The in vitro and in vivo depigmenting activity of pterostilbene through induction of autophagy in melanocytes and inhibition of UVA-irradiated α-MSH in keratinocytes via Nrf2-mediated antioxidant pathways.

Pterostilbene (Pt) is a natural polyphenol found in blueberries and several grape varieties. Pt's pharmacological importance was well documented. Nevertheless, the depigmenting effects are not demonstrated. We evaluated the Pt's depigmenting effects through autophagy induction in B16F10 cells and inhibition of UVA (3 J/cm2)-irradiated α-MSH in keratinocyte HaCaT cells via Nrf2-mediated antioxidant pathways. Pt (2.5-5µM) attenuated ROS production and downregulated the POMC/α-MSH pathway in HaCaT cells. The conditioned medium-derived from UVA-irradiated HaCaT pretreated with Pt suppressed melanogenesis in B16F10 through MITF-CREB-tyrosinase pathway downregulation. Interestingly, Pt-induced HaCaT autophagy was revealed by enhanced LC3-II accumulation, p62/SQSTM1 activation, and AVO formation. Pt significantly decreased melanosome gp100 but increased LC3-II levels in HaCaT cells exposed to B16F10-derived melanin. Pt activated and facilitated the Nrf2 antioxidant pathway in HaCaT cells leading to increased HO-1, γ-GCLC, and NQO-1 antioxidant protein expression. ERK, AMPK, and ROS pathways mediate the Nrf2 activation. However, Nrf2 knockdown suppressed Pt's antioxidant ability leading to uncontrolled ROS and α-MSH levels after UVA-irradiation suggested the essentiality of the Nrf2 pathway. Moreover, in α-MSH-stimulated B16F10 cells, Pt (10-30 µM) downregulated the MC1R, MITF, tyrosinase, TRP-1/-2, and melanin expression. Further, Pt showed potent anti-melanogenic effects through autophagy induction mechanism in B16F10 cells, verified by increased LC3-II/p62 levels, AVO formation, and Beclin-1/Bcl-2 ratio, decreased ATG4B levels and PI3K/AKT/mTOR pathway. Transmission electron microscopy provided direct evidence by showing autophagosomes engulfing melanosomes following Pt treatment in α-MSH-stimulated B16F10 cells. Moreover, Pt-induced anti-melanogenic activity through the downregulation of CREB-MITF pathway-mediated TRP-1/-2, tyrosinase expressions, melanosome formation, and melanin synthesis was substantially reversed due to 3-MA (autophagy inhibitor) pretreatment or LC3 silencing in B16F10 cells. In vivo results also confirmed that Pt-inhibited tyrosinase expression/activity and endogenous pigmentation in the zebrafish model. Therefore, pterostilbene is a potent skin-whitening and antioxidant agent and could be used in skin-whitening formulations as a topical applicant.

The Antiaging Activity of Ergothioneine in UVA-Irradiated Human Dermal Fibroblasts via the Inhibition of the AP-1 Pathway and the Activation of Nrf2-Mediated Antioxidant Genes.

UVA irradiation induced ROS-mediated photo damage to the human skin leading to coarseness, wrinkling, pigmentation, and cutaneous malignancies. We investigated the dermatoprotective efficacies of submicromolar concentrations of ergothioneine (EGT, 0.125-0.5 µM), which occurs naturally as a sulfur-containing amino acid, in the mechanisms in human skin fibroblast (HSF) cells. UVA-induced AP-1 (c-Fos and c-Jun) translocation was found to be inhibited by EGT treatments with the parallel inhibition of the collagenolytic matrix metalloproteinase- (MMP-) 1 activation and type I procollagen degradation. Moreover, EGT mitigated UVA-induced ROS generation. An increase in the amount of antioxidant genes (HO-1, NQO-1, and γ-GCLC) from EGT and were associated with upregulated Nrf2 expressions in a dose-dependent or time-dependent manner. We confirmed this from Nrf2 translocation and increased nuclear ARE promoter activity that underlie EGT dermatoprotective activities. Also, glutathione (GSH) levels (from γ-GCLC) were significantly increased. Moreover, we showed that mediated by ERK, JNK, and PKC, signaling cascades mediate Nrf2 translocation. We confirmed this phenomenon by the suppressed nuclear Nrf2 activation in cells that were treated with respective inhibitors (PD98059, SP600125, and GF109203X). However, antioxidant protein expressions were impaired in Nrf2 knockdown cells to confirm that ARE/Nrf2 pathways and the inhibition of AP-1 had significant roles in EGT-mediated protective effects. We can conclude that ergothioneine ameliorated UVA-induced skin aging and is a useful food supplement for skin care products.

The Skin-Whitening Effects of Ectoine via the Suppression of α-MSH-Stimulated Melanogenesis and the Activation of Antioxidant Nrf2 Pathways in UVA-Irradiated Keratinocytes.

Ultraviolet A (UVA)-irradiation induced reactive oxygen species (ROS) production mediates excessive melanogenesis in skin cells leading to pigmentation. We demonstrated the depigmenting and anti-melanogenic effects of Ectoine, a natural bacterial osmolyte, in UVA-irradiated human (HaCaT) keratinocytes, and the underlying molecular mechanisms were elucidated. HaCaT cells were pre-treated with low concentrations of Ectoine (0.5-1.5 µM) and assayed for various depigmenting and anti-melanogenic parameters. This pre-treatment significantly downregulated ROS generation, α-melanocyte-stimulating hormone (α-MSH) production, and proopiomelanocortin (POMC) expression in UVA-irradiated HaCaT cells. Also, antioxidant heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase [quinone 1] (NQO-1), and γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC) protein expressions were mediated via the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) whose knockdown indeed impaired this effect signifying the importance of the Nrf2 pathway. Ectoine was mediating the activation of Nrf2 via the p38, protein kinase B (also known as AKT), protein kinase C (PKC), and casein kinase II protein kinase (CKII) pathways. The conditioned medium obtained from the Ectoine pre-treated and UVA-irradiated HaCaT cells downregulated the tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1/-2), cyclic AMP (c-AMP) protein kinase, c-AMP response element-binding protein (CREB), and microphthalmia-associated transcription factor (MITF) expressions leading to melanoma B16F10 cells having inhibited melanin synthesis. Interestingly, this anti-melanogenic effect in α-MSH-stimulated B16F10 cells was observable only at 50-400 µM concentrations of Ectoine, signifying the key role played by Ectoine (0.5-1 µM)-treated keratinocytes in skin whitening effects. We concluded that Ectoine could be used as an effective topical natural cosmetic agent with depigmenting and anti-melanogenic efficacy.

Zerumbone Exhibits Antiphotoaging and Dermatoprotective Properties in Ultraviolet A-Irradiated Human Skin Fibroblast Cells via the Activation of Nrf2/ARE Defensive Pathway.

Ultraviolet A (UVA) irradiation (320-400 nm range) triggers deleterious consequences in skin cell microenvironment leading to skin damage, photoaging (premature skin aging), and cancer. The accumulation of intracellular reactive oxygen species (ROS) plays a key role in this effect. With rapid progress in cosmetic health and quality of life, use of safe and highly effective phytochemicals has become a need of the hour. Zerumbone (ZER), a natural sesquiterpene, from Zingiber zerumbet rhizomes is well-known for its beneficial effects. We investigated the antiphotoaging and dermatoprotective efficacies of ZER (2-8 µM) against UVA irradiation (3 J/cm2) and elucidated the underlying molecular mechanisms in human skin fibroblast (HSF) cells. ZER treatment prior to low dose of UVA exposure increased cell viability. UVA-induced ROS generation was remarkably suppressed by ZER with parallel inhibition of MMP-1 activation and collagen III degradation. This was due to the inhibition of AP-1 (c-Fos and c-Jun) translocation. Furthermore, ZER alleviated UVA-induced SA-ß-galactosidase activity. Dose- or time-dependent increase of antioxidant genes, HO-1 and γ-GCLC by ZER, was associated with increased expression and nuclear accumulation of Nrf2 as well as decreased cytosolic Keap-1 expressions. Altered luciferase activity of ARE could explain the significance of Nrf2/ARE pathway underlying the dermatoprotective properties of ZER. Pharmacological inhibition of various signaling pathways suppressed nuclear Nrf2 activation in HSF cells confirming that Nrf2 translocation was mediated by ERK, JNK, PI3K/AKT, PKC, AMPK, casein kinase II, and ROS signaling pathways. Moreover, increased basal ROS levels and Nrf2 translocation seem crucial in ZER-mediated Nrf2/ARE signaling pathway. This was also evidenced from Nrf2 knocked-out studies in which ZER was not able to suppress the UVA-induced ROS generation in the absence of Nrf2. This study concluded that in the treatment of UVA-induced premature skin aging, ZER may consider as a desirable food supplement for skin protection and/or preparation of skin care products.

Kalantuboside B induced apoptosis and cytoprotective autophagy in human melanoma A2058 cells: An in vitro and in vivo study.

Kalantuboside B (KB), a natural bufadienolide derivative extracted from the succulent plant Kalanchoe tubiflora, is well-known for its cardiotonic, immunomodulatory, and anti-inflammatory properties. In this study, we tested in vitro and in vivo anti-cancer efficacy with low concentrations of KB (5-30 ng/mL; 8.7-52.2 nM) on A2058 melanoma cells; and for the molecular mechanisms that underlie them. KB significantly inhibited the cell viability and colony formation via arresting the cell cycle at G2/M phase. There was an association with a decrease in Cyclin A/B1, Cdc25C, and Cdc2 expressions. Further, this treatment indicated the induction of apoptosis, DNA fragmentation, cytochrome c release, and caspase-3, -8, -9, and -12 activation, and PARP cleavage, which shows that mitochondrial, death-receptor, and ER-stress signaling pathways are involved. KB-induced autophagy was apparent from enhanced LC3-II accumulation, GFP-LC3 puncta, and AVO formation. Surprisingly, KB-mediated cell death was potentiated by 3-MA and CQ to suggest the role of autophagy as a cytoprotective mechanism. Moreover, KB-treated A2058 cells enhanced intracellular ROS generation and antioxidant NAC prevented apoptosis and reversed cytoprotective autophagy. Interestingly, KB-induced apoptosis (PARP cleavage) and cytoprotective autophagy (LC3-II accumulation) were mediated by the up-regulation of the ERK signaling pathway. It was also shown that KB promoted cytoprotective autophagy by a calcium dependent-p53 downregulation pathway. In vivo data showed that KB suppressed tumor growth significantly in A2058-xenografted nude mice. A Western blot indicated cell-cycle inhibition (cyclin A reduction), apoptosis induction (PARP cleavage and Bcl-2 inhibition), and cytoprotective autophagy (LC3-II upregulation and p53 downregulation) in KB-treated A2058-xenografted mice. Our findings suggested that KB-induced ROS pathway plays a role in mediating the apoptosis and cytoprotective autophagy in human melanoma cells. Thus, KB is considered to be a putative anti-tumor agent.

The in vitro and in vivo depigmenting activity of Coenzyme Q10 through the down-regulation of α-MSH signaling pathways and induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated skin keratinocytes.

Coenzyme CoQ10 (CoQ10), a ubiquinone compound, has been reported to inhibit tyrosinase activity and melanin production in melanoma B16F10 cells. However, the molecular mechanism underlying this inhibitory effect is poorly understood. In this paper we aimed to investigate the molecular mechanisms involved in the anti-melanogenic activity of CoQ10 (1-2 µM) in UVA (5 J/cm2)-irradiated keratinocyte HaCaT cells and α-MSH stimulated B16-F10 cells. It was observed that CoQ10 suppressed p53/POMC, α-MSH production as well as inhibited ROS generation in UVA-irradiated keratinocyte HaCaT cells. CoQ10 down-regulated the melanin synthesis in α-MSH-stimulated B16-F10 cells by suppressing the MITF expression by down regulating the cAMP mediated CREB signaling cascades. Furthermore, in vivo evidence demonstrated the inhibitory effect of CoQ10 on endogenous pigmentation in zebrafish. Increased nuclear Nrf2 translocation accompanied by the induction of HO-1 and γ-GCLC genes were observed in CoQ10 treated keratinocyte HaCaT cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished CoQ10-mediated anti-melanogenic activity, as evidenced by impaired antioxidant HO-1 gene, uncontrolled ROS generation, and α-MSH production following UVA irradiation. To conclude, CoQ10 is an effective de-pigmention or skin-whitening agent and could be used in cosmetics for topical application.