Discovery of (-)-epigallocatechin gallate, a novel olfactory receptor 2AT4 agonist that regulates proliferation and apoptosis in leukemia cells.

Olfactory receptors (ORs), the largest family of G protein-coupled receptors (GPCRs), are ectopically expressed in cancer cells and are involved in cellular physiological processes, but their function as anticancer targets is still potential. OR2AT4 is expressed in leukemia cells, influencing the proliferation and apoptosis, yet the limited number of known OR2AT4 agonists makes it challenging to fully generalize the receptor's function. In this study, we aimed to identify new ligands for OR2AT4 and to investigate their functions and mechanisms in K562 leukemia cells. After producing the recombinant OR2AT4 protein, immobilizing it on a surface plasmon resonance chip, and conducting screening to confirm binding activity using 258 chemicals, five novel OR2AT4 ligands were discovered. As a result of examining changes in intracellular calcium by five ligands in OR2AT4-expressing cells and K562 cells, (-)-epigallocatechin gallate (EGCG) was identified as an OR2AT4 agonist in both cells. EGCG reduced the viability of K562 cells and induced apoptosis in K562 cells. EGCG increased the expression of cleaved caspase 3/8 and had no effect on the expression of Bax and Bcl-2, indicating that it induced apoptosis through the extrinsic pathway. Additionally, the initiation of the extrinsic apoptosis pathway in EGCG-induced K562 cells was due to the activation of OR2AT4, using an OR2AT4 antagonist. This study highlights the potential of EGCG as an anti-cancer agent against leukemia and OR2AT4 as a target, making it a new anti-cancer drug.

Isoeugenol Inhibits Adipogenesis in 3T3-L1 Preadipocytes with Impaired Mitotic Clonal Expansion.

Isoeugenol (IEG), a natural component of clove oil, possesses antioxidant, anti-inflammatory, and antibacterial properties. However, the effects of IEG on adipogenesis have not yet been elucidated. Here, we showed that IEG blocks adipogenesis in 3T3-L1 cells at an early stage. IEG inhibits lipid accumulation in adipocytes in a concentration-dependent manner and reduces the expression of mature adipocyte-related factors including PPARγ, C/EBPα, and FABP4. IEG treatment at different stages of adipogenesis showed that IEG inhibited adipocyte differentiation by suppressing the early stage, as confirmed by lipid accumulation and adipocyte-related biomarkers. The early stage stimulates growth-arrested preadipocytes to enter mitotic clonal expansion (MCE) and initiates their differentiation into adipocytes by regulating cell cycle-related factors. IEG arrested 3T3-L1 preadipocytes in the G0/G1 phase of the cell cycle and attenuated cell cycle-related factors including cyclinD1, CDK6, CDK2, and cyclinB1 during the MCE stage. Furthermore, IEG suppresses reactive oxygen species (ROS) production during MCE and inhibits ROS-related antioxidant enzymes, including superoxide dismutase1 (SOD1) and catalase. The expression of cell proliferation-related biomarkers, including pAKT and pERK1/2, was attenuated by the IEG treatment of 3T3-L1 preadipocytes. These findings suggest that it is a potential therapeutic agent for the treatment of obesity.

Correction: Choi et al. Genistin: A Novel Potent Anti-Adipogenic and Anti-Lipogenic Agent. Molecules 2020, 25, 2042.

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Cinnamyl Alcohol Attenuates Adipogenesis in 3T3-L1 Cells by Arresting the Cell Cycle.

Cinnamyl alcohol (CA) is an aromatic compound found in several plant-based resources and has been shown to exert anti-inflammatory and anti-microbial activities. However, the anti-adipogenic mechanism of CA has not been sufficiently studied. The present study aimed to investigate the effect and mechanism of CA on the regulation of adipogenesis. As evidenced by Oil Red O staining, Western blotting, and real-time PCR (RT-PCR) analyses, CA treatment (6.25-25 µM) for 8 d significantly inhibited lipid accumulation in a concentration-dependent manner and downregulated adipogenesis-related markers (peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid binding protein 4 (FABP4), adiponectin, fatty acid synthase (FAS)) in 3-isobutyl-1-methylxanthine, dexamethasone, and insulin(MDI)-treated 3T3-L1 adipocytes. In particular, among the various differentiation stages, the early stage of adipogenesis was critical for the inhibitory effect of CA. Cell cycle analysis using flow cytometry and Western blotting showed that CA effectively inhibited MDI-induced initiation of mitotic clonal expansion (MCE) by arresting the cell cycle in the G0/G1 phase and downregulating the expression of C/EBPß, C/EBPδ, and cell cycle markers (cyclin D1, cyclin-dependent kinase 6 (CDK6), cyclin E1, CDK2, and cyclin B1). Moreover, AMP-activated protein kinase α (AMPKα), acetyl-CoA carboxylase (ACC), and extracellular signal-regulated kinase 1/2 (ERK1/2), markers of upstream signaling pathways, were phosphorylated during MCE by CA. In conclusion, CA can act as an anti-adipogenic agent by inhibiting the AMPKα and ERK1/2 signaling pathways and the cell cycle and may also act as a potential therapeutic agent for obesity.

Discovery of Novel Stimulators of Pax7 and/or MyoD: Enhancing the Efficacy of Cultured Meat Production through Culture Media Enrichment.

The principles of myogenesis play crucial roles in the production of cultured meat, and identifying protein stimulators associated with myogenesis holds great potential to enhance the efficiency of this process. In this study, we used surface plasmon resonance (SPR)-based screening of a natural product library to discover ligands for Pax7 and MyoD, key regulators of satellite cells (SCs), and performed cell-based assays on Hanwoo SCs (HWSCs) to identify substances that promote cell proliferation and/or differentiation. Through an SPR analysis, we found that six chemicals, including one Pax7+/MyoD- chemical, four Pax7+/MyoD+ chemicals, and one Pax7-/MyoD+ chemical, bound to Pax7 and/or MyoD proteins. Among four Pax7+/MyoD+ chemicals, parthenolide (0.5 and 1 µM) and rutin (100 and 200 µM) stimulated cell proliferation in the medium with 10% FBS similar to the medium with 20% FBS, without affecting differentiation. Adenosine, a Pax7-/MyoD+ chemical, accelerated differentiation. These chemicals could be potential additives to reduce the reliance of FBS required for HWSC proliferation and differentiation in cultured meat production.

Discovery of Orphan Olfactory Receptor 6M1 as a New Anticancer Target in MCF-7 Cells by a Combination of Surface Plasmon Resonance-Based and Cell-Based Systems.

Olfactory receptors (ORs) account for 49% of all G protein-coupled receptors (GPCRs), which are important targets for drug discovery, and hence ORs may also be potential drug targets. Various ORs are expressed in breast cancer cells; however, most of them are orphan receptors, and thus, their functions are unknown. Herein, we present an experimental strategy using a surface plasmon resonance (SPR) system and a cell-based assay that allowed the identification of orphan OR6M1 as a new anticancer target in the MCF-7 breast cancer cell line. After the construction of stable OR6M1-expressing cells, the SPR-based screening of 108 chemicals for ligand activity was performed against OR6M1-expressing whole cells (primary screening) or membrane fragments (secondary screening). As a result, anthraquinone (AQ) and rutin were discovered to be new OR6M1 ligands. Based on calcium imaging in OR6M1-expressing Hana3A cells, AQ and rutin were classified as an OR6M1 agonist and antagonist, respectively. Cell viability and live/dead assays showed that AQ induced the death of MCF-7 cells, which was inhibited by rutin. Therefore, OR6M1 may be considered an anticancer target, and AQ may be considered a chemotherapeutic agent. This combined method can be widely used to discover the ligands and functions of other orphan GPCRs.

Genistin: A Novel Potent Anti-Adipogenic and Anti-Lipogenic Agent.

Soy isoflavones are popular ingredients with anti-adipogenic and anti-lipogenic properties. The anti-adipogenic and anti-lipogenic properties of genistein are well-known, but those of genistin and glycitein remain unknown, and those of daidzein are characterized by contrasting data. Therefore, the purpose of our study was to investigate the effects of daidzein, glycitein, genistein, and genistin on adipogenesis and lipogenesis in 3T3-L1 cells. Proliferation of 3T3-L1 preadipocytes was unaffected by genistin and glycitein, but it was affected by 50 and 100 µM genistein and 100 µM daidzein for 48 h. Among the four isoflavones, only 50 and 100 µM genistin and genistein markedly suppressed lipid accumulation during adipogenesis in 3T3-L1 cells through a similar signaling pathway in a dose-dependent manner. Genistin and genistein suppress adipocyte-specific proteins and genes, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and adipocyte binding protein 2 (aP2)/fatty acid-binding protein 4 (FABP4), and lipogenic enzymes such as ATP citrate lyase (ACL), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FAS). Both isoflavones also activate AMP-activated protein kinase α (AMPKα), an essential factor in adipocyte differentiation, and inhibited sterol regulatory element-binding transcription factor 1c (SREBP-1c). These results indicate that genistin is a potent anti-adipogenic and anti-lipogenic agent.

Suppressive Effect of Arctium Lappa L. Leaves on Retinal Damage Against A2E-Induced ARPE-19 Cells and Mice.

Age-related macular degeneration (AMD) is a major cause of irreversible loss of vision with 80-90% of patients demonstrating dry type AMD. Dry AMD could possibly be prevented by polyphenol-rich medicinal foods by the inhibition of N-retinylidene-N-retinylethanolamine (A2E)-induced oxidative stress and cell damage. Arctium lappa L. (AL) leaves are medicinal and have antioxidant activity. The purpose of this study was to elucidate the protective effects of the extract of AL leaves (ALE) on dry AMD models, including in vitro A2E-induced damage in ARPE-19 cells, a human retinal pigment epithelial cell line, and in vivo light-induced retinal damage in BALB/c mice. According to the total phenolic contents (TPCs), total flavonoid contents (TFCs) and antioxidant activities, ALE was rich in polyphenols and had antioxidant efficacies on 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and 2',7'-dichlorofluorescin diacetate (DCFDA) assays. The effects of ALE on A2E accumulation and A2E-induced cell death were also monitored. Despite continued exposure to A2E (10 µM), ALE attenuated A2E accumulation in APRE-19 cells with levels similar to lutein. A2E-induced cell death at high concentration (25 µM) was also suppressed by ALE by inhibiting the apoptotic signaling pathway. Furthermore, ALE could protect the outer nuclear layer (ONL) in the retina from light-induced AMD in BALB/c mice. In conclusion, ALE could be considered a potentially valuable medicinal food for dry AMD.