Breast cancer is the most prevalent cancer among women. In triple-negative breast cancer (TNBC) cells, a novel quinone derivative, coenzyme Q
(CoQ
), promotes apoptosis and cell-cycle arrest. This ...study explored the anti-epithelial-mesenchymal transition (EMT) and antimetastatic attributes of CoQ
in TNBC (MDA-MB-231).
Invasion, as well as MTT assays were conducted. Lipofectamine RNAiMAX was used to transfect cells with β-catenin siRNA. Through Western blotting and RT-PCR, the major signaling pathways' protein expressions were examined, and the biopsied tumor tissues underwent immunohistochemical and hematoxylin and eosin staining as well as Western blotting.
CoQ
(0.5-2 μM) hindered tumor migration, invasion, and progression. Additionally, it caused MMP-2/- 9, uPA, uPAR, and VEGF downregulation. Furthermore, in highly metastatic MDA-MB-231 cells, TIMP-1/2 expression was subsequently upregulated and MMP-9 expression was downregulated. In addition, CoQ
inhibited metastasis and EMT in TGF-β/TNF-α-stimulated non-tumorigenic MCF-10A cells. Bioluminescence imaging of MDA-MB-231 luciferase-injected live mice demonstrated that CoQ
significantly inhibited metastasis of the breast cancer to the lungs and inhibited the development of tumors in MDA-MB-231 xenografted nude mice. Silencing of β-catenin with siRNA stimulated CoQ
-inhibited EMT. Western blotting as well as histological analysis established that CoQ0 reduced xenografted tumor development because apoptosis induction, cell-cycle inhibition, E-cadherin upregulation, β-catenin downregulation, and metastasis and EMT regulatory protein modulation were observed.
CoQ
inhibited the progression of metastasis as well as EMT (in vitro and in vivo). The described approach has potential in treating human breast cancer metastasis.
Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of ...Alpinia pricei Hayata by examining key molecular events in non‐small‐cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0–15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B‐cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase‐9/‐3 and poly ADP ribose polymerase PARP) signaling was found to be involved in FKB‐induced apoptosis. In addition, FKB‐induced reactive oxygen species (ROS) generation, and N‐acetylcysteine attenuated FKB‐induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule‐related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS‐mediated ATG4B expression. Inhibiting autophagy using 3‐methyladenine/chloroquine diminished FKB‐induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS‐mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.
Flavokawain B (FKB)‐induced autophagy was evidenced by a large accumulation of lipidated light chain 3 and acidic vesicular organelles. Mitochondrial (caspase‐9, capase‐3, and PARP) signaling was found to be critically involved in FKB‐induced apoptosis. FKB plays a crucial role in the execution and propagation of ROS‐mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.
TGF‐β1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Chalcones are reportedly cancer chemo‐preventive food components that are rich in fruits and vegetables. In this study, ...flavokawain A (FKA, 2‐30 μM), a naturally occurring chalcone in kava extracts, was evaluated for its anti‐fibrotic and antioxidant properties in TGF‐β1‐stimulated vascular smooth muscle (A7r5) cells, as well as its underlying molecular mechanism of action. Immunofluorescence data showed down‐regulated F‐actin expression with FKA treatment in TGF‐β1‐stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of α‐SMA and fibronectin proteins under TGF‐β1 stimulation. Findings from wound‐healing and invasion experiments showed that FKA inhibits TGF‐β1‐mediated migration and invasion. Western blotting demonstrated that treatment with FKA down‐regulated MMP‐9 and MMP‐2 and up‐regulated TIMP‐1 expression. Further evidence showed that FKA decreased TGF‐β1‐mediated phosphorylation and the transcriptional activity of Smad3. TGF‐β1‐induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N‐acetylcysteine (NAC) substantially diminished TGF‐β1‐induced p‐Smad3 activation and wound‐healing migration. Interestingly, FKA‐mediated antioxidant properties were associated with increased nuclear translocation of Nrf2 and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by the induction of HO‐1, NQO‐1 and γ‐GCLC genes in FKA‐treated A7r5 cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished the FKA‐mediated antioxidant effects, indicating that FKA may inhibit TGF‐β1‐induced fibrosis through suppressing ROS generation in A7r5 cells. Our results suggested that anti‐fibrotic and antioxidant activities of the chalcone flavokawain A may contribute to the development of food‐based chemo‐preventive drugs for fibrotic diseases.
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Ultraviolet A (UVA) irradiation is toxic to skin as it penetrates deep into the dermis and damages cellular components through excessive reactive oxygen species (ROS) production, ...which accelerates photoaging and skin cancer. We evaluated the dermato-protective efficacies of zerumbone (natural sesquiterpene of Zingiber zerumbet) in UVA-irradiated human skin keratinocyte (HaCaT) cells and mouse epidermis. Zerumbone pretreatment (2–10 μM) substantially suppressed UVA (15 J/cm2)-induced HaCaT cell death and lactate dehydrogenase release in a dose-dependent manner. UVA-induced excessive ROS production, DNA single-strand breaks, apoptotic DNA fragmentation and a dysregulated Bax/Bcl-2 ratio were remarkably reversed by zerumbone in keratinocytes. Zerumbone-mediated cytoprotective properties were associated with increased nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2) and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by induction of heme oxygenase-1 (HO-1) and γ-glutamyl cysteine ligase (γ-GCLC) genes in zerumbone-treated keratinocytes. Zerumbone-induced Nrf2 transcriptional activation was mediated by the p38 MAPK, PI3K/AKT and PKC signaling cascades. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished zerumbone-mediated cytoprotective effects, as evidenced by impaired antioxidant genes, uncontrolled ROS/apoptotic DNA fragmentation and keratinocytes death, following UVA irradiation. In vivo evidence demonstrated that zerumbone treatment to nude mice (55 and 110 μg/day) significantly ameliorated UVA (15 J/cm2/every 2-day/14-day) cytotoxicity via increased nuclear localization of Nrf2 and Nrf2-dependent antioxidant genes (HO-1 and γ-GCLC) in UVA-treated skin tissues. Our findings emphasized the significance of Nrf2/ARE-signaling in zerumbone-mediated induction of antioxidant genes against UVA-toxicity. The molecular evidence suggests zerumbone can be a natural medicine to treat/prevent UVA-induced skin damage/photoaging.
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 Q (CoQ) analogs with variable numbers of isoprenoid units have been demonstrated as anticancer and antioxidant/pro-oxidant molecules. This study examined the in vitro and in vivo antitumor ...and apoptosis activities of CoQ
0
(2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero isoprenoid side-chains) through upregulation of the Voltage‐dependent anion channel 1 (VDAC1) signaling pathway on human promyelocytic leukemia. CoQ
0
(0–40 μg/mL) treatment significantly reduced HL-60 cell viability, and up-regulated mitochondrial VDAC1 expression. CoQ
0
treatment triggers intracellular ROS generation, calcium release, ΔΨm collapse and PTP opening in HL-60 cells. CoQ
0
treatment induced apoptosis, which was associated with DNA fragmentation, cytochrome
c
release, caspase-3 and PARP activation, and Bax/Bcl-2 dysregulation. Annexin V-PI staining indicated that CoQ
0
promotes late apoptosis. Furthermore, the blockade of CoQ
0
-induced ROS production by antioxidant NAC pretreatment substantially attenuated CoQ
0
-induced apoptosis. The activation of p-GSK3β expression, cyclophilin D inhibition, and p53 activation through ROS are involved in CoQ
0
-induced HL-60 apoptotic cell death. Notably, ROS-independent p38 activation is involved in CoQ
0
-mediated apoptosis in HL-60 cells. In addition, the silencing of VDAC1 also prevented CoQ
0
-induced mitochondrial translocation of Bax, activation of caspase-3, and reduction in Bcl-2. Intriguingly, VDAC1 silencing did not prevent ROS production induced by CoQ
0
, which in turn indicates that CoQ
0
induced ROS-mediated VDAC1 and then mitochondrial apoptosis in HL-60 cells. In vivo results revealed that CoQ
0
is effective in delaying tumor incidence and reducing the tumor burden in HL-60-xenografted nude mice. Taken together, CoQ
0
could be a promising anticancer agent for the treatment of human promyelocytic leukemia through upregulation of VDAC1 signaling pathways.
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.
UVA irradiation-induced skin damage and redox imbalance have been shown to be ameliorated by ergothioneine (EGT), a naturally occurring sulfur-containing amino acid. However, the responsible ...molecular mechanism with nanomolar concentrations of EGT remains unclear. We investigated the dermato protective efficacies of EGT (125–500nM) against UVA irradiation (15J/cm2), and elucidated the underlying molecular mechanism in human keratinocyte-derived HaCaT cells. We found that EGT treatment prior to UVA exposure significantly increased the cell viability and prevented lactate dehydrogenase release into the medium. UVA-induced ROS and comet-like DNA formation were remarkably suppressed by EGT with a parallel inhibition of apoptosis, as evidenced by reduced DNA fragmentation (TUNEL), caspase-9/-3 activation, and Bcl-2/Bax dysregulation. Furthermore, EGT alleviated UVA-induced mitochondrial dysfunction. Dose-dependent increases of antioxidant genes, HO-1, NQO-1, and γ-GCLC and glutathione by EGT were associated with upregulated Nrf2 and downregulated Keap-1 expressions. This was confirmed by increased nuclear accumulation of Nrf2 and inhibition of Nrf2 degradation. Notably, augmented luciferase activity of ARE may explain Nrf2/ARE-mediated signaling pathways behind EGT dermato-protective properties. We further demonstrated that Nrf2 translocation was mediated by PI3K/AKT, PKC, or ROS signaling cascades. This phenomenon was confirmed with suppressed nuclear Nrf2 activation, and consequently diminished antioxidant genes in cells treated with respective pharmacological inhibitors (LY294002, GF109203X, and N-acetylcysteine). Besides, increased basal ROS by EGT appears to be crucial for triggering the Nrf2/ARE signaling pathways. Silencing of Nrf2 or OCTN1 (EGT carrier protein) signaling with siRNA showed no such protective effects of EGT against UVA-induced cell death, ROS, and apoptosis, which is evidence of the vitality of Nrf2 translocation and protective efficacy of EGT in keratinocytes. Our findings conclude that EGT at nanomolar concentrations effectively ameliorated UVA-induced skin damage, and may be considered as a desirable food supplement for skin protection and/or preparation of skin care products.
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•EGT (nM) attenuated UVA-induced ROS, apoptosis, and mitochondrial dysfunction.•EGT-mediated Nrf2/ARE signaling pathway triggered the antioxidant genes.•EGT increased Nrf2 nuclear accumulation, and inhibited degradation in keratinocytes.•Activation of Nrf2 relies on PI3K/AKT, PKC, and ROS signaling cascades.•Nrf2 and OCTN1 knockdown cells showed no EGT protection on UVA irradiation.
Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone) has been reported to exert anticancer properties against human breast/lung cancer cells. This study investigated the in vitro and in vivo ...anticancer properties of CoQ0 on human ovarian carcinoma (SKOV-3) cells and xenografted nude mice, and revealed the underlying molecular mechanism. CoQ0 induced G2/M arrest through downregulation of cyclin B1/A and CDK1/K2 expressions. CoQ0-induced autophagy as a survival mechanism was evidenced by increased accumulation of LC3-II, GFP-LC3 puncta, AVOs formation and Beclin-1/Bcl-2 dysregulation. Increased TUNEL-positive cells and Annexin-V/PI stained cells indicated CoQ0-induced late apoptosis. Both mitochondrial (caspase-3, PARP and Bax/Bcl-2 dysregulation) and ER stress (caspase-12 and Hsp70) signals are involved in execution of apoptosis. Interestingly, CoQ0-induced apoptosis/autophagy is associated with suppression of HER-2/neu and PI3K/AKT signalling cascades. CoQ0 triggered intracellular ROS production, whereas antioxidant N-acetylcysteine prevented CoQ0-induced apoptosis, but not autophagy. Inhibition of apoptosis by Z-VAD-FMK suppressed CoQ0-induced autophagy (diminished LC3-II/AVOs), indicates CoQ0-induced apoptosis led to evoke autophagy. Contrary, inhibition of autophagy by 3-MA/CQ potentiated CoQ0-induced apoptosis (increased DNA fragmentation/PARP cleavage). Furthermore, CoQ0 treatment to SKOV-3 xenografted nude mice reduced tumor incidence and burden. Histopathological analyses confirmed that CoQ0 modulated xenografted tumor progression by apoptosis induction. Our findings emphasize that CoQ0 triggered ROS-mediated apoptosis and cytoprotective autophagy.
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.
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