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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 IC50 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 G1 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. DiOC6 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 Ca2+ 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 G1 arrest and apoptosis in NPC cells. Naringin-induced ROS-mediated G1 arrest and mitochondrial-, death receptor-, and endoplasmic reticulum stress-mediated apoptosis may be a promising strategy for treating NPC.
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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-1ß 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-ß 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.
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Transição Epitelial-Mesenquimal , Subunidade alfa do Fator 1 Induzível por Hipóxia , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ubiquinona , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Animais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Camundongos , Inflamassomos/metabolismo , Efeito Warburg em Oncologia/efeitos dos fármacos , Camundongos Nus , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológicoRESUMO
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.
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Benzoquinonas , Melaninas , Polyporales , Ubiquinona , Animais , Humanos , Ubiquinona/farmacologia , Ubiquinona/metabolismo , Melaninas/metabolismo , Peixe-Zebra/metabolismo , Monofenol Mono-Oxigenase/metabolismo , alfa-MSH/metabolismo , Proteína Beclina-1/metabolismo , Melanócitos/metabolismo , Queratinócitos/metabolismo , Autofagia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linhagem Celular TumoralRESUMO
The chronic disease psoriasis is associated with severe inflammation and abnormal keratinocyte propagation in the skin. Tranexamic acid (TXA), a plasmin inhibitor, is used to cure serious bleeding. We investigated whether TXA ointment mitigated Imiquimod (IMQ)-induced psoriasis-like inflammation. Furthermore, this study investigated the effect of noncytotoxic concentrations of TXA on IL-17-induced human keratinocyte (HaCaT) cells to determine the status of proliferative psoriatic keratinocytes. We found that TXA reduced IMQ-induced psoriasis-like erythema, thickness, scaling, and cumulative scores (erythema plus thickness plus scaling) on the back skin of BALB/c mice. Additionally, TXA decreased ear thickness and suppressed hyperkeratosis, hyperplasia, and inflammation of the ear epidermis in IMQ-induced BALB/c mice. Furthermore, TXA inhibited IMQ-induced splenomegaly in BALB/c mouse models. In IL-17-induced HaCaT cells, TXA inhibited ROS production and IL-8 secretion. Interestingly, TXA suppressed the IL-17-induced NFκB signaling pathway via IKK-mediated IκB degradation. TXA inhibited IL-17-induced activation of the NLRP3 inflammasome through caspase-1 and IL1ß expression. TXA inhibited IL-17-induced NLRP3 inflammasome activation by enhancing autophagy, as indicated by LC3-II accumulation, p62/SQSTM1 expression, ATG4B inhibition, and Beclin-1/Bcl-2 dysregulation. Notably, TXA suppressed IL-17-induced Nrf2-mediated keratin 17 expression. N-acetylcysteine pretreatment reversed the effects of TXA on NFκB, NLRP3 inflammasomes, and the Nrf2-mediated keratin 17 pathway in IL-17-induced HaCaT cells. Results further confirmed that in the ear skin of IMQ-induced mice, psoriasis biomarkers such as NLRP3, IL1ß, Nrf2, and keratin 17 expression were downregulated by TXA treatment. TXA improves IMQ-induced psoriasis-like inflammation in vivo and psoriatic keratinocytes in vitro. Tranexamic acid is a promising future treatment for psoriasis.
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Dermatite , Psoríase , Ácido Tranexâmico , Humanos , Animais , Camundongos , Interleucina-17/metabolismo , Ácido Tranexâmico/farmacologia , Ácido Tranexâmico/uso terapêutico , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Queratina-17 , Fator 2 Relacionado a NF-E2 , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Psoríase/metabolismo , Pele , Queratinócitos , Inflamação/tratamento farmacológico , Inflamação/induzido quimicamente , Imiquimode/farmacologia , NF-kappa B/metabolismo , Camundongos Endogâmicos BALB C , Modelos Animais de DoençasRESUMO
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.
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Neoplasias de Cabeça e Pescoço , Ubiquinona , Humanos , Animais , Camundongos , Ubiquinona/farmacologia , Ubiquinona/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Camundongos Nus , Carcinoma de Células Escamosas de Cabeça e Pescoço , Morte Celular , Apoptose , Linhagem Celular Tumoral , Autofagia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Nucleares , Proteína 1 Relacionada a TwistRESUMO
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.
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Morte Celular Autofágica , Glioblastoma , Animais , Camundongos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Glioblastoma/tratamento farmacológico , Anexina A5 , Apoptose , Autofagia , Linhagem Celular TumoralRESUMO
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.
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Glioblastoma , beta Catenina , Humanos , Animais , Camundongos , beta Catenina/metabolismo , Ubiquinona/farmacologia , Vimentina/metabolismo , Transição Epitelial-Mesenquimal , Glioblastoma/tratamento farmacológico , Invasividade Neoplásica/patologia , Caderinas/genética , Linhagem Celular Tumoral , Subunidade alfa do Fator 1 Induzível por Hipóxia , Movimento CelularRESUMO
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.
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Neoplasias de Mama Triplo Negativas , Ubiquinona , Humanos , Trifosfato de Adenosina , Caderinas/genética , Linhagem Celular Tumoral , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamassomos , Inflamação , Interleucina-18 , Lactato Desidrogenase 5 , Lactatos , Lipopolissacarídeos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ubiquinona/farmacologiaRESUMO
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.
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Apoptose , Neoplasias de Cabeça e Pescoço , Animais , Camundongos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Camundongos Nus , Autofagia , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/tratamento farmacológicoRESUMO
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.
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Fator A de Crescimento do Endotélio VascularRESUMO
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.
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Trifosfato de Adenosina/metabolismo , Inflamassomos/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Mitofagia/imunologia , Ubiquinona/uso terapêutico , Animais , Humanos , Camundongos , Transfecção , Ubiquinona/farmacologiaRESUMO
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.
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Neoplasias de Mama Triplo Negativas , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Diarileptanoides , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Sirolimo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
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.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Benzoquinonas/uso terapêutico , Glioblastoma/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Glioblastoma/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Necrose/induzido quimicamente , Polyporales/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Melaninas , Melanoma Experimental , Animais , Ácido Ascórbico , Autofagia , Linhagem Celular Tumoral , Queratinócitos , Melanócitos , Melanoma Experimental/tratamento farmacológico , Fator 2 Relacionado a NF-E2/genética , Peixe-Zebra , alfa-MSHRESUMO
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.
Assuntos
Apoptose , Autofagia , Neoplasias do Colo/patologia , Citoproteção , Polyporales/química , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Azoximetano , Proteína Beclina-1/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Colite/induzido quimicamente , Colite/complicações , Neoplasias do Colo/etiologia , Citoproteção/efeitos dos fármacos , Sulfato de Dextrana , Progressão da Doença , Humanos , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos ICR , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
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.
Assuntos
Fator 2 Relacionado a NF-E2 , alfa-MSH , Animais , Antioxidantes , Autofagia , Linhagem Celular Tumoral , Queratinócitos , Melaninas , Melanócitos , Fosfatidilinositol 3-Quinases , Estilbenos , Peixe-ZebraRESUMO
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.
Assuntos
Antioxidantes/farmacologia , Ocimum/química , Extratos Vegetais/farmacologia , Terapia Ultravioleta/efeitos adversos , Cicatrização/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células HaCaT , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Terapia Ultravioleta/métodos , Cicatrização/efeitos da radiaçãoRESUMO
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.
Assuntos
Autofagia/efeitos dos fármacos , Ácido Elágico/farmacologia , Melanócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Raios Ultravioleta/efeitos adversos , Proteínas de Peixe-Zebra/antagonistas & inibidores , alfa-MSH/antagonistas & inibidores , Animais , Autofagia/fisiologia , Relação Dose-Resposta a Droga , Ácido Elágico/química , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Melaninas/antagonistas & inibidores , Melaninas/metabolismo , Melaninas/efeitos da radiação , Melanócitos/metabolismo , Melanócitos/efeitos da radiação , Melanoma Experimental , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/efeitos da radiação , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/efeitos da radiação , alfa-MSH/metabolismo , alfa-MSH/efeitos da radiaçãoRESUMO
In the original article [...].
RESUMO
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.