Taichi Hara

Objective: Garlic exerts protective effects against skin stress, including oxidative damage and trauma. Iodized garlic extract (IGEx) is a cosmetic ingredient developed through iodination of garlic odor components to achieve odor reduction. Although IGEx is expected to exhibit skin stress protection properties similar to garlic, the molecular mechanisms underlying the effects of IGEx on skin cells remain unclear. Therefore, this study comprehensively analyzed the effects of IGEx on human epidermal keratinocytes (HaCaT cells), with a particular focus on the stress response mechanisms. Methods: Transcriptome analysis was conducted on HaCaT cells treated with IGEx. Nrf2 protein expression was assessed by western blotting, whereas target gene expression was evaluated by qRT‐PCR. Dehydrogenase activity was measured using Cell Counting Kit‐8. Functional analyses included hydrogen peroxide‐induced oxidative stress testing and in vitro wound healing assays, and pathway specificity was validated using Nrf2 knockout cells. Results: IGEx increased Nrf2 protein expression 1.62‐fold and induced the expression of downstream target genes, including HMOX1 , AKR1C1 , and AKR1C3 . Additionally, intracellular NAD(P)H‐dependent dehydrogenase activity was enhanced in an Nrf2‐dependent manner. Furthermore, IGEx demonstrated protective effects against hydrogen peroxide toxicity and promoted wound healing. Conclusion: IGEx activated Nrf2 and regulated stress responses in HaCaT cells. The control of intracellular NAD(P)H‐dependent dehydrogenase activity has been suggested to contribute to these effects. Additionally, the involvement of AHR was indicated, suggesting the coordinated regulation of Nrf2 in stress responses. This study provides a molecular foundation for the potential applications of IGEx as a cosmetic ingredient.

Novel anticancer agents are needed to overcome therapeutic resistance, and autophagy represents a promising target. The chlorinated alkylphenones Dictyostelium differentiation-inducing factors 1 and 3 (DIF-1 and DIF-3) and their derivatives exert anti-proliferative and anti-metastatic effects; however, their molecular mechanisms remain incompletely understood. Using the GFP-LC3-RFP-LC3ΔG autophagic flux reporter, we identified Bu-DIF-3 and CP-DIF-3 as autophagy-promoting derivatives and characterized their actions principally in HeLa cells, with selected autophagy and AMP-activated protein kinase (AMPK) measurements in HAP1 cells. Both suppressed mTORC1 signaling, activated AMPK, and reduced mitochondrial membrane potential (MMP). Bu-DIF-3 additionally induced reactive oxygen species and redox stress response genes, including SLC7A11 and CHAC1. SLC7A11 induction may reflect a compensatory antioxidant response. Unlike CP-DIF-3, Bu-DIF-3 increased the FerroOrange-detectable labile Fe²⁺ signal, and deferoxamine partially restored the reduced MTT signal, supporting possible iron involvement in reduced metabolic activity. Thus, modification of the DIF-3 methoxy group modulates autophagy-promoting activity and associated stress responses. Bu-DIF-3 was associated with oxidative stress and altered labile Fe²⁺ homeostasis; whether a ferroptosis-related mechanism contributes remains unresolved. These findings provide a cellular basis for further evaluation of Bu-DIF-3 and related DIF derivatives.

Modulating autophagy has emerged as a potential strategy for treating age-related diseases. However, commonly used pharmacological approaches to induce autophagy, particularly inhibition of mechanistic target of rapamycin complex 1 (mTORC1), can be associated with adverse effects, including immunosuppression and insulin resistance. This has prompted interest in autophagy modulators that act without directly inhibiting mTORC1. 2,5-Diketopiperazines (DKPs) are bioactive cyclic dipeptide scaffolds with diverse biological activities. However, systematic evaluation of their structure-activity relationships has been hindered by racemization during conventional chemical synthesis, leaving the contribution of stereochemistry to autophagy regulation poorly understood. Here, we used a stereoselective one-pot chemoenzymatic synthesis based on the adenylation domain of tyrocidine synthetase A to generate a DKP library with defined stereochemistry. Phenotypic screening in Caco-2 cells stably expressing the GFP-LC3-RFP autophagic flux probe identified four DKPs that increased autophagic flux: c(DW-DP), c(DW-LP), c(DF-DP), and c(DM-LP). Structure-activity analysis revealed stereochemistry-dependent effects associated with amino acid side-chain properties: D-configured residues were favored among DKPs containing aromatic amino acids or methionine, whereas L-configured residues were favored among those containing branched-chain amino acids. Substitution of the proline residue further altered activity, with glycine substitution tending to increase autophagic flux in some DKP scaffolds. Importantly, the active DKPs did not detectably reduce the phosphorylation of the mTORC1 downstream targets p70 S6K and 4EBP1, indicating that their autophagy-inducing effects do not require detectable suppression of canonical mTORC1 signaling. These findings establish stereochemically defined DKPs as candidate scaffolds for the development of autophagy inducers that act through mechanisms distinct from direct mTORC1 inhibition.

Autophagy is an intracellular recycling system that contributes to the maintenance of cellular homeostasis. However, by providing tolerance to various stressors, autophagy promotes the survival and proliferation of cancer cells and confers resistance to chemotherapy. Anticancer drugs activate autophagy, leading to drug resistance in cancer cells. Autophagy inhibitors, such as chloroquine and hydroxychloroquine, enhance the antitumor effects of anticancer drugs when used in combination with them. However, these inhibitors are associated with several adverse effects. Here, using genome‐wide RNA sequencing analysis, we deciphered a novel function of electrolyzed hydrogen water (EHW) in suppressing autophagy by activating mammalian target of rapamycin complex 1 signalling. The combination of EHW with anticancer drugs, such as 5‐fluorouracil or paclitaxel, which activate autophagy, significantly decreased the viability of cervical and colorectal cancer cells. Mechanistically, molecular hydrogen and trace elements in EHW may suppress autophagy and potentiate anticancer effects. Considering its safety, we propose that EHW can act as a novel adjuvant to anticancer therapy.

Spermine (SPM) and spermidine (SPD) are polyamines found in all organisms, and their concentrations can be regulated by ingestion. We demonstrated that yeast‐fermented garlic (YF) extract significantly increased autophag flux in OUMS‐36T‐1 and HeLa cells expressing the fluorescent probe (GFP‐LC3‐RFP‐LC3ΔG). YF‐induced increase of autophagy occurred independently of mTORC1 signaling, and RNA‐sequencing analysis revealed that EGR1 was the most significantly altered gene in YF‐treated OUMS‐36T‐1 cells. YF‐treated EGR1‐deficient HAP1 cells displayed reduced autophagic flux (p < 0.05). YF‐induced increasing of autophagic flux occurred via a specific SPM/SPD ratio. HAP1 cells treated with equivalent amounts of SPD or SPM as that found in YF did not increase autophagic flux (p > 0.05); however, treatment with SPD and SPM in the same ratio as that found in YF increased autophagic flux (p < 0.05). This specific SPM/SPD ratio reduced MG132‐induced proteostress via EGR1‐dependent pathways (p < 0.05). Thus, the SPM/SPD balance may regulate autophagy via EGR1‐dependent pathways, and controlling this balance may provide a strategy to maintain cellular homeostasis.

Strawberry (Fragaria × ananassa) is a fruit that is rich in bioactive components and nutrients. A previous study revealed that strawberries offer beauty benefits, including anti-inflammatory effects. In this study, we investigated the potential hair growth-promoting properties of strawberries by measuring dehydrogenase activity as an indicator of cellular metabolism, and assessed the expression of hair growth-related genes in human follicle dermal papilla cells (HFDPC). Additionally, we performed chemical analyses to characterize the active compounds contributing to the observed effects. The results indicated that strawberry water-extract promotes the metabolism of HFDPC, and that this effect was reduced with heat treatment. The expression levels of hair growth-related genes (FGF7, VCAN, and TGFB2) were examined, but no significant differences were detected. Although further investigation into the components and mechanisms underlying this bioactivity is required, these results lay a foundation for the development of hair-care cosmetics.

Ultraviolet A (UVA) radiation can pass through the epidermis and reach the dermal skin layer, contributing to photoaging, DNA damage, and photocarcinogenesis in dermal fibroblasts. High-dose UVA exposure induces erythema, whereas low-dose, long-term UVA exposure causes skin damage and cell senescence. Biomarkers for evaluating damage caused by low-dose UVA in fibroblasts are lacking, making it difficult to develop therapeutic agents for skin aging and aging-associated diseases. We performed RNA-sequencing to investigate gene and pathway alterations in low-dose UVA-irradiated human skin-derived NB1RGB primary fibroblasts. Differentially expressed genes were identified and subjected to Gene Ontology and reactome pathway analysis, which revealed enrichment in genes in the senescence-associated secretory phenotype, apoptosis, respiratory electron transport, and transcriptional regulation by tumor suppressor p53 pathways. Insulin-like growth factor binding protein 7 (IGFBP7) showed the lowest p-value in RNA-sequencing analysis and was associated with the senescence-associated secretory phenotype. Protein–protein interaction analysis revealed that Fos proto-oncogene had a high-confidence network with IGFBP7 as transcription factor of the IGFBP7 gene among SASP hit genes, which were validated using RT-qPCR. Because of their high sensitivity to low-dose UVA radiation, Fos and IGFBP7 show potential as biomarkers for evaluating the effect of low-dose UVA radiation on dermal fibroblasts.

Excessive alcohol consumption can cause multi-systemic diseases. Among them, alcoholic liver disease is the most frequent and serious disease. Electrolytic hydrogen water (EHW) is produced at the cathode during electrolysis of water and contains a large amount of molecular hydrogen and a low content of platinum nanoparticles with alkaline properties. In this study, we found that EHW inhibits ethanol-induced cytotoxicity by decreasing the intracellular acetaldehyde, a toxic substance produced by ethanol degradation, in hepatocyte cell lines HepG2. Analysis of the mechanism of action revealed that EHW inhibits the metabolism of ethanol to acetaldehyde by suppressing alcohol dehydrogenase. EHW also promotes the metabolism of acetaldehyde to acetic acid by activating aldehyde dehydrogenase, which plays to reduce aldehyde toxicity and intracellular reactive oxygen species in HepG2 cells. These functions were correlated with the concentration of molecular hydrogen in EHW, and were abolished by degassing treatment, suggesting that molecular hydrogen may contribute as a functional factor in the suppression of ethanol-induced hepatocellular damage. Furthermore, hydrogen water with high dissolved hydrogen molecule showed the same hepatocellular protective effect against ethanol as the EHW. These results suggest that EHW may be useful in the prevention of alcoholic liver disease.