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 anti-melanogenic effects of ellagic acid through induction of autophagy in melanocytes and suppression of UVA-activated α-MSH pathways via Nrf2 activation in keratinocytes.

Ellagic acid (EA) is a natural phenol antioxidant in different fruits, vegetables, and nuts. As a copper iron chelator from the tyrosinase enzyme's active site, EA was reported to inhibit melanogenesis in melanocytes. Here, we demonstrated the anti-melanogenic mechanisms of EA through autophagy induction in melanoma B16F10 cells and the role of Nrf2 and UVA (3 J/cm2)-activated α-melanocyte stimulating hormone (α-MSH) pathways in keratinocyte HaCaT cells. In vitro data showed that EA suppressed the tyrosinase activity and melanogenesis by suppressing cAMP-mediated CREB and MITF signaling mechanisms in α-MSH-stimulated B16F10 cells. ERK, JNK, and AKT pathways were involved in this EA-regulated MITF downregulation. Notably, EA induced autophagy in B16F10 cells was evidenced from increased LC3-II accumulation, p62/SQSTM1 activation, ATG4B downregulation, acidic vesicular organelle (AVO) formation, PI3K/AKT/mTOR inhibition, and Beclin-1/Bcl-2 dysregulation. Interestingly, 3-MA (an autophagy inhibitor) pretreatment or LC3 silencing (siRNA transfection) of B16F10 cells significantly reduced EA-induced anti-melanogenic activity. Besides this, in UVA-irradiated keratinocyte HaCaT cells, EA suppressed ROS production and α-MSH generation. Moreover, EA mediated the activation and nuclear translocation of Nrf2, leading to antioxidant γ-GCLC, HO-1, and NQO-1 protein expression in HaCaT cells. However, Nrf2 knockdown has significantly impaired this effect, and there was an uncontrolled ROS generation following UVA irradiation. JNK, PKC, and ROS pathways were involved in the activation of Nrf2 in HaCaT cells. In vivo experiments using the zebrafish model confirmed that EA inhibited tyrosinase activity and endogenous pigmentation. In conclusion, ellagic acid is an effective skin-whitening agent and might be used as a topical applicant.

Correction: Hseu, Y.-C., et al. The In Vitro and In Vivo Anticancer Properties of Chalcone Flavokawain B through Induction of ROS-Mediated Apoptotic and Autophagic Cell Death in Human Melanoma Cells. Cancers 2020, 12, 2936.

In the original article [...].

Staphylococcus aureus grown in anaerobic conditions exhibits elevated glutamine biosynthesis and biofilm units.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus, sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited Km of 11.06 ± 0.05 mmol·L-1 for glutamate and 2.4 ± 0.03 mmol·L-1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus.

The In Vitro and In Vivo Anticancer Properties of Chalcone Flavokawain B through Induction of ROS-Mediated Apoptotic and Autophagic Cell Death in Human Melanoma Cells.

Melanoma is the most prevalent type of skin cancer with high mortality rates. This study demonstrates the in vitro and in vivo anticancer properties of chalcone flavokawain B (FKB) induced ROS-mediated apoptosis and autophagy in human melanoma (human epithelial melanoma cell line A375 and/or human skin lymph node derived melanoma cell line A2058) cells. Cell viability was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression patterns of various apoptosis, autophagy-associated proteins were determined by Western blot methods. Annexin V was detected by flow cytometry, whereas acidic vesicular organelles (AVOs) and intracellular ROS levels were measured by fluorescence microscopy. The in vivo anticancer properties of FKB were evaluated by xenografting the A375 cells into nude mice. The results convey that FKB inhibited cell viability, B-Raf proto-oncogene, serine/threonine kinase (BRAF)/extracellular signal-regulated kinase (ERK) expression in human melanoma cells. Caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage pathway, and Bcl2 associated X (Bax)/B-cell lymphoma 2 (Bcl-2) dysregulation were involved in the execution of apoptosis. Moreover, FKB-induced autophagy was observed through increased microtubule-associated protein 1A/1B-light chain 3B (LC3-II) accumulation and AVOs formation, which was also associated with an increase in sequestosome 1 (SQSTM1/p62), decreased protein kinase B (AKT)/mammalian target of rapamycin (mTOR) expressions, and dysregulated Beclin-1/Bcl-2 levels. Autophagy inhibitors [3-methyladenine (3-MA)/chloroquine (CQ)] and LC3 silencing suppressed FKB-induced apoptosis by decreasing caspase-3 in melanoma cells. The antioxidant N-acetylcysteine (NAC) diminished FKB-induced apoptotic and autophagic cell death. However, the inhibition of apoptosis decreased FKB-induced autophagy (LC3-I/II). The in vivo study confirmed that FKB inhibited melanoma growth in A375-xenografted nude mice. This study concluded that FKB is critically associated with the execution and generation of ROS-modulated apoptotic and autophagic cell death of melanoma cells. FKB also repressed tumor growth in xenografted nude mice. Therefore, flavokawain B might be a potential anti-tumor agent in human melanoma treatment.

The Leaf Extracts of Toona sinensis and Fermented Culture Broths of Antrodia camphorata Synergistically Cause Apoptotic Cell Death in Promyelocytic Leukemia Cells.

Toona sinensis is a common edible vegetable that is used in certain Chinese dishes and has importance in folk medicine. The leaf extracts of T sinensis possess and exhibit anticancer efficacy against various cancer cell types. In Taiwanese folklore, Antrodia camphorata, also known as "Niu-Cheng-Zi," is used in traditional medicine to treat various illnesses. Its fruit and mycelium possess various potent antiproliferative properties. Two studies from our group have reported that T sinensis or A camphorata has the ability to cause apoptosis in various cancer cells. Conversely, underlying molecular mechanisms and any beneficial effects remain unknown. This study shows anticancer efficacy for both T sinensis and A camphorata co-treatments that target HL-60 cells. The combination index values indicate that 40 µg/mL of T sinensis and 25 µg/mL of A camphorata as a combined treatment shows a synergetic effect, which reduces HL-60 cell proliferation. Alternately, this treatment exhibited no cytotoxic effects for human umbilical vein endothelial cells. Western blot data showed that T sinensis and A camphorata as a combined treatment result in augmented expression of apoptosis, cytochrome c release, Bcl-2 inhibition, expression of Bax, Fas, and FasL, as well as the cleavage of Bid in HL-60 cells. Moreover, this combined treatment overshadowed monotherapy in its ability to inhibit uPAR, MMP-9, MMP-2, COX-2 expression, and PGE2 secretions. Our study strongly implies that this combined treatment offers more beneficial effects to suppress and treat leukemia due to apoptosis-mediated cell inhibition. Further in vivo studies related to the combined treatment could establish its future potential.

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.