Mebendazole Increases Anticancer Activity of Radiotherapy in Radiotherapy-Resistant Triple-Negative Breast Cancer Cells by Enhancing Natural Killer Cell-Mediated Cytotoxicity.

Breast cancer is the most commonly diagnosed cancer worldwide and ranks first in terms of both prevalence and cancer-related mortality in women. In this study, we aimed to evaluate the anticancer effect of mebendazole (MBZ) and radiotherapy (RT) concomitant use in triple-negative breast cancer (TNBC) cells and elucidate the underlying mechanisms of action. Breast cancer mouse models and several types of breast cancer cells, including TNBC-derived RT-resistant (RT-R) MDA-MB-231 cells, were treated with MBZ and/or RT. In mice, changes in body weight, renal and liver toxicity, tumor volume, and number of lung metastases were determined. In cells, cell viability, colony formation, scratch wound healing, Matrigel invasion, and protein expression using western blotting were determined. Our findings showed that MBZ and RT combined treatment increased the anticancer effect of RT without additional toxicity. In addition, we noted that cyclin B1, PH2AX, and natural killer (NK) cell-mediated cytotoxicity increased following MBZ + RT treatment compared to unaided RT. Our results suggest that MBZ + RT have an enhanced anticancer effect in TNBC which acquires radiation resistance through blocking cell cycle progression, initiating DNA double-strand breaks, and promoting NK cell-mediated cytotoxicity.

NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations.

Cancer stem cells (CSCs) can be induced from differentiated cancer cells in the tumor microenvironment or in response to treatments and exhibit chemo- and radioresistance, leading to tumor recurrence and metastasis. We previously reported that triple negative breast cancer (TNBC) cells with acquired radioresistance exhibited more aggressive features due to an increased CSC population. Therefore, here, we isolated CSCs from radiotherapy-resistant (RT-R)-TNBC cells and investigated the effects of these CSCs on tumor progression and NK cell-mediated cytotoxicity. Compared to MDA-MB-231 and RT-R-MDA-MB-231 cells, CD24-/low/CD44+ cells isolated from RT-R-MDA-MB-231 cells showed increased proliferation, migration and invasion abilities, and induced expression of tumor progression-related molecules. Moreover, similar to MDA-MB-231 cells, CD24-/low/CD44+ cells recruited NK cells but suppressed NK cell cytotoxicity by regulating ligands for NK cell activation. In an in vivo model, CD24-/low/CD44+ cell-injected mice showed enhanced tumor progression and lung metastasis via upregulation of tumor progression-related molecules and altered host immune responses. Specifically, NK cells were recruited into the peritumoral area tumor but lost their cytotoxicity due to the altered expression of activating and inhibitory ligands on tumors. These results suggest that CSCs may cause tumor evasion of immune cells, resulting in tumor progression.

Rosmarinic Acid Increases Macrophage Cholesterol Efflux through Regulation of ABCA1 and ABCG1 in Different Mechanisms.

Lipid dysregulation in diabetes mellitus escalates endothelial dysfunction, the initial event in the development and progression of diabetic atherosclerosis. In addition, lipid-laden macrophage accumulation in the arterial wall plays a significant role in the pathology of diabetes-associated atherosclerosis. Therefore, inhibition of endothelial dysfunction and enhancement of macrophage cholesterol efflux is the important antiatherogenic mechanism. Rosmarinic acid (RA) possesses beneficial properties, including its anti-inflammatory, antioxidant, antidiabetic and cardioprotective effects. We previously reported that RA effectively inhibits diabetic endothelial dysfunction by inhibiting inflammasome activation in endothelial cells. However, its effect on cholesterol efflux remains unknown. Therefore, in this study, we aimed to assess the effect of RA on cholesterol efflux and its underlying mechanisms in macrophages. RA effectively reduced oxLDL-induced cholesterol contents under high glucose (HG) conditions in macrophages. RA enhanced ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) expression, promoting macrophage cholesterol efflux. Mechanistically, RA differentially regulated ABCA1 expression through JAK2/STAT3, JNK and PKC-p38 and ABCG1 expression through JAK2/STAT3, JNK and PKC-ERK1/2/p38 in macrophages. Moreover, RA primarily stabilized ABCA1 rather than ABCG1 protein levels by impairing protein degradation. These findings suggest RA as a candidate therapeutic to prevent atherosclerotic cardiovascular disease complications related to diabetes by regulating cholesterol efflux in macrophages.

Abscopal Effect of Radiotherapy Enhanced with Immune Checkpoint Inhibitors of Triple Negative Breast Cancer in 4T1 Mammary Carcinoma Model.

Local radiotherapy (RT) is important to manage metastatic triple-negative breast cancer (TNBC). Although RT primarily reduces cancer cells locally, this control can be enhanced by triggering the immune system via immunotherapy. RT and immunotherapy may lead to an improved systemic effect, known as the abscopal effect. Here, we analyzed the antitumor effect of combination therapy using RT with an anti-programmed cell death-1 (PD-1) antibody in primary tumors, using poorly immunogenic metastatic mouse mammary carcinoma 4T1 model. Mice were injected subcutaneously into both flanks with 4T1 cells, and treatment was initiated 12 days later. Mice were randomly assigned to three treatment groups: (1) control (no treatment with RT or immune checkpoint inhibitor (ICI)), (2) RT alone, and (3) RT+ICI. The same RT dose was prescribed in both RT-alone and RT+ICI groups as 10Gy/fx in two fractions and delivered to only one of the two tumor burdens injected at both sides of flanks. In the RT+ICI group, 200 µg fixed dose of PD-1 antibody was intraperitoneally administered concurrently with RT. The RT and ICI combination markedly reduced tumor cell growth not only in the irradiated site but also in non-irradiated sites, a typical characteristic of the abscopal effect. This was observed only in radiation-sensitive cancer cells. Lung metastasis development was lower in RT-irradiated groups (RT-only and RT+ICI groups) than in the non-irradiated group, regardless of the radiation sensitivity of tumor cells. However, there was no additive effect of ICI on RT to control lung metastasis, as was already known regarding the abscopal effect. The combination of local RT with anti-PD-1 blockade could be a promising treatment strategy against metastatic TNBC. Further research is required to integrate our results into a clinical setting.

Anticancer Effect of Benzimidazole Derivatives, Especially Mebendazole, on Triple-Negative Breast Cancer (TNBC) and Radiotherapy-Resistant TNBC In Vivo and In Vitro.

In this study, we aimed to evaluate the anticancer effect of benzimidazole derivatives on triple-negative breast cancer (TNBC) and investigate its underlying mechanism of action. Several types of cancer and normal breast cells including MDA-MB-231, radiotherapy-resistant (RT-R) MDA-MB-231, and allograft mice were treated with six benzimidazole derivatives including mebendazole (MBZ). Cells were analyzed for viability, colony formation, scratch wound healing, Matrigel invasion, cell cycle, tubulin polymerization, and protein expression by using Western blotting. In mice, liver and kidney toxicity, changes in body weight and tumor volume, and incidence of lung metastasis were analyzed. Our study showed that MBZ significantly induced DNA damage, cell cycle arrest, and downregulation of cancer stem cell markers CD44 and OCT3/4, and cancer progression-related ESM-1 protein expression in TNBC and RT-R-TNBC cells. In conclusion, MBZ has the potential to be an effective anticancer agent that can overcome treatment resistance in TNBC.

NLRC4, ASC and Caspase-1 Are Inflammasome Components that Are Mediated by P2Y2R Activation in Breast Cancer Cells.

The inflammasomes are reported to be associated with tumor progression. In our previous study, we determined that extracellular ATP enhances invasion and tumor growth by inducing inflammasome activation in a P2Y purinergic receptor 2 (P2Y2R)-dependent manner. However, it is not clear which inflammasome among the diverse complexes is associated with P2Y2R activation in breast cancer. Thus, in this study, we determined which inflammasome components are regulated by P2Y2R activation and are involved in tumor progression in breast cancer cells and radiotherapy-resistant (RT-R)-breast cancer cells. First, we found that NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3); NLR family caspase activation and recruitment domain (CARD) containing 4 (NLRC4); apoptosis-associated speck-like protein containing a CARD complex (ASC); and caspase-1 mRNA levels were upregulated in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells, whereas tumor necrosis factor-α (TNF-α) or ATP treatment induced NLRC4, ASC, and caspase-1 but not NLRP3 protein levels. Moreover, TNF-α or ATP increased protein levels of NLRC4, ASC, and caspase-1 in a P2Y2R-dependent manner in MDA-MB-231 and RT-R-MDA-MB-231 cells. In addition, P2Y2R activation by ATP induced the secretion of IL-1ß and VEGF-A, as well as invasion, in MDA-MB-231 and RT-R-MDA-MB-231 cells, which was inhibited by NLRC4, ASC, and caspase-1 small interfering RNA (siRNA). Taken together, this report suggests that P2Y2R activation by ATP induces tumor invasion and angiogenesis through inflammasome activation, specifically by regulating the inflammasome components NLRC4, ASC, and caspase-1.

Radiotherapy-Resistant Breast Cancer Cells Enhance Tumor Progression by Enhancing Premetastatic Niche Formation through the HIF-1α-LOX Axis.

Cancer stem cells (CSCs) exist in solid tumors and contribute to therapeutic resistance and disease recurrence. Previously, we reported that radiotherapy-resistant (RT-R)-MDA-MB-231 cells from highly metastatic MDA-MB-231 cells produced more CSCs than any other RT-R-breast cancer cells and showed therapeutic resistance and enhanced invasiveness. Hypoxia inducible factor-1α (HIF-1α) induced in the tumor microenvironment leads to the release of lysyl oxidase (LOX), which mediates collagen crosslinking at distant sites to facilitate environmental changes that allow cancer cells to easily metastasize. Therefore, in this study, we investigated whether RT-R-MDA-MB-231 cells induce greater HIF-1α expression, LOX secretion, and premetastatic niche formation than MDA-MB-231 cells do. RT-R-MDA-MB-231 cells increased HIF-1α expression and LOX secretion compared with MDA-MB-231 cells. Mice harboring RT-R-MDA-MB-231 cell xenografts showed enhanced tumor growth and higher expression of the CSC markers, CD44, Notch-4, and Oct3/4. In addition, mice injected with RT-R-MDA-MB-231 cells exhibited a higher level of HIF-1α in tumor tissue, increased secretion of LOX in plasma, higher induced levels of crosslinked collagen, and a higher population of CD11b+ BMDC recruitment around lung tissue, compared with those injected with MDA-MB-231 cells. These results suggest that RT-R-MDA-MB-231 cells contribute to tumor progression by enhancing premetastatic niche formation through the HIF-1α-LOX axis.

13-Ethylberberine Induces Apoptosis through the Mitochondria-Related Apoptotic Pathway in Radiotherapy-Resistant Breast Cancer Cells.

Berberine is reported to have multiple biological effects, including antimicrobial, anti-inflammatory, and antitumor activities, and 13-alkyl-substituted berberines show higher activity than berberine against certain bacterial species and human cancer cell lines. In particular, 13-ethylberberine (13-EBR) was reported to have anti-inflammatory effects in endotoxin-activated macrophage and septic mouse models. Thus, in this study, we aimed to examine the anticancer effects of 13-EBR and its mechanisms in radiotherapy-resistant (RT-R) MDA-MB-231 cells derived from the highly metastatic MDA-MB-231 cells. When we compared the gene expression between MDA-MB-231 and RT-R MDA-MB-231 cells with an RNA microarray, RT-R MDA-MB-231 showed higher levels of anti-apoptotic genes and lower levels of pro-apoptotic genes compared to MDA-MB-231 cells. Accordingly, we examined the effect of 13-EBR on the induction of apoptosis in RT-R MDA-MB-231 and MDA-MB-231 cells. The results showed that 13-EBR reduced the proliferation and colony-forming ability of both MDA-MB-231 and RT-R MDA-MB-231 cells. Moreover, 13-EBR induced apoptosis by promoting both intracellular and mitochondrial reactive oxygen species (ROS) and by regulating the apoptosis-related proteins involved in the intrinsic pathway, not in the extrinsic pathway. These results suggest that 13-EBR has pro-apoptotic effects in RT-R MDA-MB-231 and MDA-MB-231 cells by inducing mitochondrial ROS production and activating the mitochondrial apoptotic pathway, providing useful insights into new potential therapeutic strategies for RT-R breast cancer treatment.

Oleandrin and Its Derivative Odoroside A, Both Cardiac Glycosides, Exhibit Anticancer Effects by Inhibiting Invasion via Suppressing the STAT-3 Signaling Pathway.

The cardiac glycosides oleandrin and odoroside A, polyphenolic monomer compounds extracted from Nerium oleander, have been found to have antitumor effects on various tumors at low doses. However, the mechanisms of anticancer effects of oleandrin and odoroside A are not well known. Therefore, in this study, we aimed to investigate the anticancer effects of oleandrin and odoroside A and their associated mechanisms in highly metastatic MDA-MB-231 breast cancer cells and radiotherapy-resistant (RT-R) MDA-MB-231 cells. Our results showed that oleandrin and odoroside A dose-dependently decreased the colony formation and the invasion of both cell lines at nanomolar ranges. Furthermore, oleandrin (50 nM) and odoroside A (100 nM) reduced octamer-binding transcription factor 3/4 (OCT3/4) and ß-catenin levels and matrix metalloproteinase-9 (MMP-9) activity. Finally, we found that phospho-STAT-3 levels were increased in MDA-MB-231 and RT-R-MDA-MB-231, but not in endothelial cells (ECs), and that the levels were significantly decreased by oleandrin (50 nM) and odoroside A (100 nM). Inhibition of phospho-signal transducer and activator of transcription (STAT)-3 significantly reduced OCT3/4 and ß-catenin levels and MMP-9 activity, ultimately resulting in reduced invasion. These results suggest that the anticancer effects of oleandrin and odoroside A might be due to the inhibition of invasion through of phospho-STAT-3-mediated pathways that are involved in the regulation of invasion-related molecules.

P2Y2 receptor activation by nucleotides released from highly metastatic breast cancer cells increases tumor growth and invasion via crosstalk with endothelial cells.

INTRODUCTION: Extracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y2R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y2R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs). METHODS: ATP release and P2Y2R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y2R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y2R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y2R-shRNA-transfected MDA-MB-231 cells. RESULTS: We found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y2R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y2R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y2R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y2R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y2R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y2R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells. CONCLUSION: This study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.

P2Y2 R activation by nucleotides promotes skin wound-healing process.

P2Y2 R has been shown to be upregulated in a variety of tissues in response to stress or injury and to mediate tissue regeneration through its ability to activate multiple signalling pathways. This study aimed to investigate the role of P2Y2 R in the wound-healing process and the mechanisms by which P2Y2 R activation promotes wound healing in fibroblasts. The role of P2Y2 R in skin wound healing was examined using a full-thickness skin wound model in wildtype (WT) and P2Y2 R(-/-) mice and an in vitro scratch wound model in control or P2Y2 R siRNA-transfected fibroblasts. WT mice showed significantly decreased wound size compared with P2Y2 R(-/-) mice at day 14 post-wounding, and immunohistochemical analysis showed that a proliferation marker Ki67 and extracellular matrix (ECM)-related proteins VEGF, collagen I, fibronectin and α-SMA were overexpressed in WT mice, which were reduced in P2Y2 R(-/-) mice. Scratch-wounded fibroblasts increased ATP release, which peaked at 5 min. In addition, scratch wounding increased the level of P2Y2 R mRNA. Activation of P2Y2 R by ATP or UTP enhanced proliferation and migration of fibroblasts in in vitro scratch wound assays and were blocked by P2Y2 R siRNA. Finally, ATP or UTP also increased the levels of ECM-related proteins through the activation of P2Y2 R in fibroblasts. This study suggests that P2Y2 R may be a potential therapeutic target to promote wound healing in chronic wound diseases.

Dexmedetomidine-induced contraction involves c-Jun NH2 -terminal kinase phosphorylation through activation of the 5-lipoxygenase pathway in the isolated endothelium-denuded rat aorta.

Vasoconstriction induced by dexmedetomidine, a highly selective alpha-2 adrenoceptor agonist, mainly involves c-Jun NH2 -terminal kinase (JNK) phosphorylation in the isolated endothelium-denuded aorta. We carried out an in vitro study to determine the main arachidonic acid metabolic pathway that is involved in dexmedetomidine-induced JNK activation. Cumulative dexmedetomidine concentration-contractile response curves were generated in the endothelium-denuded rat aorta in the presence or absence of the following inhibitors: the JNK inhibitor SP600125, the phospholipase A2 inhibitor quinacrine dihydrochloride, the non-specific lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid, the 5-LOX inhibitor AA-861, the dual 5-LOX and cyclooxygenase (COX) inhibitor phenidone, the non-specific COX inhibitor indomethacin, the cytochrome p450 epoxygenase inhibitor fluconazole, the COX-1 inhibitor SC-560, and the COX-2 inhibitor NS-398. The effect of the alpha-2 adrenoceptor inhibitor rauwolscine and other inhibitors, such as quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, indomethacin and the protein kinase C inhibitor GF 109203X, on dexmedetomidine-induced JNK phosphorylation was investigated in rat aortic vascular smooth muscle cells with western blotting. The effect of dexmedetomidine on 5-LOX and COX-2 expression was investigated in vascular smooth muscle cells. SP600125, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, rauwolscine and chelerythrine attenuated dexmedetomidine-induced contraction. Indomethacin slightly attenuated dexmedetomidine-induced contraction. Fluconazole and SC-560 had no effect on dexmedetomidine-induced contraction, whereas NS-398 attenuated contraction. SP600125, rauwolscine, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone and GF 109203X attenuated dexmedetomidine-induced JNK phosphorylation. 5-LOX and COX-2 were upregulated by dexmedetomidine. Thus, dexmedetomidine-induced alpha-2 adrenoceptor-mediated contraction is mediated mainly by 5-LOX and partially by COX-2, which leads to JNK phosphorylation.

P2Y2R­mediated transactivation of VEGFR2 through Src phosphorylation is associated with ESM­1 overexpression in radiotherapy­resistant­triple negative breast cancer cells.

We previously reported that radiotherapy­resistant (RT­R) triple negative breast cancer (TNBC) cells upregulate the expression of endothelial­specific molecule­1 (ESM­1) compared with TNBC cells. In addition, ESM­1 is involved in an increased proliferation and invasion of RT­R­TNBC cells compared with TNBC cells. It was further identified that, in RT­R­TNBC cells, P2Y2 purinergic receptor (P2Y2R)­mediated activation of p21­activated kinase 1 (PAK1), protein kinase C (PKC), c­Jun N­terminal kinase (JNK) and p38 MAPKs is related to ESM­1 expression via forkhead box O1 (FoxO1) regulation. Notably, it has been reported that P2Y2R mediates the transactivation of vascular epithelial growth factor receptor 2 (VEGFR2), and VEGFR2 is known to be involved in ESM­1 expression. Therefore, in the present study, the involvement of VEGFR2 in the P2Y2R­mediated ESM­1 upregulation in RT­R­TNBC cells and the relationship between P2Y2R and VEGFR2 activation was further examined. Western blotting and reverse transcription­PCR were used to monitor the expression of ESM­1, and the results demonstrated that extracellular ATP treatment regulated the expression of ESM­1 in a P2Y2R­dependent manner in RT­R­MDA­MB­231 cells. In addition, extracellular ATP activated Src and VEGFR2 after 5 min of incubation, which was abolished by knockdown of P2Y2R expression. VEGFR2 activation in response to ATP was also decreased by inhibiting Src activity, suggesting that ATP­activated P2Y2R regulates VEGFR2 phosphorylation via Src activation. Furthermore, ATP­induced ESM­1 expression was decreased by transfection with VEGFR2 small interfering RNA (siRNA). ESM­1­related signaling molecules, PAK1, PKC, JNK and p38 MAPKs, and the transcriptional regulator, FoxO1, which were activated by ATP, were also decreased following transfection with VEGFR2 siRNA. These results suggest that P2Y2R­mediated transactivation of VEGFR2 through Src phosphorylation is associated with ESM­1 overexpression in RT­R­TNBC cells.

Activation of peroxisome proliferator-activated receptor-δ attenuates glutamate-induced neurotoxicity in HT22 mouse hippocampal cells.

Glutamate-induced neurotoxicity has been implicated in the pathogenesis of neurodegenerative disorders; however, little is known about the cellular events that underlie neurotoxicity or how to impede these events. This study demonstrates that peroxisome proliferator-activated receptor (PPAR)-δ regulates glutamate-induced neurotoxicity in HT22 mouse hippocampal cells. Activation of PPARδ by GW501516, a specific ligand, significantly inhibited glutamate-induced cell death and reactive oxygen species (ROS) production in HT22 cells. The siRNA-mediated knockdown of PPARδ abrogated the effects of GW501516 in neuronal toxicity and ROS production induced by glutamate. In addition, ligand-activated PPARδ reduced the glutamate-induced level of intracellular calcium ions (Ca(2+)) by modulating the influx of Ca(2+) from the extracellular space. Similarly, glutamate-induced cell death and intracellular Ca(2+) levels were attenuated in the presence of LY83583, an inhibitor of soluble guanylyl cyclase. Taken together, these results suggest that PPARδ plays an important role in glutamate-induced neurotoxicity by modulating oxidative stress and Ca(2+) influx.

P2Y2R-Mediated PAK1 Activation Is Involved in ESM-1 Overexpression in RT-R-MDA-MB-231 through FoxO1 Regulation.

ESM-1, overexpressed in several cancer types, is a potential cancer diagnostic and prognostic indicator. In our previous study, we determined that RT-R-TNBC cells were more aggressive than TNBC cells, and this difference was associated with ESM-1 overexpression. However, the mechanism explaining upregulated ESM-1 expression in RT-R-TNBC cells compared to TNBC cells was unclear. Therefore, we aimed to identify the mechanism by which ESM-1 is overexpressed in RT-R-MDA-MB-231 cells. RT-R-MDA-MB-231 cells were treated with various ESM-1 transcription factor inhibitors, and only the FoxO1 inhibitor downregulated ESM-1 expression. FoxO1 nuclear localization was modulated by JNK and p38 MAPKs, which were differentially regulated by PKC, PDK1 and PAK1. PAK1 profoundly modulated JNK and p38 MAPKs, whereas PKC and PDK1 affected only p38 MAPK. P2Y2R activated by ATP, which is highly released from RT-R-BC cells, was involved in PAK1 activation, subsequent JNK and p38 MAPK activation, FoxO1 induction, and ESM-1 expression in RT-R-MDA-MB-231 cells. These findings suggest for the first time that ESM-1 was overexpressed in RT-R-MDA-MB-231 cells and regulated through the P2Y2R-PAK1-FoxO1 signaling pathway.

Similar long-term outcomes between endovascular aneurysm repair and open surgical repair for abdominal aortic aneurysms in octogenarian patients: a single-center study.

Purpose: Endovascular aneurysm repair (EVAR) has lower perioperative mortality and morbidity rates and shorter hospital stays when compared to open surgical repair (OSR) in octogenarian patients. However, its long-term results remain unclear. Hence, we aimed to analyze and compare the long-term outcomes of OSR and EVAR in this aging population. Methods: This single-center, retrospective, observational study analyzed the data of patients older than 80 years who underwent primary repair of an abdominal aortic aneurysm (AAA) between 2011 and 2016 in our hospital. The primary outcomes were in-hospital complications and 30-day mortality, while the secondary outcomes included all-cause mortality and reintervention rate. Results: Among the 48 patients with elective AAA repair, 13 underwent OSR and 35 underwent EVAR. In-hospital complications occurred in 10 patients (20.8%), 5 for OSR (38.5%) and 5 for EVAR (14.3%) with no significant difference between the groups (P = 0.067). In the OSR group, pulmonary complications were the most common events; in the EVAR group, 2 patients had ischemic colitis diagnosed with sigmoidoscopy and recovered by conservative treatment. The 1- and 5-year survival rates were 77.8% and 55.6% in the OSR group, and 66.0% and 54.9% in the EVAR group, respectively. The reintervention rate was 8.6% for the EVAR group; none of the OSR group were readmitted. Conclusion: The difference in procedures did not affect patient survival. Therefore, OSR does not necessarily have a worse prognosis than EVAR. Individual risk stratification must be preceded before the selection of an appropriate treatment method.

Rosmarinic acid downregulates the oxLDL­induced interaction between monocytes and endothelial cells, in addition to monocyte diapedesis, under high glucose conditions.

Endothelial dysfunction during diabetes has been previously reported to be at least in part attributed to increased oxidized low­density lipoprotein (oxLDL) levels mediated by high glucose (HG) levels. Endothelial inflammation increases the adhesiveness of monocytes to the endothelium in addition to increasing vascular permeability, promoting diabetic atherogenesis. In a previous study, it was reported that oxLDL treatment induced nucleotide­binding domain and leucine­rich repeat containing family, pyrin domain­containing 3 inflammasome activation in endothelial cells (ECs) under HG conditions, in a manner that could be effectively reversed by rosmarinic acid. However, it remains unclear whether oxLDL­mediated inflammasome activation can regulate the interaction between monocytes and ECs. The effects of oxLDL­mediated inflammasome activation on endothelial permeability under HG conditions, in addition to the effects of rosmarinic acid on these oxLDL­mediated processes, also remain poorly understood. Therefore, the present study aimed to elucidate the mechanisms involved in oxLDL­induced endothelial permeability and monocyte diapedesis under HG conditions, in addition to the potential effects of rosmarinic acid. ECs were treated with oxLDL under HG conditions in the presence or absence of ROS scavengers mitoTEMPO and NAC, p38 inhibitor SB203580, FOXO1 inhibitor AS1842856 or transfected with the TXNIP siRNA, before protein expression levels of intercellular adhesion molecule 1 (ICAM­1), vascular cell adhesion molecule­1 (VCAM­1), phosphorylated vascular endothelial­cadherin (VE­cadhedrin), VE­cadherin and zonula occludens­1 (ZO­1) were measured by western blotting. In addition, adhesion assay and Transwell assays were performed. oxLDL was found to significantly increase the expression of ICAM­1 and VCAM­1 in ECs under HG conditions whilst also enhancing the adhesion of monocytes to ECs. This was found to be dependent on the reactive oxygen species (ROS)/p38 MAPK/forkhead box O1 (FOXO1)/thioredoxin interacting protein (TXNIP) signaling pathway. In addition, oxLDL­stimulated ECs under HG conditions exhibited increased phosphorylated VE­cadherin protein levels and decreased ZO­1 protein expression levels compared with those in untreated ECs, suggesting increased endothelial permeability. Furthermore, monocyte transmigration through the endothelial monolayer was significantly increased by oxLDL treatment under HG conditions. These oxLDL­mediated effects under HG conditions were also demonstrated to be dependent on this ROS/p38 MAPK/FOXO1/TXNIP signaling pathway. Subsequently, rosmarinic acid treatment significantly reversed oxLDL­induced overexpression of adhesion molecules and monocyte­EC adhesion, oxLDL­induced endothelial junction hyperpermeability and monocyte transmigration through the endothelial monolayer under HG conditions, in a dose­dependent manner. These results suggest that rosmarinic acid can exert a protective effect against oxLDL­mediated endothelial dysfunction under HG conditions by reducing the interaction between monocytes and ECs in addition to preventing monocyte diapedesis.

Peroxisome proliferator-activated receptor {delta} regulates extracellular matrix and apoptosis of vascular smooth muscle cells through the activation of transforming growth factor-{beta}1/Smad3.

Homeostasis of the extracellular matrix and apoptosis of vascular smooth muscle cells (VSMCs) are key components in the regulation of the stability of atherosclerotic plaques. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR)delta regulates extracellular matrix synthesis and degradation through transforming growth factor-beta1 and its effector, Smad3. Activation of PPARdelta strongly amplified the expression of types I and III collagen, fibronectin, elastin, and TIMP-3 (tissue inhibitor of metalloproteinases 3), but not of TIMP-1, matrix metalloproteinase-2 or -9. The effect of PPARdelta on the expression of type III collagen was dually regulated by the direct binding of PPARdelta and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, in the type III collagen gene promoter. The activation of PPARdelta attenuated apoptotic cell death in VSMCs induced by oxidized low-density lipoprotein, and similar antiapoptotic effects were observed on treatment of cells with exogenous type I and/or III collagen. Administration of a PPARdelta ligand GW501516 to mice also suppressed elastase-induced cell death of aortic VSMCs. These results suggest that PPARdelta-induced upregulation of extracellular matrix proteins exerts an antiapoptotic effect, thereby maintaining the stability of atherosclerotic plaques. Specific ligands of PPARdelta may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.

Rosmarinic Acid Exhibits a Lipid-Lowering Effect by Modulating the Expression of Reverse Cholesterol Transporters and Lipid Metabolism in High-Fat Diet-Fed Mice.

Hyperlipidemia is a potent risk factor for the development of cardiovascular diseases. The reverse cholesterol transport (RCT) process has been shown to alleviate hyperlipidemia and protect against cardiovascular diseases. Recently, rosmarinic acid was reported to exhibit lipid-lowering effects. However, the underlying mechanism is still unclear. This study aims to investigate whether rosmarinic acid lowers lipids by modulating the RCT process in high-fat diet (HFD)-induced hyperlipidemic C57BL/6J mice. Our results indicated that rosmarinic acid treatment significantly decreased body weight, blood glucose, and plasma total cholesterol and triglyceride levels in HFD-fed mice. Rosmarinic acid increased the expression levels of cholesterol uptake-associated receptors in liver tissues, including scavenger receptor B type 1 (SR-B1) and low-density lipoprotein receptor (LDL-R). Furthermore, rosmarinic acid treatment notably increased the expression of cholesterol excretion molecules, ATP-binding cassette G5 (ABCG5) and G8 (ABCG8) transporters, and cholesterol 7 alpha-hydroxylase A1 (CYP7A1) as well as markedly reduced cholesterol and triglyceride levels in liver tissues. In addition, rosmarinic acid facilitated fatty acid oxidation through AMP-activated protein kinase (AMPK)-mediated carnitine palmitoyltransferase 1A (CPT1A) induction. In conclusion, rosmarinic acid exhibited a lipid-lowering effect by modulating the expression of RCT-related proteins and lipid metabolism-associated molecules, confirming its potential for the prevention or treatment of hyperlipidemia-derived diseases.

Increased Extracellular Adenosine in Radiotherapy-Resistant Breast Cancer Cells Enhances Tumor Progression through A2AR-Akt-ß-Catenin Signaling.

Recently, we found that the expressions of adenosine (ADO) receptors A2AR and A2BR and the ectonucleotidase CD73 which is needed for the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and the extracellular ADO level are increased in TNBC MDA-MB-231 cells and RT-R-MDA-MB-231 cells compared to normal cells or non-TNBC cells. The expression of A2AR, but not A2BR, is significantly upregulated in breast cancer tissues, especially TNBC tissues, compared to normal epithelial tissues. Therefore, we further investigated the role of ADO-activated A2AR and its signaling pathway in the progression of RT-R-TNBC. ADO treatment induced MDA-MB-231 cell proliferation, colony formation, and invasion, which were enhanced in RT-R-MDA-MB-231 cells in an A2AR-dependent manner. A2AR activation by ADO induced AKT phosphorylation and then ß-catenin, Snail, and vimentin expression, and these effects were abolished by A2AR-siRNA transfection. In an in vivo animal study, compared to 4T1-injected mice, RT-R-4T1-injected mice exhibited significantly increased tumor growth and lung metastasis, which were decreased by A2AR-knockdown. The upregulation of phospho-AKT, ß-catenin, Snail, and vimentin expression in mice injected with RT-R-4T1 cells was also attenuated in mice injected with RT-R-4T1-A2AR-shRNA cells. These results suggest that A2AR is significantly upregulated in BC tissues, especially TNBC tissues, and ADO-mediated A2AR activation is involved in RT-R-TNBC invasion and metastasis through the AKT-ß-catenin pathway.