You-Cheng Hseu

Zerumbone (Zer), a natural sesquiterpene of Zingiber zerumbet , exerts apoptosis in human melanoma cells. We evaluated the anticancer efficacies and the underlying molecular mechanisms of Zer through autophagic cell death in both in vitro melanoma (A375 and/or A2058) cells and in vivo xenografted athymic nude mice. We found that Zer inhibited cell growth and EGFR/BRAF expression in A375 and A2058 cells. Additionally, Zer‐induced autophagy was apparent from enhanced LC3‐II accumulation, p62 expression, ATG4B reduction, Beclin‐1/Bcl‐2 ratio, AKT/mTOR suppression, and AVO production in A375 and A2058 cells. Surprisingly, Zer‐triggered cell death was prevented by autophagic inhibitors 3‐MA and CQ, suggesting the contribution of autophagy as a death mechanism. TEM data displayed that Zer provoked autophagosome generation in A375 and A2058 cells. However, obstruction of autophagy by 3‐MA or CQ, and LC3 silencing curbed Zer‐incited apoptosis in A375 cells, which suggests autophagy induction enhanced apoptosis in Zer‐treated A375 cells. Interestingly, reduction of apoptosis by caspase blocker Z‐VAD‐FMK enhanced Zer‐provoked autophagy in A375 cells, indicating induction of apoptosis inhibited autophagy in Zer‐treated A375 cells. Moreover, Zer instigated intracellular ROS generation, whereas antioxidant NAC or Mito‐Tempo reversed autophagic or apoptotic cell death in A375 and/or A2058 cells. In vivo results displayed that Zer significantly inhibited tumor development in A375 and A2058‐xenografted athymic nude mice. Our findings implied that Zerumbone is a potent antitumor agent that triggered ROS‐mediated autophagic cell death in human melanoma cells.

Objective: We investigated skin hydration and anti‐ageing efficacies of Ectoine, a natural bacterial osmolyte, utilising human keratinocyte (HaCaT) and fibroblast (Hs68) cells under non‐ or UVB (30 mJ/cm ² ) exposure. Methods: We incorporated various experimental techniques, including MTT assay (cell viability), small interfering RNA (siRNA) transfection, and immunoblotting analysis, to assess the molecular mechanisms of skin hydration and anti‐ageing efficacies of Ectoine. Results: The findings exhibited that Ectoine improved cell viability in the presence or absence of UVB exposure in HaCaT cells. Ectoine upregulated Src and HAS‐2 expression in the presence or absence of UVB irradiation in HaCaT cells. Src knockdown reduced Ectoine‐increased HAS‐2 expression, implying that Ectoine provoked Src‐mediated HAS‐2 expression in the presence of or without UVB irradiation in HaCaT cells. Ectoine enhanced phosphorylated‐ERK expression in a time‐ and dose‐dependent manner in HaCaT cells. In addition, Ectoine increased phosphorylated‐ERK expression in UVB‐irradiated HaCaT cells. The ERK inhibitor (PD98059) remarkably decreased Ectoine‐provoked HAS‐2 expression, suggesting that Ectoine triggered ERK‐mediated HAS‐2 expression in HaCaT cells. Furthermore, Ectoine amplified JNK and AQP‐3 levels in the presence or lack of UVB exposure in HaCaT cells. The JNK inhibitor (SP600125) significantly reduced Ectoine‐triggered AQP‐3 expression, suggesting that Ectoine provoked JNK‐mediated AQP‐3 expression in HaCaT cells. Ectoine increased collagen‐I expression in the presence or lack of UVB irradiation in HaCaT cells. Notably, Ectoine enhanced collagen‐I expression and inhibited MMP‐1 expression in a dose‐dependent manner in fibroblast (Hs68) cells. Conclusion: We demonstrated that Ectoine exerts skin hydration effects without or with UVB exposure in human skin keratinocyte (HaCaT) cells and anti‐ageing in fibroblast (Hs68) cells. Therefore, Ectoine could serve as a potential natural compound in cosmetic preparations for skin hydration and anti‐ageing.

Dihydromikanolide (DHK) is a natural product in Mikania species. We examined the anti‐inflammatory molecular mechanisms of DHK employing in vitro RAW264.7 macrophages and in vivo BALB/c mice under LPS/ATP stimulation. We found that DHK suppressed NLRP3 inflammasome, procaspase‐1 activation and then pro‐inflammatory IL1β expression in LPS/ATP‐stimulated RAW264.7 cells. Notably, DHK‐triggered autophagy in RAW264.7 cells was demonstrated by increased LC3‐II accumulation, p62/SQSTM1 expression, Beclin‐1/Bcl‐2 ratio and PI3K/AKT/mTOR phosphorylation. Besides, DHK increased Parkin and Pink‐1 protein expressions implying mitophagy induction in RAW264.7 cells. Interestingly, DHK enhanced Nrf2 nuclear translocation and provoked antioxidant HO‐1, NQO‐1 and γ‐GCLC expressions in RAW264.7 cells. Nrf2 knockdown reversed DHK‐inhibited LPS/ATP‐stimulated IL1β expression in RAW264.7 cells. Interestingly, LPS/ATP‐stimulated NLRP3 inflammasome and IL1β expression were inhibited by DHK, Mito‐TEMPO (a mitochondrial ROS inhibitor), or N‐acetylcysteine (a ROS inhibitor). In vivo study revealed that DHK attenuated wet/dry weight ratio of lung tissue, lung neutrophil intrusions and pulmonary oedema, and reduced the increased total cells, neutrophils, TNFα and IL1β expression in bronchoalveolar lavage fluid (BALF) in LPS‐stimulated BALB/c mice. DHK alleviated LPS‐induced pathological alterations of lung through inhibiting NLRP3 inflammation, enhancing antioxidant Nrf2 pathway and inducing mitophagy in LPS‐stimulated BALB/c mice. Dihydromikanolide may be a potential therapeutic agent for inflammatory diseases.

Ectoine, a natural bacterial osmolyte, suppressed UVA irradiated‐α‐melanocyte stimulating hormone (MSH) stimulated melanogenesis through antioxidant Nrf2 pathways in human keratinocytes; however, the underlying skin whitening mechanisms were not elucidated. The depigmenting efficiency of Ectoine (0–400 μM) through antimelanogenesis and melanin degradation by autophagy promotion was investigated in melanoma (B16F10) and melanin‐feeding keratinocyte (HaCaT) cells and in vivo zebrafish model. MTT assay, Western blotting, GFP‐LC3 puncta, AVO formation, melanin assay, immunofluorescence staining, TEM techniques, siLC3 transfection, and zebrafish model were utilized. Ectoine‐induced autophagy in B16F10 and HaCaT cells was shown by enhanced LC3‐II accumulation, autophagosome GFP‐LC3 puncta, autolysosome AVOs formation, ATG4B downregulation, and Beclin‐1/Bcl‐2 dysregulation. The immunoprecipitation data revealed that Ectoine increased the association between LC3‐II and p62 proteins in B16F10 and HaCaT cells. Importantly, antioxidant NAC pretreatment antagonized the Ectoine‐induced ATG4B diminution in B16F10 and HaCaT cells. Ectoine inhibited melanogenesis by suppressing melanosome gp100, tyrosinase, TRP‐1/‐2, and/or melanin formation via autophagy in α‐MSH‐stimulated B16F10 and melanin‐feeding HaCaT cells. TEM findings displayed that Ectoine increased melanosome‐engulfing autophagosomes and autolysosomes in α‐MSH‐stimulated B16F10 and melanin‐feeding HaCaT cells. Ectoine‐inhibited melanogenesis in α‐MSH‐stimulated B16F10 cells and melanin‐feeding HaCaT cells was reversed by pretreatment with the autophagy inhibitor 3‐MA or LC3 silencing. In vivo study demonstrated that Ectoine (5 mM) suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. The in vitro and in vivo study demonstrated that Ectoine inhibits melanogenesis and enhances melanin degradation by triggering autophagy. Ectoine could be utilized as a whitening ingredient in cosmetic formulations.

Naringin, a bioflavonoid compound from grapefruit or citrus, exerts anticancer activities on cervical, thyroid, colon, brain, liver, lung, thyroid, and breast cancers. The present investigation addressed exploring the anticancer effects of naringin on nasopharyngeal carcinoma (NPC) cells. Naringin exhibits a cytotoxic effect on NPC‐TW 039 and NPC‐TW 076 cells with IC ₅₀ 372/328 and 394/307 μM for 24 or 48 h, respectively, while causing little toxicity toward normal gingival epithelial (SG) cells (>500/500 μM). We established that naringin triggered G ₁ arrest is achieved by suppressing cyclin D1, cyclin A, and CDK2, and upregulating p21 protein in NPC cells. Exposure of NPC cells to naringin caused a series of events leading to apoptosis including morphology change (cell shrinkage and membrane blebbing) and chromatin condensation. Annexin V and PI staining indicated that naringin treatment promotes necrosis and late apoptosis in NPC cells. DiOC ₆ staining showed a decline in the mitochondrial membrane potential by naringin treatment, which was followed with cytochrome c release, Apaf‐1/caspase‐9/‐3 activation, PARP cleavage, and EndoG expression in NPC cells. Naringin upregulated proapoptotic Bax and decreased antiapoptotic Bcl‐xL expression, and dysregulated Bax/Bcl‐xL ratio in NPC cells. Notably, naringin enhanced death receptor‐related t‐Bid expression. Furthermore, an increased Ca ²⁺ release by naringin treatment which instigated endoplasmic reticulum stress‐associated apoptosis through increased IRE1, ATF‐6, GRP78, GADD153, and caspase‐12 expression in NPC cells. In addition, naringin triggers ROS production, and inhibition of naringin‐induced ROS generation by antioxidant N ‐acetylcysteine resulted in the prevention of G ₁ arrest and apoptosis in NPC cells. Naringin‐induced ROS‐mediated G ₁ arrest and mitochondrial‐, death receptor‐, and endoplasmic reticulum stress–mediated apoptosis may be a promising strategy for treating NPC.

Coenzyme Q₀ (CoQ₀), a quinone derivative from Antrodia camphorata, has antitumor capabilities. This study investigated the antitumor effect of noncytotoxic CoQ₀, which included NLRP3 inflammasome inhibition, anti-EMT/metastasis, and metabolic reprogramming via HIF-1α inhibition, in HNSCC cells under normoxia and hypoxia. CoQ₀ suppressed hypoxia-induced ROS-mediated HIF-1α expression in OECM-1 and SAS cells. Under normoxia and hypoxia, the inflammatory NLRP3, ASC/caspase-1, NFκB, and IL-1β expression was reduced by CoQ₀. CoQ₀ reduced migration/invasion by enhancing epithelial marker E-cadherin and suppressing mesenchymal markers Twist, N-cadherin, Snail, and MMP-9, and MMP-2 expression. CoQ₀ inhibited glucose uptake, lactate accumulation, GLUT1 levels, and HIF-1α-target gene (HK-2, PFK-1, and LDH-A) expressions that are involved in aerobic glycolysis. Notably, CoQ₀ reduced ECAR as well as glycolysis, glycolytic capability, and glycolytic reserve and enhanced OCR, basal respiration, ATP generation, maximal respiration, and spare capacity in OECM-1 cells. Metabolomic analysis using LC-ESI-MS showed that CoQ₀ treatment decreased the levels of glycolytic intermediates, including lactate, 2/3-phosphoglycerate, fructose 1,6-bisphosphate, and phosphoenolpyruvate, and increased the levels of TCA cycle metabolites, including citrate, isocitrate, and succinate. HIF-1α silencing reversed CoQ₀-mediated anti-metastasis (N-Cadherin, Snail, and MMP-9) and metabolic reprogramming (GLUT1, HK-2, and PKM-2) under hypoxia. CoQ₀ prevents cancer stem-like characteristics (upregulated CD24 expression and downregulated CD44, ALDH1, and OCT4) under normoxia and/or hypoxia. Further, in IL-6-treated SG cells, CoQ₀ attenuated fibrosis by inhibiting TGF-β and Collagen I expression and suppressed EMT by downregulating Slug and upregulating E-cadherin expression. Interesting, CoQ₀ inhibited the growth of OECM-1 tumors in xenografted mice. Our results advocate CoQ₀ for the therapeutic application against HNSCC.