due to its heterogeneity and the lack of well-defined molecular targets. In our endeavour towards the development of novel anti-TNBC agents, herein we report a one-pot three-component synthesis of novel spirooxindoles 6a–p, and evaluation of their potential anti-proliferative activity towards TNBC MDA-MB-231 cells. Spirooxindoles 6a, 6e and 6i emerged as the most potent analogues with IC₅₀ =?6.70, 6.40 and 6.70?µM, respectively. Compounds 6a and 6e induced apoptosis in MDA-MB-231 cells, as evidenced by the up-regulation of the Bax and down-regulation of the Bcl-2, besides boosting caspase-3 levels. Additionally, 6e displayed significant increase in the percent of annexin V-FITC positive apoptotic cells from 1.34 to 44%. Furthermore, spirooxindoles 6e and 6i displayed good inhibitory activity against EGFR (IC₅₀ =?120 and 150?nM, respectively). Collectively, these data demonstrated that 6e might be a potential lead compound for the development of effective anti-TNBC agents.     

Ectophosphatase activity in the triple-negative breast cancer cell line MDA-MB-231

Breast cancer is one of the most common cancers in the female population worldwide, and its development is thought to be associated with genetic mutations that lead to uncontrolled and accelerated growth of breast cells. This abnormal behavior requires extra energy, and indeed, tumor cells display a rewired energy metabolism compared to normal breast cells. Inorganic phosphate (Pi) is a glycolytic substrate of glyceraldehyde-3-phosphate dehydrogenase and has an important role in cancer cell proliferation. For cells to obtain Pi, ectoenzymes in the plasma membrane with their catalytic site facing the extracellular environment can hydrolyze phosphorylated molecules, and this is an initial and possibly limiting step for the uptake of Pi by carriers that behave as adjuvants in the process of energy harvesting and thus partially contributes to tumor energy requirements. In this study, the activity of an ectophosphatase in MDA-MB-231 cells was biochemically characterized, and the results showed that the activity of this enzyme was higher in the acidic pH range and that the enzyme had a K_m = 4.5 ± 0.5 mM para-nitrophenylphosphate and a V_max = 2280 ± 158 nM × h⁻¹ × mg protein⁻¹. In addition, classical acid phosphatase inhibitors, including sodium orthovanadate, decreased enzymatic activity. Sodium orthovanadate was able to inhibit ectophosphatase activity while also inhibiting cell proliferation, adhesion, and migration, which are important processes in tumor progression, especially in metastatic breast cancer MDA-MB-231 cells that have higher ectophosphatase activity than MCF-7 and MCF-10 breast cells.     

Resveratrol is an inhibitor of sodium-dependent inorganic phosphate transport in triple-negative MDA-MB-231 breast cancer cells

Metastasis is a major cause of death in patients with breast cancer. A growing body of evidence has demonstrated the antitumour effects of resveratrol, a non-flavonoid polyphenol. Resveratrol inhibits metastatic processes, such as the migration and invasion of cancer cells. In several cancer types, the importance of inorganic phosphate (Pi) for tumor progression has been demonstrated. The metastatic process in breast cancer is associated with Na⁺-dependent Pi transporters. In this study, we demonstrate, for the first time, that resveratrol inhibits the Na⁺-dependent Pi transporter. Results from kinetic analysis shows that resveratrol inhibits Na⁺-dependent Pi transport non-competitively. Resveratrol also inhibits adhesion/migration in MDA-MB-231 cells, likely related to inhibition of the Na⁺-dependent Pi transporter.     

Synergistic anticancer action of quercetin and curcumin against triple-negative breast cancer cell lines

Women with the breast cancer type 1 susceptibility protein (BRCA1) mutation and loss of BRCA1 expression are reported to have an increased risk of triple-negative breast cancer (TNBC). Targeting BRCA1 modulation might offer a therapeutic option to treat TNBC patients. Our studies detected that BRCA1 is poorly expressed in TNBC cell lines and highly expressed in ER⁺ breast cancer cell lines. To modulate BRCA1 expression, we tested two different dietary components to find out if any would induce tumor suppressor genes. We detected that quercetin and curcumin dose-dependently enhanced the BRCA1 expression. Further, a synergistic action of quercetin and curcumin was observed in modulating the BRCA1 level and in inhibiting the cell survival and migration of TNBC cell lines. Quercetin and curcumin appeared to induce BRCA1 promoter histone acetylation. Furthermore, BRCA1 knockdown induced cell survival and cell migration in ER ⁺ cells were significantly decreased by the combined treatment of quercetin and curcumin. Our present study concluded that the combination treatment of quercetin and curcumin acts synergistically to induce anticancer activity against TNBC cells by modulating tumor suppressor genes.     

磷酸化蛋白质组学在三阴性乳腺癌诊治研究中的进展

三阴性乳腺癌是乳腺癌中恶性程度最高的亚型,其治疗仍以化疗为主,但容易出现耐药,且患者预后较差。随着蛋白质组学技术的发展,磷酸化蛋白质组学研究取得了长足的进步,并在肿瘤发生发展机制和诊治研究中得到了广泛的应用。同样,磷酸化蛋白质组学在三阴性乳腺癌的发生发展、靶向治疗和耐药机制研究等方面也发挥着重要作用。本文主要对目前磷酸化蛋白质组学在三阴性乳腺癌中的研究进展进行综述,旨在为基于磷酸化蛋白质组学的三阴性乳腺癌发生发展机制和诊治研究提供指导和帮助。     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

The anti-proliferative effect of metformin in triple-negative MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: Implications for cancer therapy and prevention

Background

Metformin has been shown to have a strong anti-proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP-activated protein kinase (AMPK). MDA-MB-231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti-cancer agents as well as to the anti-proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin.

Methods

MDA-MB-231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by Western blotting.

Results

In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA-MB-231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin-treatment of MDA-MB-231 cells cultured in normoglycemic conditions and not in hyperglycemic conditions caused a striking activation of AMPK, and an AMPK-dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.

Conclusion

Our data show that normoglycemia sensitizes the triple negative MDA-MB-231 breast cancer cells to the anti-proliferative effect of metformin through an AMPK-dependent mechanism.

General significance

These findings suggest that tight normoglycemic control may enhance the anti-proliferative effect of metformin in diabetic cancer patients.     

Comprehensive analysis of novel three-long noncoding RNA signatures as a diagnostic and prognostic biomarkers of human triple-negative breast cancer

Currently, traditional predictors of prognosis (tumor size, nodal status, progesterone receptor [PR], estrogen receptor [ER], or human epidermal growth factor receptor-2 [HER2]) are insufficient for precise survival prediction for triple-negative breast cancer (TNBC). Long noncoding RNAs (lncRNAs) have been observed to exert critical functions in cancer, including in TNBC. Nevertheless, systematically tracking expression-based lncRNA biomarkers based on the sequence data for the prediction of prognosis in TNBC has not yet been investigated. To ascertain whether biomarkers exist that can distinguish TNBC from adjacent normal tissue or nTNBC, we implemented a comprehensive analysis of lncRNA expression profiles and clinical data of 1097 BC samples from The Cancer Genome Atlas database. A total of 1510 differentially expressed lncRNAs in normal and TNBC samples were extracted. Similarly, 672 differentially expressed lncRNAs between nTNBC and TNBC samples were detected. The receiver operating characteristic curve analysis indicated that three upregulated lncRNAs (AC091043.1, AP000924.1, and FOXCUT) may be of strong diagnostic value for predicting the existence of TNBC in the training and validation sets (area under the curve (AUC > 0.85). Kaplan-Meier analysis demonstrated that the other three lncRNAs (AC010343.3, AL354793.1, and FGF10-AS1) were associated with the prognosis of TNBC patients (P < 0.05). We used the three overall survival (OS)-related lncRNAs to establish a three-lncRNA signature. Multivariate Cox regression analysis suggested that the three-lncRNA signature was a prognostic factor independent of other clinical variables ( P < 0.01) for predicting OS in TNBC patients that could be utilized to classify patients into high- or low-risk subgroups. Our results might provide efficient signatures for clinical diagnosis and prognostic evaluation of TNBC.     

Discovery and SAR studies of novel 2-anilinopyrimidine-based selective inhibitors against triple-negative breast cancer cell line MDA-MB-468

Triple-negative breast cancers (TNBCs) are characterized as an invasive and intractable subtype of breast cancers. Overexpression of epidermal growth factor receptor (EGFR) has been considered to be an important target for TNBC therapy, but efficacies of EGFR inhibitors in clinical trials are elusive. In this study, novel series of 2-anilinopyrimidines were synthesized in an effort to identify selective inhibitors against an EGFR-overexpressing TNBC cell line. Biological evaluation demonstrated that compounds 21 and 38, with a 4-methylpiperidine group and a high ClogP value, exhibited good potency and selectivity for the TNBC cell line. This study has provided evidence to support further development of 2-anilinopyrimidine-based TNBC selective inhibitors and investigation of the targets of compounds 21 and 38.     

Discovering alkylamide derivatives of bexarotene as new therapeutic agents against triple-negative breast cancer

Triple-negative breast cancer (TNBC) has been reported to be correlated with high expression of proliferation markers as well as constitutive activation of metastasis-relevant signaling pathways. For many years, breast cancer researchers have been investigating specific and effective methods to treat or to control the development of TNBC, but promising therapeutic options remain elusive. In this study, we have demonstrated that alkylamide derivatives of bexarotene DK-1–150 and DK-1–166 induce apoptotic cell death in TNBC cell lines without causing cytotoxicity in the normal mammary epithelial cell line. Furthermore, the bexarotene derivatives also showed significant effects in inhibiting TNBC cell proliferation and migration, modulating cancer stem cell markers expressions, as well as limiting the epithelial-mesenchymal transition (EMT) activities of TNBC cell lines in terms of downregulating EMT marker and blocking nuclear translocation of β-catenin. Therefore, we propose the alkylamide derivatives of bexarotene as potential candidates for novel anticancer therapeutics against TNBC.     

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

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.     

Wnt pathway targeting reduces triple-negative breast cancer aggressiveness through miRNA regulation in vitro and in vivo

Triple-negative breast cancer, devoid of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER-2) expression, is deprived of commonly used targeted therapies. MicroRNAs (miRNAs) are undergoing a revolution in terms of potentially diagnostic or therapeutic elements. Combining computational approaches, we enriched miRNA binding motifs of Wnt pathway-associated upregulated genes. Our in-depth bioinformatics, in vitro and in vivo analyses indicated that miR-381 targets main genes of the Wnt signaling pathway including CTNNB1, RhoA, ROCK1, and c-MYC genes. The expression level of miR-381 and target genes was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) in MCF-7, MDA-MB-231, and MCF-10A as well as 20 breast cancer samples and normal tissues. Luciferase reporter assay was performed. Lentiviral particles containing miR-381 were used to evaluate the effect of miR-381 restoration on cell proliferation, migration, and invasion of the invasive triple-negative MDA-MB-231 cell line and also in a mouse model of breast cancer. The expression of miR-381 was lower than that of normal cells, especially in TNBC cell line and breast tissues. Luciferase assay results confirmed that miR-381 targets all the predicted 3′-untranslated regions (3′-UTRs). Upon miR-381 overexpression, the expression of target genes declined, and the migration and invasion potential of miR-381-receiving MDA-MB-231 cells decreased. In a mouse model of triple-negative breast cancer, miR-381 re-expression inhibited the invasion of cancer cells to lung and liver and prolonged the survival time of cancer cell-bearing mice. Therefore, miR-381 is a regulator of Wnt signaling and its re-expression provides a potentially effective strategy for inhibition of TNBC.     

TQFL12, a novel synthetic derivative of TQ,inhibits triple-negative breast cancer metastasis and invasion through activating AMPK/ACC pathway

Thymoquinone (TQ) has been reported as an anti-tumour drug widely studied in various tumours, and its mechanism and effect of which has become a focus of current research. However, previous studies from our laboratory and other groups found that TQ showed weak anti-tumour effects in many cancer cell lines and animal models. Therefore, it is necessary to modify and optimize the structure of TQ to obtain new chemical entities with high efficiency and low toxicity as candidates for development of new drugs in treating cancer. Therefore, we designed and synthesized several TQ derivatives. Systematic analysis, including in vitro and in vivo, was conducted on a panel of triple-negative breast cancer (TNBC) cells and mouse model to demonstrate whether TQFL12, a new TQ derivative, is more efficient than TQ. We found that the anti-proliferative effect of TQFL12 against TNBC cells is significantly stronger than TQ. We also demonstrated TQFL12 affects different aspects in breast cancer development including cell proliferation, migration, invasion and apoptosis. Moreover, TQFL12 inhibited tumour growth and metastasis in cancer cell–derived xenograft mouse model, with less toxicity compared with TQ. Finally, mechanism research indicated that TQFL12 increased AMPK/ACC activity by stabilizing AMPKα, while molecular docking supported the direct interaction between TQFL12 and AMPKα. Taken together, our findings suggest that TQFL12, as a novel chemical entity, possesses a better inhibitory effect on TNBC cells and less toxicity in both in vitro and in vivo studies. As such, TQFL12 could serve as a potential therapeutic agent for breast cancer.     

Synthesis,computational studies and antiproliferative activities of coumarin-tagged 1,3,4-oxadiazole conjugates against MDA-MB-231 and MCF-7 human breast cancer cells

A novel library of coumarin tagged 1,3,4 oxadiazole conjugates was synthesized and evaluated for their antiproliferative activities against MDA-MB-231 and MCF-7 breast cancer cell lines. The evaluation studies revealed that compound 9d was the most potent molecule with an IC₅₀ value of <5?µM against the MCF-7 cell line. Interestingly, compounds 10b and 11a showed a similar trend with lower inhibitory concentration (IC₅₀?=?7.07?µM), in Estrogen Negative (ER?) cells than Estrogen Positive (ER+) cells. Structure–activity relationship (SAR) studies revealed that conjugates bearing benzyl moieties (9b, 9c and 9d) had superior activities compared to their alkyl analogues. The most potent compound 9d showed ～1.4?times more potent activity than tamoxifen against MCF-7 cell line; while the introduction of sulfone unit in compounds 11a, 11b and 11c resulted in significant cytotoxicity against both MCF-7 and MDA-MB-231 cell lines. These results were further supported by docking studies, which revealed that the stronger binding affinity of the synthesized conjugates is due to the presence of sulfone unit attached to the substituted benzyl moiety in their pharmacophores.     

Propranolol inhibits neonatal Nav1.5 activity and invasiveness of MDA-MB-231 breast cancer cells: Effects of combination with ranolazine

The MDA-MB-231 cell line was used as a model of triple negative breast cancer to investigate the interaction of β-adrenergic receptor (β-AR) and voltage-gated sodium channel (VGSC). There was significant (86%) overlap in their expression. Short-term (acute) application of the β-AR antagonist propranolol (25 μM) led to reduction of peak current and quickening of current inactivation (the latter occurred only in 5% fetal bovine serum). Long-term (48 hr) incubation with propranolol (25 μM) resulted in several changes in VGSC characteristics: shifts in (a) current-voltage relationship and (b) steady-state inactivation, both to more negative potentials and (c) the slowing of recovery from inactivation. We then investigated the effects of propranolol and ranolazine, a blocker of VGSC activity, alone and in combination, on lateral motility and Matrigel invasion. These assays were carried out under hypoxic conditions more representative of tumor progression. Propranolol (2.5 and 25 μM) and ranolazine (5 μM), and their combination inhibited lateral motility. Also, propranolol (25 μM) and ranolazine (5 μM), and their combination inhibited invasion. However, no synergy was observed in the pharmacological combinations for both assays. Propranolol also significantly decreased total neonatal Nav1.5 protein expression, the predominant VGSC subtype expressed in these cells. We conclude (a) that β-AR and VGSC are functionally coupled in MDA-MB-231 cells; (b) that propranolol has direct blocking action on the VGSC; (c) that the action of propranolol is modulated by serum; and (d) that the antimetastatic cellular effects of propranolol and ranolazine are not additive.