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.

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.

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.

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.

Coriolic Acid (13-(S)-Hydroxy-9Z, 11E-octadecadienoic Acid) from Glasswort (Salicornia herbacea L.) Suppresses Breast Cancer Stem Cell through the Regulation of c-Myc.

Cancer stem cells have certain characteristics, such as self-renewal, differentiation, and drug resistance, which are related to tumor progression, maintenance, recurrence, and metastasis. In our study, we targeted breast cancer stem cells (BCSCs) using a natural compound, coriolic acid, from Salicornia herbacea L. This compound was isolated by mammosphere formation inhibition bioassay-guided fractionation and identified by using NMR spectroscopy and electrospray ionization mass spectrometry. Coriolic acid inhibited the formation of mammospheres and induced BCSC apoptosis. It also decreased the subpopulation of CD44high/CD24low cells, a cancer stem cell (CSC) phenotype, and specific genes related to CSCs, such as Nanog,Oct4, and CD44. Coriolic acid decreased the transcriptional and translational levels of the c-Myc gene, which is a CSC survival factor. These results indicated that coriolic acid could be a novel compound to target BCSCs via regulation of c-Myc.

Betavulgarin Isolated from Sugar Beet (Beta vulgaris) Suppresses Breast Cancer Stem Cells through Stat3 Signaling.

Breast cancer is a major health problem that affects lives worldwide. Breast cancer stem cells (BCSCs) are small subpopulations of cells with capacities for drug resistance, self-renewal, recurrence, metastasis, and differentiation. Herein, powder extracts of beetroot were subjected to silica gel, gel filtration, thin layer chromatography (TLC), and preparatory high-pressure liquid chromatography (HPLC) for isolation of one compound, based on activity-guided purification using tumorsphere formation assays. The purified compound was identified as betavulgarin, using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI) mass spectrometry. Betavulgarin suppressed the proliferation, migration, colony formation, and mammosphere formation of breast cancer cells and reduced the size of the CD44+/CD24- subpopulation and the expression of the self-renewal-related genes, C-Myc, Nanog, and Oct4. This compound decreased the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3) and reduced the mRNA and protein levels of sex determining region Y (SRY)-box 2 (SOX2), in mammospheres. These data suggest that betavulgarin inhibit the Stat3/Sox2 signaling pathway and induces BCSC death, indicating betavulgarin might be an anticancer agent against breast cancer cells and BCSCs.

Caudatin Isolated from Cynanchum auriculatum Inhibits Breast Cancer Stem Cell Formation via a GR/YAP Signaling.

In the complex tumor microenvironment, cancer stem cells (CSCs), a rare population of cells, are responsible for malignant tumor initiation, metastasis, drug resistance and recurrence. Controlling breast CSCs (BCSCs) using natural compounds is a novel potential therapeutic strategy for clinical cancer treatment. In this study, a mammosphere assay-guided isolation protocol including silica gel, a C18 column, gel filtration, and high-pressure liquid chromatography was used to isolate an inhibitory compound from Cynanchum auriculatum extracts. The isolated inhibitory compound was identified as caudatin. Caudatin inhibited breast cancer cell proliferation, mammosphere formation and tumor growth. Caudatin decreased the CD44+/CD24- and aldehyde dehydrogenase+ cell proportions and the levels of c-Myc, Oct4, Sox2, and CD44. Caudatin induced ubiquitin (Ub)-dependent glucocorticoid receptor (GR) degradation and blocked subsequent Yes-associated protein (YAP) nuclear accumulation and target gene transcription signals in BCSCs. These results show that the GR/YAP signaling pathway regulates BCSC formation and that caudatin may be a potential chemopreventive agent that targets breast cancer cells and CSCs.

Inhibitory Effects of Tangeretin, A Citrus Peel-Derived Flavonoid, on Breast Cancer Stem Cell Formation through Suppression of Stat3 Signaling.

Breast cancer stem cells (BCSCs) are responsible for tumor chemoresistance and recurrence. Targeting CSCs using natural compounds is a novel approach for cancer therapy. A CSC-inhibiting compound was purified from citrus extracts using silica gel, gel filtration and high-pressure liquid chromatography. The purified compound was identified as tangeretin by using nuclear magnetic resonance (NMR). Tangeretin inhibited cell proliferation, CSC formation and tumor growth, and modestly induced apoptosis in CSCs. The frequency of a subpopulation with a CSC phenotype (CD44+/CD24-) was reduced by tangeretin. Tangeretin reduced the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3). Our results in this study show that tangeretin inhibits the Stat3 signaling pathway and induces CSC death, indicating that tangeretin may be a potential natural compound that targets breast cancer cells and CSCs.