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.

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.

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.

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.

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.

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.

Rosmarinic acid inhibits oxLDL-induced inflammasome activation under high-glucose conditions through downregulating the p38-FOXO1-TXNIP pathway.

Elevated glucose levels in diabetes mellitus is associated with increased oxidized low density lipoprotein (oxLDL). High glucose (HG) and oxLDL are key inducers of oxidative stress and inflammatory processes responsible for diabetic vascular disorders. Rosmarinic acid is a polyphenol with antioxidant, anti-inflammatory and insulin-sensitizing effects. However, whether rosmarinic acid protects against diabetic atherosclerosis remains unknown. In this study, we aimed to investigate the protective effect of rosmarinic acid against diabetic atherosclerosis and the related signaling pathway. oxLDL-mediated oxidative stress upregulated thioredoxin-interacting protein (TXNIP) and subsequently induced binding of TXNIP to NLRP3 to mediate NLRP3 inflammasome assembly and activation under HG conditions in ECs. Reactive oxygen species (ROS) scavengers, p38 and FOXO1 inhibitors and TXNIP siRNA inhibited TXNIP protein upregulation and NLRP3 inflammasome assembly and activation. Rosmarinic acid abrogated TXNIP protein upregulation and the interaction between TXNIP and NLRP3 to attenuate NLRP3 inflammasome assembly and activation and eventually IL-1ß secretion in ECs through downregulating ROS production, p38 phosphorylation and FOXO1 protein induction in ECs. These findings show that rosmarinic acid inhibits endothelial dysfunction which is shown in diabetic atherosclerosis through downregulating the p38-FOXO1-TXNIP pathway and inhibiting inflammasome activation.

ESM-1 Overexpression is Involved in Increased Tumorigenesis of Radiotherapy-Resistant Breast Cancer Cells.

The key barrier to the effectiveness of radiotherapy remains the radioresistance of breast cancer cells, resulting in increased tumor recurrence and metastasis. Thus, in this study, we aimed to clarify the difference between radiotherapy-resistant (RT-R) breast cancer (BC) and BC, and accordingly, analyzed gene expression levels between radiotherapy-resistant (RT-R) MDA-MB-231 cells and MDA-MB-231 cells. Gene expression array showed that ESM-1 was the most upregulated in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells. Then, we aimed to investigate the role of ESM-1 in the increased tumorigenesis of RT-R-BC cells. RT-R-MDA-MB-231, which showed an increased expression level of ESM1, exhibited significantly enhanced proliferation, colony forming ability, migration, and invasion compared to MDA-MB-231 cells, and ESM-1 knockdown effectively reversed these effects. In addition, compared to MDA-MB-231 cells, RT-R-MDA-MB-231 cells displayed improved adhesion to endothelial cells (ECs) due to the induction of adhesion molecules and increased MMP-9 activity and VEGF-A production, which were decreased by ESM-1 knockdown. Moreover, the expression of HIF-1α and activation of NF-κB and STAT-3 were increased in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells, and these effects were abolished by the knockdown of ESM-1. Finally, we confirmed the role of ESM-1 in tumorigenesis in an in vivo mouse model. Tumor volume, lung metastasis, and tumorigenic molecules (VEGF-A, HIF-1α, MMP-9, ICAM-1, VCAM-1, and phospho-NF-κB and phospho-STAT-3) were significantly induced in mice injected with ESM-1-overexpressing 4T1 cells and greatly enhanced in those injected with ESM-1-overexpressing RT-R-4T1 cells. Taken together, these results suggest for the first time that ESM-1 plays a critical role in tumorigenesis of breast cancer cells, especially RT-R-breast cancer cells, through the induction of cell proliferation and invasion.

Polyphenols Extracted from Artemisia annua L. Exhibit Anti-Cancer Effects on Radio-Resistant MDA-MB-231 Human Breast Cancer Cells by Suppressing Stem Cell Phenotype, ß-Catenin, and MMP-9.

Artemisia annua L. has been reported to show anti-cancer activities. Here, we determined whether polyphenols extracted from Artemisia annua L. (pKAL) exhibit anti-cancer effects on radio-resistant MDA-MB-231 human breast cancer cells (RT-R-MDA-MB-231 cells), and further explored their molecular mechanisms. Cell viability assay and colony-forming assay revealed that pKAL inhibited cell proliferation on both parental and RT-R-MDA-MB-231 cells in a dose-dependent manner. The anti-proliferative effects of pKAL on RT-R-MDA-MB-231 cells were superior or similar to those on parental ones. Western blot analysis revealed that expressions of cluster of differentiation 44 (CD44) and Oct 3/4, matrix metalloproteinase-9 (MMP-9) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation were significantly increased in RT-R-MDA-MB-231 cells compared to parental ones, suggesting that these proteins could be associated with RT resistance. pKAL inhibited the expression of CD44 and Oct 3/4 (CSC markers), and ß-catenin and MMP-9 as well as STAT-3 phosphorylation of RT-R-MDA-MB-231. Regarding upstream signaling, the JNK or JAK2 inhibitor could inhibit STAT-3 activation in RT-R-MDA-MB-231 cells, but not augmented pKAL-induced anti-cancer effects. These findings suggest that c-Jun N-terminal kinase (JNK) or Janus kinase 2 (JAK2)/STAT3 signaling are not closely related to the anti-cancer effects of pKAL. In conclusion, this study suggests that pKAL exhibit anti-cancer effects on RT-R-MDA-MB-231 cells by suppressing CD44 and Oct 3/4, ß-catenin and MMP-9, which appeared to be linked to RT resistance of RT-R-MDA-MB-231 cells.

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.

P2Y2R activation by ATP induces oxLDL-mediated inflammasome activation through modulation of mitochondrial damage in human endothelial cells.

Oxidative stress and the related inflammatory responses are closely associated with many diseases including cardiovascular diseases such as atherosclerosis. Especially, mitochondrial damage and inflammasome activation have been reported to be directly involved in atherogenesis. In addition, we previously reported that endothelial cells (ECs) exposed to oxidized LDL (oxLDL) release ATP, which activates P2Y2R, resulting in the expression of receptors for advanced glycation end products and adhesion molecules that are involved in the pathogenesis of atherosclerosis. Therefore, it is expected that P2Y2R activation by ATP released under inflammatory conditions may be linked to the inflammasome-mediated pathogenesis of cardiovascular diseases such as atherosclerosis. However, the exact association remains unclear. Thus, in this study, we investigated the role of P2Y2R in oxLDL-mediated inflammasome activation and the related atherosclerotic pathogenesis in ECs. ECs stimulated with oxLDL demonstrated increased intracellular production and extracellular secretion of ATP. In addition, mitochondrial reactive oxygen species (mtROS) production and mitochondrial DNA (mtDNA) expression and cytosolic release were increased in ECs stimulated with oxLDL or the P2Y2R agonists ATP and UTP. Moreover, caspase-1 activity and IL-1ß production were increased in ECs stimulated with oxLDL, ATP or UTP through the modulation of mtROS production and mtDNA expression, in a P2Y2R-dependent manner. Furthermore, TLR-9 and NF-κB activation was increased in ECs in response to oxLDL, ATP or UTP, in a mtDNA-dependent manner. Taken together, our findings suggest that P2Y2R activation by ATP is involved in oxLDL-mediated inflammasome activation and subsequent IL-1ß production through the modulation of mtROS-mtDNA induction and the TLR9-NF-κB signaling pathway.

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.

Radioresistant breast cancer cells exhibit increased resistance to chemotherapy and enhanced invasive properties due to cancer stem cells.

Previous studies suggest that cancer stem cells (CSCs) exist in solid tumors, and contribute to therapeutic resistance and disease recurrence. Therefore, the present study aimed to investigate whether radioresistant (RT­R) breast cancer cells derived from breast cancer cells increase the number of CSCs, and whether these CSCs are responsible to increased invasiveness and therapeutic resistance. MCF­7, T47D and MDA­MB­231 cells were irradiated 25 times (2 Gy each; 50 Gy total) to generate radioresistant breast cancer cells (RT­R­MCF­7, RT­R­T47D and RT­R­MDA­MB­231). RT­R­breast cancer cells demonstrated increased cell viability against irradiation and increased colony forming abilities compared with parental breast cancer cells. Particularly, RT­R­MDA­MB­231 cells derived from highly metastatic MDA­MB­231 cells exhibited most radioresistance and chemoresistance of the three cell lines. In addition, MDA­MB­231 cells exhibited the most increased protein levels of CSCs markers cluster of differentiation 44, Notch­4, octamer­binding transcription factor 3/4 and aldehyde dehydrogenase 1, compared with RT­R­MCF­7 cells, suggesting highly metastatic breast cancer cells MDA­MB­231 produce more CSCs. RT­R­MDA­MB­231 cells increased intercellular adhesion molecule­1 and vascular cell adhesion molecule­1 levels, resulting in enhanced migration and adhesion to endothelial cells (ECs), and enhanced invasiveness through ECs by inducing matrix metalloproteinase­9, Snail­1 and ß­catenin, and by downregulating E­cadherin compared with MDA­MB­231 cells. These results suggest that highly metastatic breast cancer cells may increase the number of CSCs following radiation therapy, and CSCs present in RT­R­MDA­MB­231 cells contribute to the enhanced invasiveness by increasing migration, adhesion to ECs and invasion through ECs by promoting epithelial­mesenchymal transition (EMT) via the upregulation of adhesion molecules and EMT­associated proteins.

P2Y2R-mediated inflammasome activation is involved in tumor progression in breast cancer cells and in radiotherapy-resistant breast cancer.

In the tumor microenvironment, extracellular nucleotides are released and accumulate, and can activate the P2Y2 receptor (P2Y2R), which regulates various responses in tumor cells, resulting in tumor progression and metastasis. Moreover, the inflammasome has recently been reported to be associated with tumor progression. However, the role of P2Y2R in inflammasome activation in breast cancer cells is not yet well defined. Therefore, in this study, we investigated the role of P2Y2R in inflammasome-mediated tumor progression in breast cancer using breast cancer cells and radiotherapy-resistant (RT­R) breast cancer cells. We established RT­R-breast cancer cells (RT­R­MDA­MB­231, RT­R­MCF­7, and RT­R-T47D cells) by repeated irradiation (2 Gy each, 25 times) in a previous study. In this study, we found that the RT­R breast cancer cells exhibited an increased release of adenosine triphosphate (ATP) and P2Y2R activity. In particular, the RT­R­MDA­MB­231 cells derived from highly metastatic MDA­MB­231 cells, exhibited a markedly increased ATP release, which was potentiated by tumor necrosis factor (TNF)-α. The MDA­MB­231 cells exhibited inflammasome activation, as measured by caspase­1 activity and interleukin (IL)-1ß secretion following treatment with TNF­α and ATP; these effects were enhanced in the RT­R­MDA­MB­231 cells. However, the increased caspase­1 activities and IL­1ß secretion levels induced in response to treatment with TNF­α or ATP were significantly reduced by P2Y2R knockdown or the presence of apyrase in both the MDA­MB­231 and RT­R­MDA­MB­231 cells, suggesting the involvement of ATP-activated P2Y2R in inflammasome activation. In addition, TNF­α and ATP increased the invasive and colony-forming ability of the MDA­MB­231 and RT­R­MDA­MB­231 cells, and these effects were caspase­1-dependent. Moreover, matrix metalloproteinase (MMP)-9 activity was modulated by caspase-1, in a P2Y2R-dependent manner in the MDA­MB­231 and RT­R­MDA­MB­231 cells. Finally, nude mice injected with the RT­R­MDA­MB­231-EV cells (transfected with the empty vector) exhibited increased tumor growth, and higher levels of MMP-9 in their tumors and IL­1ß levels in their serum compared with the mice injected with the RT­R­MDA­MB­231-P2Y2R shRNA cells (transfected with P2Y2R shRNA). On the whole, the findings of this study suggest that extracellular ATP promotes tumor progression in RT­R-breast cancer cells and breast cancer cells by modulating invasion and associated molecules through the P2Y2R-inflammasome activation pathway.

A limited series of synthetic tetrahydroisoquinoline alkaloids reduce inflammatory gene iNOS via inhibition of p-STAT-1 and suppress HMGB1 secretion in LPS-treated mice lung tissue.

Tetrahydroisoquinoline alkaloids (THIs) have shown to increase survival and beneficial effect on animal model of sepsis, partly due to heme oxygenase-1 (HO-1) induction. Here, we aimed to compare a limited series of synthesized THIs on HO-1 induction and inhibitory effect of iNOS and COX-2 expression in lipopolysaccharide (LPS)-activated RAW264.7 cells. To the end, most promising compound (THI-61) was tested whether this compound reduces iNOS protein expression and inflammatory markers (HMGB1, TNF-α) in LPS-treated mice lung tissue. The results indicated that N-carbonyl substituted THI seem to affect HO-1 induction depending on which functional group is attached to C1 position. All compounds that reduce LPS-activated NF-κB-luciferase activity showed to preferential inhibition of iNOS/NO but not COX-2/PGE2 that was partly related to inhibition of STAT-1 phosphorylation. In particular, THI-61 induced translocation of Nrf2 from cytosol into the nucleus by an increased Nrf2-ARE binding activity, and reduced IL-1ß production in LPS-activated RAW264.7 cells. The reduced expression of iNOS/NO by THI-61 was reversed by siHO-1RNA-transfection. In LPS-treated mice, THI-61 significantly reduced iNOS protein in lung tissues, and HMGB1 and TNF-α levels in the BALF. We concluded that 1) lipophilic moiety of 1C substituent is much more important in N-carbonyl substituted THI for induction of HO-1, 2) newly synthesized THI-61 may be beneficial for treatment of lung injury.

Polyphenols from Artemisia annua L Inhibit Adhesion and EMT of Highly Metastatic Breast Cancer Cells MDA-MB-231.

Recent evidence suggests that polyphenolic compounds from plants have anti-invasion and anti-metastasis capabilities. The Korean annual weed, Artemisia annua L., has been used as a folk medicine for treatment of various diseases. Here, we isolated and characterized polyphenols from Korean A. annua L (pKAL). We investigated anti-metastatic effects of pKAL on the highly metastatic MDA-MB-231 breast cancer cells especially focusing on cancer cell adhesion to the endothelial cell and epithelial-mesenchymal transition (EMT). Firstly, pKAL inhibited cell viability of MDA-MB-231 cells in a dose-dependent manner, but not that of human umbilical vein endothelial cells (ECs). Polyphenols from Korean A. annua L inhibited the adhesion of MDA-MB-231 cells to ECs through reducing vascular cell adhesion molecule-1 expression of MDA-MB-231 and ECs, but not intracellular adhesion molecule-1 at the concentrations where pKAL did not influence the cell viability of either MDA-MB-231 cells nor EC. Further, pKAL inhibited tumor necrosis factor-activated MDA-MB-231 breast cancer cell invasion through inhibition of matrix metalloproteinase-2 and matrix metalloproteinase-9 and EMT. Moreover, pKAL inhibited phosphorylation of Akt, but not that of protein kinase C. These results suggest that pKAL may serve as a therapeutic agent against cancer metastasis at least in part by inhibiting the cancer cell adhesion to ECs through suppression of vascular cell adhesion molecule-1 and invasion through suppression of EMT. Copyright © 2016 John Wiley & Sons, Ltd.

Polyphenol mixtures of Euphorbia supina the inhibit invasion and metastasis of highly metastatic breast cancer MDA-MB-231 cells.

The Korean prostrate spurge Euphorbia supina is abundant in polyphenols and has been used as a folk medicine in Korea against a variety of diseases. Thus, we aimed to investigate the effect of polyphenol mixtures of Korean Euphorbia supina (PES) on the invasion and metastasis of highly metastatic breast cancer MDA-MB-231 cells. Firstly, PES showed no cytotoxicity on cancer cells and endothelial cells (ECs) at the doses of 0.1-10 µg/ml, but showed significant cytotoxicity from 50 µg/ml. Thus, we performed subsequent experiments with PES at doses up to 5 µg/ml. PES dose­dependently suppressed epithelial-mesenchymal transition by downregulating the mesenchymal markers, Snail1 and N-cadherin, showing significant inhibition from 1 and 5 µg/ml, respectively. In addition, PES significantly inhibited MMP-9 activity and LOX release induced by TNF-α at 5 µg/ml. Then, we determined the effect of PES on the expression of adhesion molecules and VE-cadherin phosphorylation. The results showed that PES effectively reduced TNF-α-mediated VCAM-1 expression but not ICAM expression both in the MDA-MB-231 cells and ECs, resulting in the reduced adhesion of MDA-MB-231 to ECs. Finally, PES effectively inhibited MDA-MB-231 cell invasion through ECs, suggesting that PES may serve as a therapeutic agent against cancer metastasis with minimal cytotoxicity to normal cells.

P2Y2 nucleotide receptor-mediated extracellular signal-regulated kinases and protein kinase C activation induces the invasion of highly metastatic breast cancer cells.

Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low-metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB-231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.

2-methoxycinnamaldehyde from Cinnamomum cassia reduces rat myocardial ischemia and reperfusion injury in vivo due to HO-1 induction.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Blume has been used as a traditional Chinese herbal medicine for alleviation of fever, inflammation, chronic bronchitis, and to improve blood circulation. AIM OF THE STUDY: We addressed whether 2-methoxycinnamaldehyde (2-MCA), one of active ingredients of Cinnamomum cassia, reduces vascular cell adhesion molecule-1 (VCAM-1) expression in tumor necrosis factor-alpha (TNF-α)-activated endothelial cells and protects ischemia/reperfusion (I/R)-injury due to heme oxygenase (HO)-1 induction. MATERIALS AND METHODS: Adult male rats were subjected to 30 min of ischemia by occlusion of the left anterior descending coronary artery followed by 24h of reperfusion. Rats were randomized to receive vehicle or 2-MCA (i.v.) 10 min before reperfusion. RESULTS: Administration of 2-MCA significantly improved I/R-induced myocardial dysfunction by increasing the values of the first derivative (±dp/dt) of left ventricular pressure and decreased infarct size. In addition, 2-MCA reduced the expression of high mobility group box 1 (HMGB1), an activator of the inflammatory cascade when released into the extracellular space, and VCAM-1 in I/R myocardium along with increase of HO-1 induction. The reduced injury was accompanied by significantly reduction of neutrophils infiltration and increased SOD activity in ischemic tissues and reduced serum level of cardiac troponin I (cTnI). Furthermore, 2-MCA significantly increased HO-1 induction by translocation of Nrf-2 from cytosol to nucleus in endothelial cells. Inhibition of VCAM-1 expression by 2-MCA was reversed both by SnPPIX, a HO-1 inhibitor and siHO-1 RNA trasfection in TNF-α-activated cells. In addition, 2-MCA significantly inhibited NF-κB luciferase activity in TNF-α-activated endothelial cells. As expected, 2-MCA significantly inhibited monocyte (U937) adhesion to endothelial cells. CONCLUSION: We concluded that 2-MCA protects of myocardial I/R-injury due to antioxidant and anti-inflammatory action possibly by HO-1 induction which can be explained why Cinnamomum cassia has been used in inflammatory disorders.

Induction of manganese-superoxide dismutase by YS 51, a synthetic 1-(beta-naphtylmethyl)6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline alkaloid: implication for anti-inflammatory actions.

The effect of YS 51, a synthetic 1-(beta-naphtylmethyl)6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline alkaloid, on the expression of manganese-superoxide dismutase (Mn-SOD), an antioxidant enzyme, was examined in sheep pulmonary artery endothelial cells (SPAEC) and a human cervical carcinoma cell line (Hela). YS 51 alone or in combination with cytokines enhanced the expression of Mn-SOD mRNA in SPAEC and Hela cells. YS 51 also showed synergistic effects on the induction of Mn-SOD mRNA with phorbol-12-myristate-13-acetate (TPA) and/or tumor necrosis factor-alpha (TNF-alpha). In Hela cells, the induction of Mn-SOD mRNA by YS 51 was in a time- and dose-dependent manner and the expression of Mn-SOD mRNA was increased to a maximum of 4-fold in 9 h. Enhancement of Mn-SOD mRNA by YS 51 was completely abolished by actinomycin D but not cycloheximide, suggesting that the induction of Mn-SOD mRNA byYS 51 is independent of new protein synthesis. Pretreatment of curcumin, an inhibitor of c-jun N-terminal kinase (JNK), dose-dependently suppressed the induction of Mn-SOD mRNA by YS 51, but not by 2'-amino-3'-methoxyflavone (PD98059) and 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580), inhibitors of mitogen-activated protein kinase. Also, YS 51 induced the phosphorylation activity of JNK in a time-dependent manner without affecting the phosphorylation activity of the extracellular signal-regulated kinase 1 (ERK1) and p38 MAP kinase. These results implicated that the JNK pathway appears to play a crucial role in mediating the YS 51-induced Mn-SOD gene expression, and that up-regulation of Mn-SOD would contribute to the anti-inflammatory actions mediated by YS 51.