The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes.

BACKGROUND: Coenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied. METHODS: We resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used. RESULTS: CoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α-MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α-MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α-MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. CONCLUSIONS: Our results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application.

The in vitro and in vivo anticancer activities of Antrodia salmonea through inhibition of metastasis and induction of ROS-mediated apoptotic and autophagic cell death in human glioblastoma cells.

BACKGROUND: Antrodia salmonea (AS) exhibits anticancer activities against various cancers. OBJECTIVE: This study investigated the anticancer activities of AS on human glioblastoma (GBM8401 and U87MG) cells both in vitro and in vivo and explained the underlying molecular mechanism. METHODS: MTT, colony formation, migration/invasion assay, immunoblotting, immunofluorescence, TUNEL, Annexin V/PI staining, AO staining, GFP-LC3 transfection, TEM, qPCR, siLC3, DCFH2-DA assay, and xenografted-nude mice were used to assess the potential of AS therapy. RESULTS: AS treatment retarded growth and suppressed colony formation in glioblastoma cells. AS attenuates EMT by suppressing invasion and migration, increasing E-cadherin expression, decreasing Twist, Snail, and N-cadherin expression, and inhibiting Wnt/ß-catenin pathways in GBM8401 and U87MG cells. Furthermore, AS induced apoptosis by activating caspase-3, cleaving PARP, and dysregulating Bax and Bcl-2 in both cell lines. TUNEL assay and Annexin V/PI staining indicated AS-mediated late apoptosis. Interestingly, AS induced autophagic cell death by LC3-II accumulation, AVO formation, autophagosome GFP-LC3 puncta, p62/SQSTM1 expression, and ATG4B inhibition in GBM8401 and U87MG cells. TEM data revealed that AS favored autophagosome and autolysosome formation. The autophagy inhibitors 3-MA/CQ and LC3 knockdown suppressed AS-induced apoptosis in glioblastoma cells, indicating that the inhibition of autophagy decreased AS-induced apoptosis. Notably, the antioxidant N-acetylcysteine (NAC) inhibited AS-mediated ROS production and AS-induced apoptotic and autophagic cell death. Furthermore, AS induced ROS-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. AS reduced the tumor burden in GBM8401-xenografted nude mice and significantly modulated tumor xenografts by inducing anti-EMT, apoptosis, and autophagy. AS could be a potential antitumor agent in human glioblastoma treatment.

In vitro and in vivo anti-tumor activity of Coenzyme Q0 against TWIST1-overexpressing HNSCC cells: ROS-mediated inhibition of EMT/metastasis and autophagy/apoptosis induction.

HNSCC (Head and Heck Squamous Cell Carcinoma) is a reasonably prevalent cancer with a high mortality rate. In this study, we tried to examine the anti-metastasis and apoptosis/autophagy actions of Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone), a derivative of Antrodia camphorata in HNCC TWIST1 overexpressing (FaDu-TWIST1) cells as well as in vivo tumor xenograft mice model. Using fluorescence based cellular assays, western blot and nude mice tumor xenografts, we determined that CoQ0 effectively reduced cell viability and displayed rapid morphological changes in FaDu-TWIST1 cells compared to FaDu cells. Non/sub-cytotoxic concentrations of CoQ0 treatment reduces the cell migration by downregulating TWIST1 and upregulating E-cadherin. Apoptosis produced by CoQ0 was mostly related with caspase-3 activation, PARP cleavage, and VDAC-1 expression. The FaDu-TWIST1 cells treated with CoQ0 exhibits autophagy-mediated LC3-II accumulation and acidic vesicular organelles (AVOs) formation. Pre-treatment with 3-MA and CoQ effectively prevented CoQ0-induced cell death and CoQ0-triggered autophagy in FaDu-TWIST cells as a death mechanism. CoQ0 induces ROS production in FaDu-TWIST1 cells and NAC pre-treatment significantly reduces anti-metastasis, apoptosis, and autophagy. Likewise, ROS-mediated AKT inhibition regulates CoQ0-induced apoptosis/autophagy in FaDu-TWIST1 cells. In vivo studies exhibit, CoQ0 effectively delays and reduces the tumor incidence and burden in FaDu-TWIST1-xenografted nude mice. Current findings display, CoQ0 exhibits a novel anti-cancer mechanism hence, it might be appropriate for anticancer therapy, and a new potent drug for HNSCC.

Antrodia camphorata and coenzyme Q0 , a novel quinone derivative of Antrodia camphorata, impede HIF-1α and epithelial-mesenchymal transition/metastasis in human glioblastoma cells.

Antrodia camphorata (AC) and Coenzyme Q0 (CoQ0 ), a novel quinone derivative of AC, exhibits antitumor activities. The present study evaluated EMT/metastasis inhibition and autophagy induction aspects of AC and CoQ0 in human glioblastoma (GBM8401) cells. Our findings revealed that AC treatment (0-150 µg/mL) hindered tumor cell proliferation and migration/invasion in GBM8401 cells. Notably, AC treatment inhibited HIF-1α and EMT by upregulating epithelial marker protein E-cadherin while downregulating mesenchymal proteins Twist, Slug, Snail, and ß-catenin. There was an appearance of the autophagy markers LC3-II and p62/SQSTM1, while ATG4B was downregulated by AC treatment. We also found that CoQ0 (0-10 µM) could inhibit migration and invasion in GBM8401 cells. In particular, E-cadherin was elevated and N-cadherin, Vimentin, Twist, Slug, and Snail, were reduced upon CoQ0 treatment. In addition, MMP-2/-9 expression and Wnt/ß-catenin pathways were downregulated. Furthermore, autophagy inhibitors 3-MA or CQ reversed the CoQ0 -elicited suppression of migration/invasion and metastasis-related proteins (Vimentin, Snail, and ß-catenin). Results suggested autophagy-mediated antiEMT and antimetastasis upon CoQ0 treatment. CoQ0 inhibited HIF-1α and metastasis in GBM8401 cells under normoxia and hypoxia. HIF-1α knockdown using siRNA accelerated CoQ0 -inhibited migration. Finally, CoQ0 exhibited a prolonged survival rate in GBM8401-xenografted mice. Treatment with Antrodia camphorata/CoQ0 inhibited HIF-1α and EMT/metastasis in glioblastoma.

Coenzyme Q0 defeats NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects by inhibiting HIF-1α expression in human triple-negative breast cancer cells.

Coenzyme Q0 (CoQ0) is a derivative quinone from Antrodia camphorata (AC) that exerts anticancer activities. This study examined the anticancer attributes of CoQ0 (0-4 µM) on inhibited anti-EMT/metastasis and NLRP3 inflammasome, and altered Warburg effects via HIF-1α inhibition in triple-negative breast cancer (MDA-MB-231 and 468) cells. MTT assay, cell migration/invasion assays, Western blotting, immunofluorescence, metabolic reprogramming, and LC-ESI-MS were carried out to assess the therapy potential of CoQ0. CoQ0 inhibited HIF-1α expression and suppressed the NLRP3 inflammasome and ASC/caspase-1 expression, followed by downregulation of IL-1ß and IL-18 expression in MDA-MB-231 and 468 cells. CoQ0 ameliorated cancer stem-like markers by decreasing CD44 and increasing CD24 expression. Notably, CoQ0 modulated EMT by upregulating the epithelial marker E-cadherin and downregulating the mesenchymal marker N-cadherin. CoQ0 inhibited glucose uptake and lactate accumulation. CoQ0 also inhibited HIF-1α downstream genes involved in glycolysis, such as HK-2, LDH-A, PDK-1, and PKM-2 enzymes. CoQ0 decreased extracellular acidification rate (ECAR), glycolysis, glycolytic capacity, and glycolytic reserve in MDA-MB-231 and 468 cells under normoxic and hypoxic (CoCl2) conditions. CoQ0 inhibited the glycolytic intermediates lactate, FBP, and 2/3-PG, and PEP levels. CoQ0 increased oxygen consumption rate (OCR), basal respiration, ATP production, maximal respiration, and spare capacity under normoxic and hypoxic (CoCl2) conditions. CoQ0 increased TCA cycle metabolites, such as citrate, isocitrate, and succinate. CoQ0 inhibited aerobic glycolysis and enhanced mitochondrial oxidative phosphorylation in TNBC cells. Under hypoxic conditions, CoQ0 also mitigated HIF-1α, GLUT1, glycolytic-related (HK-2, LDH-A, and PFK-1), and metastasis-related (E-cadherin, N-cadherin, and MMP-9) protein or mRNA expression in MDA-MB-231 and/or 468 cells. Under LPS/ATP stimulation, CoQ0 inhibited NLRP3 inflammasome/procaspase-1/IL-18 activation and NFκB/iNOS expression. CoQ0 also hindered LPS/ATP-stimulated tumor migration and downregulated LPS/ATP-stimulated N-cadherin and MMP-2/-9 expression. The present study revealed that suppression of HIF-1α expression caused by CoQ0 may contribute to inhibition of NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects of triple-negative breast cancers.

In vitro and in vivo anti-tumor activity of Antrodia salmonea against twist-overexpressing HNSCC cells: Induction of ROS-mediated autophagic and apoptotic cell death.

Head and neck squamous cell carcinoma (HNSCC) is a relatively common malignancy, characterized by lethal morbidity. Herein, we attempted to investigate the autophagy/apoptosis activities of the submerged fermented broths of Antrodia salmonea (AS) in HNSCC Twist-overexpressing (OECM-1 and FaDu-Twist) cells. AS (0-150 µg/mL) effectively reduced cell viability, colony formation, and downregulated Twist expression in OECM-1 and FaDu-Twist cells compared to FaDu cells. AS- induced apoptosis was mainly associated with activation of caspase-3, PARP cleavage, increased expression of VDAC-1 and disproportionation of Bax/Bcl-2. Annexin V/PI staining suggested late apoptosis induction by AS treatment. AS exhibits enhanced autophagy process mediated via LC3-I/II accumulation, increased acidic vesicular organelles (AVOs) formation and p62/SQSTM1 expression feeding into the apoptotic program. However, pre-treatment with autophagy blockers 3-MA and CQ significantly diminished AS-induced cell death. Additionally, suppression of AS-induced ROS release by treatment with antioxidant N-acetylcysteine (NAC) resulted in reduction of apoptotic and autophagic cell death. In vivo studies strengthened the above observations and showed that AS effectively reduced the tumor volume and tumor weight in OECM-1-xenografted nude mice. This study discovered that Antrodia salmonea exhibits a novel anti-cancer mechanism which could be harnessed as a new potent drug for HNSCC treatment.

Improved Wound Healing by Naringin Associated with MMP and the VEGF Pathway.

This study aims to investigate the wound-healing effectiveness of the phenolic compound, naringin, both in vitro and in vivo. Male mice were shaved on their dorsal skin under isoflurane, a biopsy punch was made in four symmetrical circular resection windows (6 mm) to induce a wound. These excision wounds were used to study the topical effects of naringin in terms of various biochemical, molecular, and histological parameters. We observed a significant recovery in the wound area. Increased levels of MMP-2, 9, 14, TIMP-2, VEGF-A, and VEGF-R1 were induced by naringin in the HaCaT cells. The time course experiments further revealed that levels of VEGF-A and B increased within 36 h; whereas levels of VEGF-C decreased. In line with this, VEGF-R3 levels, but not VEGF-R1 and 2 levels, increased soon after stimulation; although the increase subsided after 36 h. Additionally, naringin cream upregulated wound healing in vitro. The blockage of VEGF by Bevacizumab abolished the function of naringin cream on cell migration. Histological alterations in the wounded skin were restored by naringin cream, which accelerated wound healing via upregulated expression of growth factors (VEGF-A, B, and C and VEGF-R3), and thus increased MMP-2, 9, 14 expressions.

Coenzyme Q0 Inhibits NLRP3 Inflammasome Activation through Mitophagy Induction in LPS/ATP-Stimulated Macrophages.

Coenzyme Q (CoQ) analogs with a variable number of isoprenoid units have exhibited as anti-inflammatory as well as antioxidant molecules. Using novel quinone derivative CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero side chain isoprenoid), we studied its molecular activities against LPS/ATP-induced inflammation and redox imbalance in murine RAW264.7 macrophages. CoQ0's non- or subcytotoxic concentration suppressed the NLRP3 inflammasome and procaspase-1 activation, followed by downregulation of IL1ß expression in LPS/ATP-stimulated RAW264.7 macrophages. Similarly, treatment of CoQ0 led to LC3-I/II accumulation and p62/SQSTM1 activation. An increase in the Beclin-1/Bcl-2 ratio and a decrease in the expression of phosphorylated PI3K/AKT, p70 S6 kinase, and mTOR showed that autophagy was activated. Besides, CoQ0 increased Parkin protein to recruit damaged mitochondria and induced mitophagy in LPS/ATP-stimulated RAW264.7 macrophages. CoQ0 inhibited LPS/ATP-stimulated ROS generation in RAW264.7 macrophages. Notably, when LPS/ATP-stimulated RAW264.7 macrophages were treated with CoQ0, Mito-TEMPO (a mitochondrial ROS inhibitor), or N-acetylcysteine (NAC, a ROS inhibitor), there was a significant reduction of LPS/ATP-stimulated NLRP3 inflammasome activation and IL1ß expression. Interestingly, treatment with CoQ0 or Mito-TEMPO, but not NAC, significantly increased LPS/ATP-induced LC3-II accumulation indicating that mitophagy plays a key role in the regulation of CoQ0-inhibited NLRP3 inflammasome activation. Nrf2 knockdown significantly decreased IL1ß expression in LPS/ATP-stimulated RAW264.7 macrophages suggesting that CoQ0 inhibited ROS-mediated NLRP3 inflammasome activation and IL1ß expression was suppressed due to the Nrf2 activation. Hence, this study showed that CoQ0 might be a promising candidate for the therapeutics of inflammatory disorders due to its effective anti-inflammatory as well as antioxidant properties.

Bisdemethoxycurcumin Promotes Apoptosis and Inhibits the Epithelial-Mesenchymal Transition through the Inhibition of the G-Protein-Coupled Receptor 161/Mammalian Target of Rapamycin Signaling Pathway in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is one of the leading causes of cancer death in the world and lacks an effective targeted therapy. G-protein-coupled receptor 161 (GPR161) has been demonstrated to perform the functional regulations on TNBC progression and might be a potential new target for TNBC therapy. This study showed the effects of bisdemethoxycurcumin (BDMC) on GPR161 regulation, indicating that BDMC effectively inhibited GPR161 expression and downregulated GPR161-driven signaling. BDMC showed the potent inhibitory effects on TNBC proliferation through suppressing GPR161-mediated mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase (p70S6K) activation. Besides, in this study, we discover the mechanism of GPR161-driven TNBC metastasis, linking to GPR161-mediated twist-related protein 1 (Twist1)/matrix metallopeptidase 9 (MMP9) contributing to the epithelial-mesenchymal transition (EMT). BDMC effectively repressed GPR161-mediated TNBC metastasis via inhibiting Twist1/MMP9-induced EMT. The three-dimensional invasion assay also showed that BDMC significantly inhibited TNBC invasion. The combination treatment of BDMC and rapamycin enhanced the inhibition of TNBC proliferation and metastasis through increasing the blockage of mTOR activation. Furthermore, this study also observed that BDMC activated the caspase 3/9 signaling pathway to induce TNBC apoptosis. Therefore, BDMC could be applicable to anticancer therapy, especially targeting on the GPR161-driven cancer type.

Coenzyme Q0, a novel quinone derivative of Antrodia camphorata, induces ROS-mediated cytotoxic autophagy and apoptosis against human glioblastoma cells in vitro and in vivo.

Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone) derived from Antrodia camphorata exerts anticancer activities against breast, melanoma, and ovarian carcinoma. Glioblastoma multiforme is a common tumor affecting the central nervous system. This study explored anticancer properties of CoQ0 on human glioblastoma both in vitro and in vivo, and explained the molecular mechanism behind it. CoQ0 treatment retarded the growth and suppressed colony formation in glioblastoma (U87MG and GBM8401) cells. CoQ0 induced apoptosis by activation of caspase-3, cleavage of PARP, and dysregulation of Bax and Bcl-2 in both cell lines. Annexin V/PI staining indicated CoQ0 mediated necrosis and apoptosis. Interestingly, AVOs were increased trough induction of autophagy by CoQ0, LC3-II accumulation, and p62/SQSTM1 expression, leading to death mechanism. Z-VAD-FMK has no effect on CoQ0-induced autophagy but autophagy inhibition by 3-methyladenine (3-MA)/chloroquine (CQ) led to CoQ0-induced apoptosis. N-acetylcysteine (NAC) inhibited CoQ0-mediated ROS production and diminished CoQ0-induced apoptotic and autophagic cell death. Further, CoQ0 inhibited PI3K/AKT/mTOR signaling pathways. CoQ0 reduced the tumor burden in U87MG and GBM8401 xenografted athymic nude mice and significantly modulated tumor xenograft by inducing apoptosis and autophagy. CoQ0 generated ROS-mediated apoptotic and autophagic cell death for effective glioblastoma treatment.

The anti-melanogenic effects of 3-O-ethyl ascorbic acid via Nrf2-mediated α-MSH inhibition in UVA-irradiated keratinocytes and autophagy induction in melanocytes.

3-O-ethyl ascorbic acid (EAA) is an ether-derivative of ascorbic acid, known to inhibit tyrosinase activity, and is widely used in skincare formulations. Nevertheless, the molecular mechanisms underlying the EAA's effects are poorly understood. Here, the anti-melanogenic activity of EAA was demonstrated through Nrf2-mediated α-MSH inhibition in UVA-irradiated keratinocytes (HaCaT) and autophagy induction and inhibition of α-MSH-stimulated melanogenesis in melanocytes (B16F10). EAA pretreatment increased the HaCaT cell viability but suppressed ROS-mediated p53/POMC/α-MSH pathways in UVA-irradiated cells. Further, the conditioned medium from EAA-pretreated and UVA-irradiated HaCaT cells suppressed the MITF-CREB-tyrosinase pathways leading to the inhibition of melanin synthesis in B16F10 cells. EAA treatment increased nuclear Nrf2 translocation via the p38, PKC, and ROS pathways leading to HO-1, γ-GCLC, and NQO-1 antioxidant expression in HaCaT cells. However, Nrf2 silencing reduced the EAA-mediated anti-melanogenic activity, evidenced by impaired antioxidant gene expression and uncontrolled ROS (H202) generation following UVA irradiation. In B16F10 cells, EAA-induced autophagy was shown by enhanced LC3-II levels, AVO formation, Beclin-1 upregulation, and activation of p62/SQSTM1. Further, EAA-induced anti-melanogenic activity was substantially decreased in autophagy inhibitor (3-MA) pretreated or LC3 knockdown B16F10 cells. Notably, transmission electron microscopy data showed increased melanosome-engulfing autophagosomes in EAA-treated B16F10 cells. Moreover, EAA also down-regulated MC1R, TRP-1/-2, tyrosinase expressions, and melanin synthesis by suppressing the cAMP-CREB-mediated MITF expression in B16F10 cells stimulated with α-MSH. In vivo studies on the zebrafish model further confirmed that EAA inhibited tyrosinase expression/activity and endogenous pigmentation. In conclusion, 3-O-ethyl ascorbic acid is an effective skin-whitening agent and could be used as a topical agent for cosmetic purposes.

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.

Antrodia salmonea induces apoptosis and enhances cytoprotective autophagy in colon cancer cells.

A traditional Chinese medicinal fungus, Antrodia salmonea (AS), with antioxidant properties is familiar in Taiwan but anti-cancer activity of AS in human colon cancer is ambiguous. Hence, we explored the anti-cancer activity of AS in colon cancer cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that AS showed a remarkable effect on cell viability in colon cancer cells; SW620, HCT116, and HT29. Annexin V/propidium iodide (PI) stained cells indicated that AS induced both early/late apoptosis in SW620 cells. Additionally, cells treated with AS induced caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, mitochondrial dysfunction, and Bcl-2 associated X (Bax)/B-cell lymphoma (Bcl-2) dysregulation. Microtubule- associated protein 1A/1B-light chain 3B (LC3-II) accumulation, sequestosome 1 (p62/SQSTM1) activation, autophagy related 4B cysteine peptidase (ATG4B) inactivation, acidic vesicular organelles (AVOs) formation, and Beclin-1/Bcl-2 dysregulation revealed that AS-induced autophagy. Interestingly, cells pretreated with 3-methyladenine (3-MA) strengthened AS-induced caspase-3/apoptosis. Suppression of apoptosis by z-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not however block AS-induced autophagy, suggesting that autophagy was not attenuated by the AS-induced apoptosis. Application of N-acetylcysteine (NAC) prevented AS-induced cell death, caspase-3 activation, LC3-II accumulation, and AVOs formation, indicating that AS-induced apoptosis and autophagy was mediated by reactive oxygen species (ROS). Furthermore, AS-induced cytoprotective autophagy and apoptosis through extracellular signal-regulated kinase (ERK) signaling cascades. Moreover, in vivo data disclosed that AS inhibited colitis-associated tumorigenesis in azoxymethane (AOM)-dextran sodium sulphate (DSS)-treated mice. For the first time, we report the anti-cancer properties of this potentially advantageous mushroom for the treatment of human colon cancer.

Protective Effects of Ocimum gratissimumAqueous Extracts on HaCaT Cells Against UVC-Induced Inhibition of Cell Viability and Migration.

Ultraviolet C (UVC) has been applied to treatment of infections in wounds for at least the last two decades, however, cells being treated can be damaged if exposure is prolonged, which calls for protective measures, such as drug or herbal pre-treatment, to minimize damage. Ocimum gratissimum contains plant polyphenols such as isoflavones and caffeic acid, which have antioxidant effects. We hypothesize that Ocimum gratissimum aqueous extracts (OGE) can inhibit UVC-induced oxidative damage on skin cells. In this study, HaCaT skin cells are used to test the protective effects of OGE on cell proliferation and migration after exposure to UVC radiation. Pretreatment with OGE (50~150µg/mL) before 40 J/m2 UVC exposure was able to restore survival from 32.25% to between 46.77% and 68.00%, and 80 J/m2 UVC exposure from 11.49% to between 19.07% and 43.04%. Morphological observation of primarily apoptotic cell death confirms the above findings. The flow cytometry analysis revealed that UVC increased the number of cells at the sub-G1 phase in a dose dependent manner, and when pre-treated with OGE the changes were partially reversed. Moreover, the wound healing test for observing migration showed that UVC 40-80 J/m2 decreased cell migration to 47-28% activity and 100 µg/mL OGE was able to restore cell activity to81-69% at day 3. Based on the above results, we suggest that OGE has a protective effect on UVC-induced inhibition of cell proliferation and migration of skin cells and thus has potential application in wound care.

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 [...].

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.

Flavokawain B and Doxorubicin Work Synergistically to Impede the Propagation of Gastric Cancer Cells via ROS-Mediated Apoptosis and Autophagy Pathways.

Chalcone flavokawain B (FKB) possesses a chemopreventive and anti-cancer activity. Doxorubicin is a chemotherapeutic DNA intercalating agent widely used in malignancy treatment. The present study investigated whether synergistic effects exist between the combination of FKB (1.25-5 µg/mL) and doxorubicin (0.5 µg/mL) on the apoptosis and autophagy in human gastric cancer (AGS) cells, and the possible in vitro and in vivo mechanisms. The MTT assay measured cell viability. Various apoptotic-, autophagy-associated protein expression was determined by the Western blot technique. FKB+doxorubicin synergy was estimated by the Chou-Talalay combination index (CI) method. In vivo studies were performed on BALB/c mice. Results showed that compared to FKB/doxorubicin treatments, low doses of FKB+doxorubicin suppressed AGS cell growth. FKB potentiated doxorubicin-induced DNA fragmentation, apoptotic cell death, and enhanced doxorubicin-mediated mitochondrial, death receptor pathways. FKB+doxorubicin activated increased LC3-II accumulation, p62/SQSTM1 expression, and AVO formation as compared to the FKB/doxorubicin alone treatments indicating autophagy in these cells. The death mechanism in FKB+doxorubicin-treated AGS cells is due to the activation of autophagy. FKB+doxorubicin-mediated dysregulated Bax/Bcl-2, Beclin-1/Bcl-2 ratios suggested apoptosis, autophagy induction in AGS cells. FKB+doxorubicin-induced LC3-II/AVOs downregulation was suppressed due to an apoptotic inhibitor Z-VAD-FMK. Whereas, 3-methyladenine/chloroquine weakened FKB+doxorubicin-induced apoptosis (decreased DNA fragmentation/caspase-3). Activation of ERK/JNK may be involved in FKB+doxorubicin-induced apoptosis and autophagy. FKB+doxorubicin-triggered ROS generation, but NAC attenuated FKB+doxorubicin-induced autophagic (LC3 accumulation) and apoptotic (caspase-3 activation and PARP cleavage) cell death. FKB+doxorubicin blocked gastric cancer cell xenografts in nude mice in vivo as compared to FKB/doxorubicin alone treatments. FKB and doxorubicin wielded synergistic anti-tumor effects in gastric cancer cells and is a promising therapeutic approach.

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.

Aqueous Ocimum gratissimum extract induces cell apoptosis in human hepatocellular carcinoma cells.

Treatment of advanced hepatocellular carcinoma (HCC) has exhibited a poor overall survival rate of only six to ten months, and the urgency of the development of more effective novel agents is ever present. In this line of research, we aimed to investigate the effects and inhibitive mechanisms of aqueous Ocimum gratissimum leaf extract (OGE), the extract of Ocimum gratissimum, which is commonly used as a therapeutic herb for its numerous pharmacological properties, on malignant HCC cells. Our results showed that OGE decreased the cell viability of HCC SK-Hep1 and HA22T cells in a dose-dependent manner (from 400 to 800 µg/mL), while there is little effect on Chang liver cells. Moreover, cell-cycle analysis shows increased Sub-G1 cell count in SK-Hep1 and HA22T cells which is not observed in Chang liver cells. These findings raise suspicion that the OGE-induced cell death may be mediated through proteins that regulate cell cycle and apoptosis in SK-Hep1 and HA22T cells, and further experimentation revealed that OGE treatment resulted in a dose-dependent decrease in caspase 3 and PARP expressions and in CDK4and p-ERK1/2expressions. Moreover, animal tests also exhibited decreased HCC tumor growth by OGE treatment. We therefore suggest that the inhibition of cell viability and tumor growth induced by OGE may be correlated to the alteration of apoptosis-related proteins.