Takahiro Oyama

Psoriasis is a chronic inflammatory skin disease characterized by erythema, infiltration, and scaling, which is mainly caused by interleukin (IL)-17. The use of molecular targeted drugs in specific therapies offers high efficacy; however, high medical costs and a significant risk of side effects highlight the need for novel therapeutic agents. We previously observed that Morus alba extract (MAE) suppressed IL-17-induced CCL20 mRNA expression in normal human epidermal keratinocytes (NHEKs). In this study, we focused on the IL-17 signaling pathway and investigated the effects of pentacyclic triterpenoids, betulinic acid (BA), and ursolic acid (UA), which are present in MAE, on NHEK cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) revealed that both BA and UA suppressed CCL20 expression, while only UA alone inhibited CCL20 release. ELISA using specific inhibitors demonstrated that both the p38 and extracellular-signal-regulated kinase 1/2 (ERK1/2) pathways were crucial for IL-17-induced CCL20 release in NHEK. UA effectively suppressed ERK1/2 nuclear localization and moderately affected p38 phosphorylation. These results indicated that UA is a potential seed compound for psoriasis treatment through its targeting of the IL-17 pathway.

Numerous natural extracts and compounds have been evaluated for their ability to mitigate the adverse effects of ultraviolet (UV) overexposure. However, variability in the UV doses that trigger biological responses across studies likely arises from inconsistencies in UV exposure standardization. We hypothesize that these discrepancies are due to variations in culture plates and dishes. The UV dose (D) required to reduce cell viability by 50% differed by a factor of ten between 3.5 cm dishes and 96-well plates. Similarly, the EC₅₀ dose for IL-6 release (D_1/2) varied, potentially correlating with the surface area (S). UV exposure to wells with increasing height in 3.5 cm dishes resulted in a decrease in IL-6 release, suggesting that the greater the well height, the more it may influence UV exposure through reflection or shielding effects, thereby contributing to the physiological effects on the cells. To compare these differences among plates, we defined the height-to-diameter ratio (r). Analysis revealed a linear correlation between D_1/2 and S in a log-log plot, and between D_1/2 and r in a semi-log plot. From this, we defined two empirical indices σ and ρ for UV dose adjustment. A deductive model was also developed to derive a D′ value that adjusts UV doses without requiring training. As with σ and ρ, the UV dose D was effectively adjusted using D′ as well. These attempts suggest that D′ offers a foundational framework for evaluating UVB effects on cultured cells.

Nicotinamide adenine dinucleotide (NAD⁺) plays a pivotal role in various physiological processes within mammalian cells, including energy metabolism, redox homeostasis, and genetic regulation. In the majority of mammalian cellular contexts, NAD⁺ biosynthesis primarily relies on vitamin B3, including nicotinamide (NAM) and nicotinic acid (NA). The concept of NAD⁺ augmentation therapy has recently emerged as a promising strategy to mitigate aging-associated phenomena, termed rejuvenation. Despite the involvement of diverse enzymatic cascades in NAD⁺ biosynthesis, certain cellular environments exhibit deficiencies in specific enzymes, suggesting cell type-dependent variability in optimal NAD⁺ precursor selection. However, the optimization of NAD⁺ precursors for topical formulations has received scant attention thus far. In the present investigation, we sought to delineate the most efficacious precursor for augmenting NAD⁺ levels in human skin keratinocytes. Remarkably, NA supplementation led to a significant 1.3-fold elevation in intracellular NAD⁺ levels, even in the presence of nicotinamide phosphoribosyltransferase inhibition by FK866. Additionally, NA mononucleotide demonstrated a 1.5-fold increase (but not significant) in NAD⁺ levels following 100 μM application. Conversely, NAM and its derivatives failed to elicit a NAD⁺ response in keratinocytes. Notably, NA supplementation elicited up-regulation of mitochondrial superoxide dismutase (SOD2) and sirtuin 3 (SIRT3), indicative of its beneficial impact on mitochondrial function. Furthermore, NA mitigated rotenone-induced mitochondrial reactive oxygen species (ROS) accumulation. Collectively, these findings advocate for the potential utility of NA in topical applications aimed at skin rejuvenation.

Nicotinamide dinucleotide (NAD⁺) is an important component for various biological processes in mammalian cells, such as energy production, redox state maintenance, and gene regulation. In most mammalian cells, NAD⁺ is produced by vitamin B3, including nicotinamide (NAM) and nicotinic acid (NA). Recently, NAD⁺ up-regulation therapy has attracted attention for suppressing the aging processes, called rejuvenation. Although various enzymes participate in the NAD⁺ production pathway, some enzymes are lacking in particular cells. Therefore, it is thought that the suitable material for NAD⁺ production varies with the types of cells. However, the optimization of the NAD⁺-precursor for use in topical formulations has rarely been considered. In this study, we asked which precursor is suitable for application against human skin keratinocytes. As a result, NA supplementation 1.3-fold up-regulated intracellular NAD⁺ level significantly, even with a nicotinamide phosphoribosyltransferase inhibitor, FK866, and its metabolites NA mononucleotide also increased NAD⁺ level by1.5-fold with 100 μM application. Surprisingly, NAM and its derivatives could not up-regulate cellular NAD⁺ levels in keratinocytes. The NA supplementation also up-regulated mitochondrial superoxide dismutase (SOD2), which indicates the effect for mitochondria. NA also alleviated rotenone-induced mitochondrial ROS accumulation. These results suggest that NA can be used for topical application for skin rejuvenation.

The current anti-cancer treatments are not enough to eradicate tumors, and therefore, new modalities and strategies are still needed. Most tumors generate an inflammatory tumor microenvironment (TME) and maintain the niche for their development. Because of the critical role of inflammation via high-mobility group box 1 (HMGB1)–receptor for advanced glycation end-products (RAGE) signaling pathway in the TME, a novel compound possessing both anti-cancer and anti-inflammatory activities by suppressing the HMGB1-RAGE axis provides an effective strategy for cancer treatment. A recent work of our group found that some anti-cancer 3-styrylchromones have weak anti-inflammatory activities via the suppression of this axis. In this direction, we searched such anti-cancer molecules possessing potent anti-inflammatory activities and discovered 7-methoxy-3-hydroxy-styrylchromone (C6) having dual suppressive activities. Mechanism-of-action studies revealed that C6 inhibited the increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) under the stimulation of HMGB1-RAGE signaling and thereby suppressed cytokine production in macrophage-like RAW264.7 cells. On the other hand, in colorectal cancer HCT116 cells, C6 inhibited the activation of ERK1/2, cyclin-dependent kinase 1, and AKT, down-regulated the protein level of XIAP, and up-regulated pro-apoptotic Bax and caspase-3/7 expression. These alterations are suggested to be involved in the C6-induced suppression of cell cycle/proliferation and initiation of apoptosis in the cancer cells. More importantly, in cancer cells, the treatment of C6 potentiates the anti-cancer effects of DNA-damaging agents. Thus, C6 may be a promising lead for the generation of a novel class of cancer therapeutics.

Background: β-thujaplicin, a natural tropolone derivative, has anticancer effects on various cancer cells via apoptosis. However, the apoptosis regulatory proteins involved in this process have yet to be revealed. Methods: Trypan blue staining, a WST-8 assay, and a caspase-3/7 activity assay were used to investigate whether β-thujaplicin sensitizes cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Additionally, western blotting was performed to clarify the effects of β-thujaplicin on X-linked inhibitor of apoptosis protein (XIAP) in NCI-H460 cells and a fluorescence polarization binding assay was used to evaluate the binding-inhibitory activity of β-thujaplicin against XIAP-BIR3. Results: β- and γ-thujaplicins decreased the viability of NCI-H460 cells in a dose-dependent manner; they also sensitized the cells to TRAIL-induced cell growth inhibition and apoptosis. β-thujaplicin significantly potentiated the apoptosis induction effect of TRAIL on NCI-H460 cells, which was accompanied by enhanced caspase-3/7 activity. Interestingly, β-thujaplicin treatment in NCI-H460 cells decreased XIAP levels. Furthermore, β-thujaplicin was able to bind XIAP-BIR3 at the Smac binding site. Conclusions: These findings indicate that β-thujaplicin could enhance TRAIL-induced apoptosis in NCI-H460 cells via XIAP inhibition and degradation. Thus, the tropolone scaffold may be useful for designing novel nonpeptidic small-molecule inhibitors of XIAP and developing new types of anticancer drugs.

Background: High mobility group box 1 (HMGB1)-receptor for advanced glycation endo-products (RAGE) axis serves as a key player in linking inflammation and carcinogenesis. Recently, papaverine was revealed to suppress the HMGB1-RAGE inflammatory signaling pathway and cancer cell proliferation. Therefore, a dual suppressor targeting this axis is expected to become a new type of therapeutic agent to treat cancer. Methods: Papaverine 3D pharmacophore mimetic compounds were selected by the LigandScout software from our in-house, anti-cancer chemical library and assessed for their anti-inflammatory activities by a HMGB1-RAGE-mediated interleukin-6 production assay using macrophage-like RAW264.7 cells. Molecular-biological analyses, such as Western blotting, were performed to clarify the mechanism of action. Results: A unique 6-methoxy-3-hydroxy-styrylchromone was found to possess potent anti-inflammatory and anti-cancer activities via the suppression of the HMGB1-RAGE-extracellular signal-regulated kinase 1/2 signaling pathway. Furthermore, the 3D pharmacophore-activity relationship analyses revealed that the hydroxyl group at the C4′ position of the benzene ring in a 3-styryl moiety was significant in its dual suppressive effects. Conclusions: These findings indicated that this compound may provide a valuable scaffold for the development of a new type of anti-cancer drug possessing anti-inflammatory activity and as a tool for understanding the link between inflammation and carcinogenesis.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is an attractive therapeutic strategy for targeting cancer metabolism. So far, many potent NAMPT inhibitors have been developed and shown to bind to two unique tunnel-shaped cavities existing adjacent to each active site of a NAMPT homodimer. However, cytotoxicities and resistances to NAMPT inhibitors have become apparent. Therefore, there remains an urgent need to develop effective and safe NAMPT inhibitors. Thus, we designed and synthesized two close structural analogues of NAMPT inhibitors, azaindole–piperidine (3a)- and azaindole–piperazine (3b)-motif compounds, which were modified from the well-known NAMPT inhibitor FK866 (1). Notably, 3a displayed considerably stronger enzyme inhibitory activity and cellular potency than did 3b and 1. The main reason for this phenomenon was revealed to be due to apparent electronic repulsion between the replaced nitrogen atom (N1) of piperazine in 3b and the Nδ atom of His191 in NAMPT by our in silico binding mode analyses. Indeed, 3b had a lower binding affinity score than did 3a and 1, although these inhibitors took similar stable chair conformations in the tunnel region. Taken together, these observations indicate that the electrostatic enthalpy potential rather than entropy effects inside the tunnel cavity has a significant impact on the different binding affinity of 3a from that of 3b in the disparate enzymatic and cellular potencies. Thus, it is better to avoid or minimize interactions with His191 in designing further effective NAMPT inhibitors.