Lipid mediators obtained from docosahexaenoic acid by soybean lipoxygenase attenuate RANKL-induced osteoclast differentiation and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by persistent inflammation and joint destruction. A lipid mediator (LM, namely, 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA, exhibits anti-inflammatory activity. In this study, we determined the effect of LM on collagen antibody-induced arthritis (CAIA) in mice and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in RAW264.7 cells. LM effectively downregulated the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K, inhibited osteoclast formation, and suppressed the NF-κB signaling pathway in vitro. In vivo, LM at 10 µg/kg/day significantly decreased paw swelling and inhibited progression of arthritis in CAIA mice. Moreover, proinflammatory cytokine (tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, IL-17, and interferon-γ) levels in serum were decreased, whereas IL-10 levels were increased following LM treatment. Furthermore, LM alleviated joint inflammation, cartilage erosion, and bone destruction in the ankles, which may be related to matrix metalloproteinase and Janus kinase (JAK)-signal transducer and activators of transcription (STAT) signaling pathway. Our findings suggest that LM attenuates arthritis severity, restores serum imbalances, and modifies joint damage. Thus, LM represents a promising therapy for relieving RA symptoms.

BRD4/nuclear PD-L1/RelB circuit is involved in the stemness of breast cancer cells.

BACKGROUND: Breast cancer (BC) is the most common cancer diagnosed in women worldwide. BC stem cells (BCSCs) have been known to be involved in the carcinogenesis of the breast and contribute to therapeutic resistance. The programmed death-ligand 1 (PD-L1) expression of BC correlated with a poor prognosis. Immunotherapies that target PD-L1 have great potential and have been successful when applied to cancer treatment. However, whether PD-L1 regulates BCSC formation is unknown. METHODS: BCSCs were enriched by serum-free suspension culture. The properties of BCSCs were examined by mammosphere formation assay, CD44+/Cd24-, aldehyde dehydrogenase (ALDH) assay, CSC marker analysis, and mammosphere growth assay. To elucidate the functions of bromodomain-containing protein 4 (BRD4), nuclear PD-L1, and RelB proteins in the stemness of BCSCs, mammosphere formation was examined using BRD4 inhibitor and degrader, PD-L1 degrader, and RelB inhibitor. The antitumor function of 3',4',7,8-tetrahydroxyflavone (THF), a specific BRD4 inhibitor, was studied through in vivo tumor model and mouse studies, and the protein levels of c-Myc, PD-L1, and RelB were examined in tumor model under THF treatment. RESULTS: BRD4 was upregulated in breast CSCs and regulates the stemness of BCs. The downregulation of BRD4 using BRD4 PROTAC, ARV-825, and BRD4 inhibitor, (+)-JQ1, inhibits mammosphere formation and reduces the levels of breast CSC markers (CD44+/CD24- and ALDH1), stem cell marker genes, and mammosphere growth. BRD4 inhibitor (JQ1) and degrader (ARV825) downregulate membrane and nuclear fractions of PD-L1 through the inhibition of PD-L1 transcript levels. The knockdown of PD-L1 inhibits mammosphere formation. Verteporfin, a PD-L1 degrader, inhibits the transcripts and protein levels of PD-L1 and downregulates the transcript and protein levels of RelB. Calcitriol, a RelB inhibitor, and the knockdown of RelB using si-RelB regulate mammosphere formation through interleukin-6 (IL-6) expression. THF is a natural product and a potent selective BRD4 inhibitor, inhibits mammosphere formation, and reduces the levels of CD44+/CD24- and mammosphere growth by downregulating c-Myc, PD-L1, and RelB. 3',4',7,8-THF shows tumoricidal activity and increased levels of CD3+CD4+ and CD3+CD8+ T-cells in the tumor and tumor-draining lymph nodes (TDLNs) in the murine tumor model using 4T1 and MC38 cells. CONCLUSIONS: The results show the first evidence of the essential role of the BRD4/nuclear PD-L1/RelB axis in breast CSC formation. The nuclear PD-L1 regulates RelB, and the RelB/p65 complex induces IL6 and breast CSC formation. Targeting nuclear PD-L1 represents a potential and novel tool for immunotherapies of intractable BC. Video Abstract.

Lipid mediators derived from DHA alleviate DNCB-induced atopic dermatitis and improve the gut microbiome in BALB/c mice.

Atopic dermatitis (AD) is a chronic inflammatory skin condition that primarily results from immune dysregulation. We determined the potential therapeutic benefits of lipid mediators (LM, 17S-monohydroxy DHA, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA. The underlying molecular mechanisms involved in TNF-α/IFN-γ-stimulated HaCaT cells as well as its effect in an AD mouse model induced by DNCB in BALB/c mice were examined. The results indicated that LM effectively attenuates the production of inflammatory cytokines (IL-6 and IL-1ß) and chemokines (IL-8 and MCP-1) by inhibiting the NF-κB signaling pathway in TNF-α/IFN-γ-stimulated HaCaT cells. The oral administration of LM at 5 or 10 µg/kg/day significantly reduced skin lesions, epidermal thickness, and mast cell infiltration in AD mice. Furthermore, LM reduced the production of IgE and inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in the serum, modulated gut microbiota diversity, and restored the microbial composition. Overall, our findings suggest that LM represents a potential therapeutic agent for improving AD symptoms through its ability to suppress inflammatory cytokines and alter the composition of gut microbiota.

13-Oxo-9Z,11E-octadecadienoic Acid Down-regulates c-Myc and Attenuates Breast Cancer Cell Stemness.

BACKGROUND/AIM: Breast cancer stem cells (BCSCs) are involved in the development of breast cancer and contribute to therapeutic resistance. This study aimed to investigate the anticancer stem cell (CSC) mechanism of 13-Oxo-9Z,11E-octadecadienoic acid (13-Oxo-ODE) as a potent CSC inhibitor in breast cancer. MATERIALS AND METHODS: The effects of 13-Oxo-ODE on BCSCs were evaluated using a mammosphere formation assay, CD44high/CD24low analysis, aldehyde dehydrogenase (ALDH) assay, apoptosis assay, quantitative real-time PCR, and western blotting. RESULTS: We found that 13-Oxo-ODE suppressed cell proliferation, CSC formation, and mammosphere proliferation and increased apoptosis of BCSCs. Additionally, 13-Oxo-ODE reduced the subpopulation of CD44high/CD24low cells and ALDH expression. Furthermore, 13-Oxo-ODE decreased c-myc gene expression. These results suggest that 13-Oxo-ODE has potential as a natural inhibitor targeting BCSCs through the degradation of c-Myc. CONCLUSION: In summary, 13-Oxo-ODE induced CSC death possibly through reduced c-Myc expression, making it a promising natural inhibitor of BCSCs.

Isophysalin A Inhibits the Stemness of Breast Cancer Cells Possibly via Stat3 and IL-6 Signaling.

BACKGROUND/AIM: Breast cancer stem cells (BCSCs) are involved in carcinogenesis of the breast and contribute to therapeutic resistance. In the present study, we found that isophysalin A acts as a potent cancer stem cell inhibitor and investigated the anti-CSC mechanism of action of isophysalin A on breast cancer. MATERIALS AND METHODS: The effect of isophysalin A on BCSCs was examined using a mammosphere formation, a colony formation and a cell migration assay, as well as CD44 (Cluster of differentiation 44)high/CD24 (Cluster of differentiation 24)low analysis, an apoptosis assay, quantitative real-time PCR, western blotting, an electrophoretic mobility shift assay, and a cytokine profiling assay. RESULTS: Isophysalin A inhibited cell proliferation, colony formation, cell migration, CSC formation, and mammosphere proliferation and increased BCSC apoptosis. The subpopulation of CD44high/CD24low was decreased by isophysalin A, which also reduced the DNA binding of Stat3 and the total and nuclear protein expression levels of Stat3 and phosphorylated Stat3. Furthermore, the mRNA and media IL-6/IL-8 levels of the mammosphere were also reduced by isophysalin A. CONCLUSION: Isophysalin A inhibited the Stat3 and IL-6 signaling pathways and induced CSC death; thus, isophysalin A may be a potential natural inhibitor of BCSCs.

7S,15R-Dihydroxy-16S,17S-epoxy-docosapentaenoic Acid Overcomes Chemoresistance of 5-Fluorouracil by Suppressing the Infiltration of Tumor-Associated Macrophages and Inhibiting the Activation of Cancer Stem Cells in a Colorectal Cancer Xenograft Model.

Although the tumor bulk is initially reduced by 5-fluorouracil (5-FU), chemoresistance developed due to prolonged chemotherapy in colorectal cancer (CRC). The enrichment of cancer stem cells (CSCs) and the infiltration of tumor-associated macrophages (TAMs) contribute to chemoresistance and poor outcomes. A docosahexaenoic acid derivative developed by our group, 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), exerts antitumor effects against TAMs infiltration and CSCs enrichment in our previous study. The current study aimed to investigate whether diHEP-DPA was able to overcome chemoresistance to 5-FU in CRCs, together with the potential synergistic mechanisms in a CT26-BALB/c mouse model. Our results suggested that although 5-FU inhibited tumor growth, 5-FU enriched CSCs via the WNT/ß-catenin signaling pathway, resulting in chemoresistance in CRCs. However, we revealed that 5-FU promoted the infiltration of TAMs via the NF-kB signaling pathway and improved epithelial-mesenchymal transition (EMT) via the signal transducer and activator of the transcription 3 (STAT3) signaling pathway; these traits were believed to contribute to CSC activation. Furthermore, supplementation with diHEP-DPA could overcome drug resistance by decreasing the CSCs, suppressing the infiltration of TAMs, and inhibiting EMT progression. Additionally, the combinatorial treatment of diHEP-DPA and 5-FU effectively enhanced phagocytosis by blocking the CD47/signal regulatory protein alpha (SIRPα) axis. These findings present that diHEP-DPA is a potential therapeutic supplement to improve drug outcomes and suppress chemoresistance associated with the current 5-FU-based therapies for colorectal cancer.

Effects of Fish Oil, Lipid Mediators, Derived from Docosahexaenoic Acid, and Their Co-Treatment against Lipid Metabolism Dysfunction and Inflammation in HFD Mice and HepG2 Cells.

Although fish oil (FO) and lipid mediators (LM) derived from polyunsaturated fatty acids can prevent obesity, their combined effects and cellular metabolism remain unclear. Therefore, this study aimed to examine the potential protective and metabolic effects of FO in combination with LM (a mixture of 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX [3:47:50], derived from docosahexaenoic acid (DHA)) on palmitic acid (PA)-induced HepG2 cells and high-fat- diet (HFD)-induced C57BL/6J mice after 9-week treatment. Lipid metabolism disorders and inflammation induced by HFD and PA were substantially reduced after FO and LM treatment. Further, FO and LM treatments reduced lipid accumulation by increasing fatty acid oxidation via peroxisome proliferator-activated receptor α and carnitine-palmitoyl transferase 1 as well as by decreasing fatty acid synthesis via sterol regulatory element-binding protein-1c and fatty acid synthase. Finally, FO and LM treatment reduced inflammation by blocking the NF-κB signaling pathway. Importantly, the combination of FO and LM exhibited more robust efficacy against nonalcoholic fatty liver disease, suggesting that FO supplemented with LM is a beneficial dietary strategy for treating this disease.

Protective effect of 17S­epoxy­docosapentaenoic acid against dextran sulfate sodium induced ulcerative colitis in BALB/c mice.

Ulcerative colitis (UC) is difficult to eradicate as it leads to chronic inflammation in the digestive tract due to immune system malfunction. The present study demonstrated the protective effect of 7S,15R­dihydroxy­16S,17S­epoxy­docosapentaenoic acid (diHEP­DPA), which had been previously synthesized, on a dextran sulfate sodium (DSS)­induced BALB/c mouse model of UC. UC was induced with 4% DSS drinking water for 7 days. Initially, the anti­inflammatory effect of diHEP­DPA was confirmed by demonstrating that lipopolysaccharide­stimulated THP1 cells treated with diHEP­DPA decreased IL­6, TNF­α and nitrite levels by fluorescence­activated cell sorting (FACS) and Griess reagent kit. The results indicated that the administration of diHEP­DPA at 20 µg/kg significantly reduced the severity of colitis, as determined by hematoxylin and eosin staining. The levels of TNF­α, IL­6 and IL­1ß in the colon tissue and serum were significantly reduced in the diHEP­DPA + DSS­treated group compared with in the control group, as determined by FACS and ELISA kit. It was also observed that diHEP­DPA decreased myeloperoxidase (MPO) and nitrite levels in the colon tissues of diHEP­DPA + DSS­treated mice, as indicated using commercial MPO and nitric oxide kits. The diHEP­DPA+DSS­treated mice also exhibited decreased expression levels of phosporylated (p)­inhibitor κB protein, p­p65 and inducible nitric oxide synthase in the colon tissue by inhibiting inflammation, which were measured by reverse transcription­quantitative PCR and weatern blot analysis. Overall, the present study demonstrated the protective effect of diHEP­DPA against a severe colitis condition in vivo.

Dihydroconiferyl Ferulate Isolated from Dendropanax morbiferus H.Lév. Suppresses Stemness of Breast Cancer Cells via Nuclear EGFR/c-Myc Signaling.

Breast cancer is the leading cause of global cancer incidence and breast cancer stem cells (BCSCs) have been identified as the target to overcome breast cancer in patients. In this study, we purified a BCSC inhibitor from Dendropanax morbiferus H.Lév. leaves through several open column and high-performance liquid chromatography via activity-based purification. The purified cancer stem cell (CSC) inhibitor was identified as dihydroconiferyl ferulate using nuclear magnetic resonance and mass spectrometry. Dihydroconiferyl ferulate inhibited the proliferation and mammosphere formation of breast cancer cells and reduced the population of CD44high/CD24low cells. Dihydroconiferyl ferulate also induced apoptosis, inhibited the growth of mammospheres and reduced the level of total and nuclear EGFR protein. It suppressed the EGFR levels, the interaction of Stat3 with EGFR, and c-Myc protein levels. Our findings show that dihydroconiferyl ferulate reduced the level of nuclear epidermal growth factor receptor (EGFR) and induced apoptosis of BCSCs through nEGFR/Stat3-dependent c-Myc deregulation. Dihydroconiferyl ferulate exhibits potential as an anti-CSC agent through nEGFR/Stat3/c-Myc signaling.

Anti-Inflammatory Effects of (9Z,11E)-13-Oxooctadeca-9,11-dienoic Acid (13-KODE) Derived from Salicornia herbacea L. on Lipopolysaccharide-Stimulated Murine Macrophage via NF-kB and MAPK Inhibition and Nrf2/HO-1 Signaling Activation.

Glasswort (Salicornia herbacea L.) is a halophyte that exhibits antioxidant and antidiabetic effects. Only a few studies have been conducted on its antioxidant effects. Here, we isolated an antioxidant using an activity-based purification method, and the resulting compound was identified as (9Z,11E)-13-Oxooctadeca-9,11-dienoic acid (13-KODE). We investigated its ability to suppress inflammatory responses and the molecular mechanisms underlying these abilities using lipopolysaccharide-stimulated RAW 264.7 macrophage cells. We studied the anti-inflammatory effects of 13-KODE derived from S. herbacea L on RAW 264.7 macrophages. 13-KODE inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production by suppressing inducible NO synthase and suppressed LPS-induced tumor necrosis factor and interleukin-1ß expression in RAW 264.7 macrophages. LPS-mediated nuclear localization of NF-κB and mitogen-activated protein kinase activation were inhibited by 13-KODE. 13-KODE significantly reduced LPS-induced production of reactive oxygen species and increased the expression of nuclear factor erythroid-2 like 2 (Nfe2I2) and heme oxygenase 1. Overall, our results indicate that 13-KODE may have potential for treating inflammation.

The docosahexaenoic acid derivatives, diHEP-DPA and TH-DPA, synthesized via recombinant lipoxygenase, ameliorate disturbances in lipid metabolism and liver inflammation in high fat diet-fed mice.

7S,15R-Dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA) and 7S,15R,16S,17S-tetrahydroxy-docosapentaenoic acid (TH-DPA) are two novel lipid mediators derived from docosahexaenoic acid (DHA) that we previously synthesized via combined enzymatic and chemical reactions. In the present study, we investigated the effects of these compounds on disturbances in lipid metabolism and liver inflammation induced by a high fat diet (HFD) in mice. Male BALB/c mice were randomly divided into four groups (n = 10/group): controls, HFD only, HFD + diHEP-DPA, and HFD + TH-DPA. Mice in HFD + diHEP-DPA and HFD + TH-DPA groups were orally administered 20 µg/kg of diHEP-DPA or TH-DPA, respectively. Measurements of adipose accumulation and liver inflammation showed that both diHEP-DPA and TH-DPA decreased adipose tissue mass and liver color depth, as well as total cholesterol, triglycerides, and low-density lipoprotein-cholesterol in the serum of HFD-fed mice compared with mice in the HFD-only group, while elevating high-density lipoprotein-cholesterol. Both of them also decreased hepatic expression of genes encoding lipid synthesis-related proteins (PPARγ, SIRT1, SREBP-1c and FASN) and increased the expression of genes encoding proteins involved in lipid degradation (PPARα and CPT-1) in the liver. Western blotting and quantitative RT-PCR confirmed that diHEP-DPA or TH-DPA administration modulated the expression of inflammation-related genes (TNF-α and IL-6) and inhibited activation of the NF-κB signaling pathway in livers of HFD-fed mice. Taken together, our data indicate that diHEP-DPA and TH-DPA ameliorate liver inflammation and inhibit HFD-induced obesity in mice.

Primaquine Inhibits the Endosomal Trafficking and Nuclear Localization of EGFR and Induces the Apoptosis of Breast Cancer Cells by Nuclear EGFR/Stat3-Mediated c-Myc Downregulation.

Triple-negative breast cancer (TNBC) cells overexpress the epidermal growth factor receptor (EGFR). Nuclear EGFR (nEGFR) drives resistance to anti-EGFR therapy and is correlated with poor survival in breast cancer. Inhibition of EGFR nuclear translocation may be a reasonable approach for the treatment of TNBC. The anti-malarial drugs chloroquine and primaquine have been shown to promote an anticancer effect. The aim of the present study was to investigate the effect and mechanism of chloroquine- and primaquine-induced apoptosis of breast cancer cells. We showed that primaquine, a malaria drug, inhibits the growth, migration, and colony formation of breast cancer cells in vitro, and inhibits tumor growth in vivo. Primaquine induces damage to early endosomes and inhibits the nuclear translocation of EGFR. Primaquine inhibits the interaction of Stat3 and nEGFR and reduces the transcript and protein levels of c-Myc. Moreover, primaquine and chloroquine induce the apoptosis of breast cancer cells through c-Myc/Bcl-2 downregulation, induce early endosome damage and reduce nEGFR levels, and induce apoptosis in breast cancer through nEGFR/Stat3-dependent c-Myc downregulation. Our study of primaquine and chloroquine provides a rationale for targeting EGFR signaling components in the treatment of breast cancer.

Extracellular Vesicles from Korean Codium fragile and Sargassum fusiforme Negatively Regulate Melanin Synthesis.

Although various marine ingredients have been exploited for the development of cosmetic products, no previous study has examined the potential of seaweed extracellular vesicles (EV) in such applications. Our results revealed that EV from Codium fragile and Sargassum fusiforme effectively decreased α-MSH-mediated melanin synthesis in MNT-1 human melanoma cells, associated with downregulation of MITF (microphthalmia-associated transcription factor), tyrosinase and TRP1 (tyrosinase-related proteins 1). The most effective inhibitory concentrations of EV were 250 µg/ml for S. fusiforme and 25 µg/ml for C. fragile, without affecting the viability of MNT-1 cells. Both EV reduced melanin synthesis in the epidermal basal layer of a three-dimensional model of human epidermis. Moreover, the application of the prototype cream containing C. fragile EV (final 5 µg/ml) yielded 1.31% improvement in skin brightness in a clinical trial. Together, these results suggest that EV from C. fragile and S. fusiforme reduce melanin synthesis and may be potential therapeutic and/or supplementary whitening agents.

7S,15R-Dihydroxy-16S,17S-Epoxy-Docosapentaenoic Acid, a Novel DHA Epoxy Derivative, Inhibits Colorectal Cancer Stemness through Repolarization of Tumor-Associated Macrophage Functions and the ROS/STAT3 Signaling Pathway.

Colorectal cancer is a highly malignant cancer that is inherently resistant to many chemotherapeutic drugs owing to the complicated tumor-supportive microenvironment (TME). Tumor-associated macrophages (TAM) are known to mediate colorectal cancer metastasis and relapse and are therefore a promising therapeutic target. In the current study, we first confirmed the anti-inflammatory effect of 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), a novel DHA dihydroxy derivative synthesized in our previous work. We found that diHEP-DPA significantly reduced lipopolysaccharide (LPS)-induced inflammatory cytokines secretion of THP1 macrophages, IL-6, and TNF-α. As expected, diHEP-DPA also modulated TAM polarization, as evidenced by decreased gene and protein expression of the TAM markers, CD206, CD163, VEGF, and TGF-ß1. During the polarization process, diHEP-DPA treatment decreased the concentration of TGF-ß1, IL-1ß, IL-6, and TNF-α in culture supernatants via inhibiting the NF-κB pathway. Moreover, diHEP-DPA blocked immunosuppression by reducing the expression of SIRPα in TAMs and CD47 in colorectal cancer cells. Knowing that an inflammatory TME largely serves to support epithelial-mesenchymal transition (EMT) and cancer stemness, we tested whether diHEP-DPA acted through polarization of TAMs to regulate these processes. The intraperitoneally injected diHEP-DPA inhibited tumor growth when administered alone or in combination with 5-fluorouracil (5-FU) chemotherapy in vivo. We further found that diHEP-DPA effectively reversed TAM-conditioned medium (TCCM)-induced EMT and enhanced colorectal cancer stemness, as evidenced by its inhibition of colorectal cancer cell migration, invasion and expression of EMT markers, as well as cancer cell tumorspheres formation, without damaging colorectal cancer cells. DiHEP-DPA reduced the population of aldehyde dehydrogenase (ALDH)-positive cells and expression of colorectal stemness marker proteins (CD133, CD44, and Sox2) by modulating TAM polarization. Additionally, diHEP-DPA directly inhibited cancer stemness by inducing the production of reactive oxygen species (ROS), which, in turn, reduced the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). These data collectively suggest that diHEP-DPA has the potential for development as an anticancer agent against colorectal cancer.

5-Hydroxymaltol Derived from Beetroot Juice through Lactobacillus Fermentation Suppresses Inflammatory Effect and Oxidant Stress via Regulating NF-kB, MAPKs Pathway and NRF2/HO-1 Expression.

Inflammation is the first response of the immune system against bacterial pathogens. This study isolated and examined an antioxidant derived from Lactobacillus fermentation products using cultured media with 1% beet powder. The antioxidant activity of the beet culture media was significantly high. Antioxidant activity-guided purification and repeated sample isolation yielded an isolated compound, which was identified as 5-hydoxymaltol using nuclear magnetic resonance spectrometry. We examined the mechanism of its protective effect on lipopolysaccharide (LPS)-induced inflammation of macrophages. 5-Hydroxymaltol suppressed nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells. It also suppressed tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, and inducible nitric oxide synthase (iNOS) in the messenger RNA and protein levels in LPS-treated RAW 264.7 cells. Moreover, it suppressed LPS-induced nuclear translocation of NF-κB (p65) and mitogen-activated protein kinase activation. Furthermore, 5-hydroxymaltol reduced LPS-induced reactive oxygen species (ROS) production as well as increased nuclear factor erythroid 2-related factor 2 and heme oxygenase 1 expression. Overall, this study found that 5-hydroxymaltol has anti-inflammatory activities in LPS-stimulated RAW 264.7 macrophage cells based on its inhibition of pro-inflammatory cytokine production depending on the nuclear factor κB signaling pathway, inhibition of LPS-induced reactive oxygen species production, inhibition of LPS-induced mitogen-activated protein kinase induction, and induction of the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 signaling pathway. Our data showed that 5-hydroxymaltol may be an effective compound for treating inflammation-mediated diseases.

Physalin A, 13,14-Seco-16, 24-Cyclo-Steroid, Inhibits Stemness of Breast Cancer Cells by Regulation of Hedgehog Signaling Pathway and Yes-Associated Protein 1 (YAP1).

The Hedgehog (HH) signaling pathway plays an important role in embryonic development and adult organ homeostasis. Aberrant activity of the Hedgehog signaling pathway induces many developmental disorders and cancers. Recent studies have investigated the relationship of this pathway with various cancers. GPCR-like protein Smoothened (SMO) and the glioma-associated oncogene (GLI1) are the main effectors of Hedgehog signaling. Physalin A, a bioactive substance derived from Physalis alkekengi, inhibits proliferation and migration of breast cancer cells and mammospheres formation. Physalin A-induced apoptosis and growth inhibition of mammospheres, and reduced transcripts of cancer stem cell (CSC) marker genes. Physalin A reduced protein expressions of SMO and GLI1/2. Down-regulation of SMO and GLI1 using siRNA inhibited mammosphere formation. Physalin A reduced mammosphere formation by reducing GLI1 gene expression. Down-regulation of GLI1 reduced CSC marker genes. Physalin A reduced protein level of YAP1. Down-regulation of YAP1 using siRNA inhibited mammosphere formation. Physalin A reduced mammosphere formation through reduction of YAP1 gene expression. Down-regulation of YAP1 reduced CSC marker genes. We showed that treatment of MDA-MB-231 breast cancer cells with GLI1 siRNA induced inhibition of mammosphere formation and down-regulation of YAP1, a Hippo pathway effector. These results show that Hippo signaling is regulated by the Hedgehog signaling pathway. Physalin A also inhibits the canonical Hedgehog and Hippo signaling pathways, CSC-specific genes, and the formation of mammospheres. These findings suggest that physalin A is a potential therapeutic agent for targeting CSCs.

5-Desmethylsinensetin isolated from Artemisia princeps suppresses the stemness of breast cancer cells via Stat3/IL-6 and Stat3/YAP1 signaling.

AIMS: To study 5-desmethylsinensetin exhibiting potential anticancer activity against breast cancer stem cells and the related molecular mechanism. MAIN METHODS: In this study, isolation of a cancer stem cell (CSC) inhibitor of Artemisia princeps was performed using a silica gel column, a Sephadex gel column, and high-performance liquid chromatography. A single compound was purified via activity-based isolation using mammosphere formation assays. An MTS was used to examine the proliferation of breast cancer cells, and flow cytometry was used to analyze apoptosis and cancer stem cell markers. Western blotting was used to detect the signaling pathway. RESULTS: The isolated compound was identified as 5-desmethylsinensetin using nuclear magnetic resonance and mass spectrometry. 5-Desmethylsinensetin suppresses the proliferation and mammosphere formation of breast cancer cells, reduces the subpopulations of CD44+/CD24- and ALDH1+ cancer cells, and reduces the transcription of the stemness markers Oct4, c-Myc, Nanog and CD44 in Breast CSCs. 5-Desmethylsinensetin inhibits the total and nuclear expression of Stat3 and p-Stat3, as well as the translocation of YAP1. Additionally, 5-desmethylsinensetin reduces the mRNA and protein levels of IL-6. CONCLUSION: Our results show that 5-desmethylsinensetin exhibits potential anticancer activity against breast cancer stem cells via Stat3-IL-6 and Stat3-YAP1 signaling.

13R,20-Dihydroxydocosahexaenoic Acid, a Novel Dihydroxy- DHA Derivative, Inhibits Breast Cancer Stemness through Regulation of the Stat3/IL-6 Signaling Pathway by Inducing ROS Production.

Breast cancer is a major health problem worldwide. Cancer stem cells (CSCs) are known to mediate breast cancer metastasis and recurrence and are therefore a promising therapeutic target. In this study, we investigated the anti-inflammatory effect of 13R,20-dihydroxydocosahexaenoic acid (13R,20-diHDHA), a novel dihydroxy-DHA derivative, which was synthesized through an enzymatic reaction using cyanobacterial lipoxygenase. We found that 13R,20-diHDHA reduced the macrophage secretion of the inflammatory cytokines, IL-6 and TNF-α, and thus appeared to have anti-inflammatory effects. As the inflammatory tumor microenvironment is largely devoted to supporting the cancer stemness of breast cancer cells, we investigated the effect of 13R,20-diHDHA on breast cancer stemness. Indeed, 13R,20-diHDHA effectively inhibited breast cancer stemness, as evidenced by its ability to dose-dependently inhibit the mammospheres formation, colony formation, migration, and invasion of breast CSCs. 13R,20-diHDHA reduced the populations of CD44high/CD24low and aldehyde dehydrogenase (ALDH)-positive cells and the expression levels of the cancer stemness-related self-renewal genes, Nanog, Sox2, Oct4, c-Myc, and CD44. 13R,20-diHDHA increased reactive oxygen species (ROS) production, and the generated ROS reduced the phosphorylation of nuclear signal transducer and activator of transcription 3 (Stat3) and the secretion of IL-6 by mammospheres. These data collectively suggest that 13R,20-diHDHA inhibits breast cancer stemness through ROS production and downstream regulation of Stat3/IL-6 signaling, and thus might be developed as an anti-cancer agent acting against CSCs.

The Antiasthma Medication Ciclesonide Suppresses Breast Cancer Stem Cells through Inhibition of the Glucocorticoid Receptor Signaling-Dependent YAP Pathway.

Ciclesonide is an FDA-approved glucocorticoid used to treat asthma and allergic rhinitis. However, whether it has anticancer and anti-cancer stem cell (CSC) effects is unknown. This study focused on investigating the effect of ciclesonide on breast cancer and CSCs and determining its underlying mechanism. Here, we showed that ciclesonide inhibits breast cancer and CSC formation. Similar glucocorticoids-dexamethasone and prednisone-did not inhibit CSC formation. Ciclesonide-induced glucocorticoid receptor (GR) degradation was dependent on ubiquitination. We showed via GR small interfering RNA (siRNA) that GR plays an important role in CSC formation. We showed via western blot and immunofluorescence assays that ciclesonide reduces the nuclear level of GR. The GR antagonist RU-486 also inhibited CSC formation. Ciclesonide reduced the protein level of the Hippo transducer Yes-associated protein (YAP). GR siRNA induced a decrease in YAP protein expression and inhibited mammosphere formation. The YAP inhibitor verteporfin inhibited CSC formation and transcription of the connective tissue growth factor and cysteine-rich protein 61 genes. The GR/YAP1 pathway regulated breast CSC formation. We showed that the GR/YAP signaling pathway regulates breast CSC formation and revealed a new approach for targeting GR and YAP to inhibit CSC formation.

Plant Volatile, Phenylacetaldehyde Targets Breast Cancer Stem Cell by Induction of ROS and Regulation of Stat3 Signal.

Cancer stem cells (CSCs) are undifferentiated cells that give rise to tumor and resistance to chemotherapy. This study reports that phenylacetaldehyde (PAA), a flower flavor, inhibits formation on breast CSCs. PAA showed anti-proliferation and increased apoptosis of breast cancer. PAA also reduced tumor growth in an in vivo mice model. PAA reduced the CD44+/CD24- and ALDH1-expressing cells, mammosphere formation, and CSC marker genes. PAA preferentially induced reactive oxygen species (ROS) production and combined treatment with PAA and N-acetyl cysteine (NAC) decreased inhibition of mammosphere formation. PAA reduced phosphorylation of nuclear Stat3. PAA inhibited Stat3 signaling through de-phosphorylation of Stat3 and reduced secretory IL-6. Our results suggest that the PAA-induced ROS deregulated Stat3/IL-6 pathway and PAA may be a potential agent targeting breast cancer and CSCs.