USP7 Induces Chemoresistance in Triple-Negative Breast Cancer via Deubiquitination and Stabilization of ABCB1.

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer. TNBC does not express the estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. Cytotoxic chemotherapy and surgery are the current therapeutic strategies for TNBC patients, but the chemoresistance of TNBC limits the efficiency of this strategy and shortens the lifespan of patients. The exploration of targeted therapy is ongoing in TNBC research. The aim of the present study was to identify the mechanism underlying acquired resistance in TNBC through the exploration of the relationship between the expression of USP7 and of ABCB1. We found that ubiquitin specific protease 7 (USP7) is a potential therapeutic target for overcoming the chemoresistance of TNBC. USP7 overexpression increased the chemoresistance of TNBC, while the knockdown of USP7 effectively increased the chemosensitivity of chemoresistant TNBC. A USP7 inhibitor effectively induced apoptosis and suppressed metastasis in chemoresistant TNBC. We further clarified that USP7 is a specific deubiquitinating enzyme for ABCB1 that plays an essential role in drug resistance. USP7 directly interacted with ABCB1 and regulated its stability. We concluded that USP7 promotes the chemoresistance of TNBC by stabilizing the ABCB1 protein.

Nafamostat mesylate overcomes endocrine resistance of breast cancer through epigenetic regulation of CDK4 and CDK6 expression.

Breast cancer is common worldwide, and the estrogen receptor-positive subtype accounts for approximately 70% of breast cancer in women. Tamoxifen and fulvestrant are drugs currently used for endocrinal therapy. Breast cancer exhibiting endocrine resistance can undergo metastasis and lead to the death of breast cancer patients. Drug repurposing is an active area of research in clinical medicine. We found that nafamostat mesylate, clinically used for patients with pancreatitis and disseminated intravascular coagulation, acts as an anti-cancer drug for endocrine-resistant estrogen receptor-positive breast cancer (ERPBC). Epigenetic repression of CDK4 and CDK6 by nafamostat mesylate induced apoptosis and suppressed the metastasis of ERPBC through the deacetylation of Histone 3 Lysine 27. A combination of nafamostat mesylate and CDK4/6 inhibitor synergistically overcame endocrine resistance in ERPBC. Nafamostat mesylate might be an essential adjuvant or alternative drug for the treatment of endocrine-resistant ERPBC due to the low cost-efficiency of the CDK4/6 inhibitor.

Luteolin suppresses androgen receptor-positive triple-negative breast cancer cell proliferation and metastasis by epigenetic regulation of MMP9 expression via the AKT/mTOR signaling pathway.

BACKGROUND: Triple-negative breast cancer (TNBC) represents up to 20% of all breast cancers. This cancer lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The current therapeutic strategy for patients with this subtype is the use of cytotoxic chemotherapy and surgery. Luteolin is a natural herbal flavonoid and a potential therapeutic candidate for multiple diseases. The use of a treatment that combines Chinese herbal medicine and western medicine is rising in Asia. PURPOSE: The present study evaluates the effects and molecular mechanisms involved with luteolin treatment and evaluates whether this herb affects androgen receptor-positive breast cancer cell proliferation or metastasis. STUDY DESIGN: In vitro evaluation of the effect of luteolin on androgen receptor-positive TNBC cell proliferation and metastasis METHODS: Cell viability analysis was used for the cytotoxicity test. Colony formation and Bromodeoxyuridine (BrdU) staining-based proliferation experiments were used for cell proliferation. Wound healing and transwell assays were used for in vitro migration/invasion. The RT-qPCR analysis was used for gene expression. Furthermore, ChIP-qPCR analysis was used for epigenetic modification of gene promoters. RESULTS: Luteolin significantly inhibited the proliferation and metastasis of androgen receptor-positive TNBC. Furthermore, luteolin inactivated the AKT/mTOR signaling pathway and reversed the epithelial-mesenchymal transition (EMT). The combination of luteolin and inhibitors of AKT/mTOR synergistically repressed an androgen receptor-positive TNBC cell proliferation and metastasis. Luteolin also downregulated MMP9 expression by decreasing the levels of the AKT/mTOR promoting H3K27Ac and H3K56A on the MMP9 promoter region. CONCLUSION: Our findings indicate that luteolin inhibited the proliferation and metastasis of androgen receptor-positive TNBC by regulating MMP9 expression through a reduction in the levels of AKT/mTOR-inducing H3K27Ac and H3K56Ac.

MLL3 Induced by Luteolin Causes Apoptosis in Tamoxifen-Resistant Breast Cancer Cells through H3K4 Monomethylation and Suppression of the PI3K/AKT/mTOR Pathway.

Tamoxifen is one of the most common hormone therapy drug for estrogen receptor (ER)-positive breast cancer. Tumor cells with drug resistance often cause recurrence and metastasis in cancer patients. Luteolin is a natural compound found from various types of vegetables and exhibit anticancer activity in different cancers. This study demonstrated that luteolin inhibits the proliferation and induces apoptosis of tamoxifen-resistant ER-positive breast cancer cells. Luteolin also causes cell cycle arrest at the G2/M phase and decreases mitochondrial membrane potential. Besides, luteolin reduces the levels of activated PI3K/AKT/mTOR signaling pathway. The combination treatment of luteolin and PI3K, AKT, or mTOR inhibitors synergistically increases apoptosis in tamoxifen-resistant ER-positive breast cancer cells. Ras gene family (K-Ras, H-Ras, and N-Ras), an activator of PI3K, was transcriptionally repressed by luteolin via induction of tumor suppressor mixed-lineage leukemia 3 (MLL3) expression. MLL3 increases the level of monomethylation of Histone 3 Lysine 4 on the enhancer and promoter region of Ras genes, thus causes repression of Ras expressions. Our finding implies that luteolin was a promising natural agent against tamoxifen resistance of breast cancer.

Effect of spatial heterogeneity and colocalization of eNOS and capacitative calcium entry channels on shear stress-induced NO production by endothelial cells: A modeling approach.

INTRODUCTION: Colocalization of endothelial nitric oxide synthase (eNOS) and capacitative Ca2+ entry (CCE) channels in microdomains such as cavaeolae in endothelial cells (ECs) has been shown to significantly affect intracellular Ca2+ dynamics and NO production, but the effect has not been well quantified. METHODS: We developed a two-dimensional continuum model of an EC integrating shear stress-mediated ATP production, intracellular Ca2+ mobilization, and eNOS activation to investigate the effects of spatial colocalization of plasma membrane eNOS and CCE channels on Ca2+ dynamics and NO production in response to flow-induced shear stress. Our model examines the hypothesis that subcellular colocalization of cellular components can be critical for optimal coupling of NO production to blood flow. RESULTS: Our simulations predict that heterogeneity of CCE can result in formation of microdomains with significantly higher Ca2+ compared to the average cytosolic Ca2+. Ca2+ buffers with lower or no mobility further enhanced Ca2+ gradients relative to mobile buffers. Colocalization of eNOS to CCE channels significantly increased NO production. CONCLUSIONS: Our results provide quantitative understanding for the role of spatial heterogeneity and the compartmentalization of signals in regulation of shear stress-induced NO production.

A dynamic computational network model for the role of nitric oxide and the myogenic response in microvascular flow regulation.

OBJECTIVES: The effect of NO on smooth muscle cell contractility is crucial in regulating vascular tone, blood flow, and O2 delivery. Quantitative predictions for interactions between the NO production rate and the myogenic response for microcirculatory blood vessels are lacking. METHODS: We developed a computational model of a branching microcirculatory network with four representative classes of resistance vessels to predict the effect of endothelium-derived NO on the microvascular pressure-flow response. Our model links vessel scale biotransport simulations of NO and O2 delivery to a mechanistic model of autoregulation and myogenic tone in a simplified microcirculatory network. RESULTS: The model predicts that smooth muscle cell NO bioavailability significantly contributes to resting vascular tone of resistance vessels. Deficiencies in NO seen during hypoxia or ischemia lead to a decreased vessel diameter for all classes at a given intravascular pressure. At the network level, NO deficiencies lead to an increase in pressure drop across the vessels studied, a downward shift in the pressure-flow curve, and a decrease in the effective range of the autoregulatory response. CONCLUSIONS: Our model predicts the steady state and transient behavior of resistance vessels to perturbations in blood pressure, including effects of NO bioavailability on vascular regulation.

Nitric oxide release by deoxymyoglobin nitrite reduction during cardiac ischemia: A mathematical model.

Interactions between cardiac myoglobin (Mb), nitrite, and nitric oxide (NO) are vital in regulating O2 storage, transport, and NO homeostasis. Production of NO through the reduction of endogenous myocardial nitrite by deoxygenated myoglobin has been shown to significantly reduce myocardial infarction damage and ischemic injury. We developed a mathematical model for a cardiac arteriole and surrounding myocardium to examine the hypothesis that myoglobin switches functions from being a strong NO scavenger to an NO producer via the deoxymyoglobin nitrite reductase pathway. Our results predict that under ischemic conditions of flow, blood oxygen level, and tissue pH, deoxyMb nitrite reduction significantly elevates tissue and smooth muscle cell NO. The size of the effect is consistent at different flow rates, increases with decreasing blood oxygen and tissue pH and, in extreme pathophysiological conditions, NO can even be elevated above the normoxic levels. Our simulations suggest that cardiac deoxyMb nitrite reduction is a plausible mechanism for preserving or enhancing NO levels using endogenous nitrite despite the rate-limiting O2 levels for endothelial NO production. This NO could then be responsible for mitigating deleterious effects under ischemic conditions.

Nitrite-Mediated Hypoxic Vasodilation Predicted from Mathematical Modeling and Quantified from in Vivo Studies in Rat Mesentery.

Nitric oxide (NO) generated from nitrite through nitrite reductase activity in red blood cells has been proposed to play a major role in hypoxic vasodilation. However, we have previously predicted from mathematical modeling that much more NO can be derived from tissue nitrite reductase activity than from red blood cell nitrite reductase activity. Evidence in the literature suggests that tissue nitrite reductase activity is associated with xanthine oxidoreductase (XOR) and/or aldehyde oxidoreductase (AOR). We investigated the role of XOR and AOR in nitrite-mediated vasodilation from computer simulations and from in vivo exteriorized rat mesentery experiments. Vasodilation responses to nitrite in the superfusion medium bathing the mesentery equilibrated with 5% O2 (normoxia) or zero O2 (hypoxia) at either normal or acidic pH were quantified. Experiments were also conducted following intraperitoneal (IP) injection of nitrite before and after inhibiting XOR with allopurinol or inhibiting AOR with raloxifene. Computer simulations for NO and O2 transport using reaction parameters reported in the literature were also conducted to predict nitrite-dependent NO production from XOR and AOR activity as a function of nitrite concentration, PO2 and pH. Experimentally, the largest arteriolar responses were found with nitrite >10 mM in the superfusate, but no statistically significant differences were found with hypoxic and acidic conditions in the superfusate. Nitrite-mediated vasodilation with IP nitrite injections was reduced or abolished after inhibiting XOR with allopurinol (p < 0.001). Responses to IP nitrite before and after inhibiting AOR with raloxifene were not as consistent. Our mathematical model predicts that under certain conditions, XOR and AOR nitrite reductase activity in tissue can significantly elevate smooth muscle cell NO and can serve as a compensatory pathway when endothelial NO production is limited by hypoxic conditions. Our theoretical and experimental results provide further evidence for a role of tissue nitrite reductases to contribute additional NO to compensate for reduced NO production by endothelial nitric oxide synthase during hypoxia. Our mathematical model demonstrates that under extreme hypoxic conditions with acidic pH, endogenous nitrite levels alone can be sufficient for a functionally significant increase in NO bioavailability. However, these conditions are difficult to achieve experimentally.

A mathematical model for the role of N2O3 in enhancing nitric oxide bioavailability following nitrite infusion.

Nitrite infusion into the bloodstream has been shown to elicit vasodilation and protect against ischemia-reperfusion injury through nitric oxide (NO) release in hypoxic conditions. However, the mechanism by which nitrite-derived NO escapes scavenging by hemoglobin in the erythrocyte has not been fully elucidated, owing in part to the difficulty in measuring the reactions and transport on NO in vivo. We developed a mathematical model for an arteriole and surrounding tissue to examine the hypothesis that dinitrogen trioxide (N2O3) acts as a stable intermediate for preserving NO. Our simulations predict that with hypoxia and moderate nitrite concentrations, the N2O3 pathway can significantly preserve the NO produced by hemoglobin nitrite reductase in the erythrocyte and elevate NO reaching the smooth muscle cells. Nitrite retains its ability to increase NO bioavailability even at varying flow conditions, but there is minimal effect under normoxia or very low nitrite concentrations. Our model demonstrates a viable pathway for reconciling experimental findings of potentially beneficial effects of nitrite infusions despite previous models showing negligible NO elevation associated with hemoglobin nitrite reductase. Our results suggest that additional mechanisms may be needed to explain the efficacy of nitrite-induced vasodilation at low infusion concentrations.