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Ototoxicity refers to damage of sensory hair cells and functional hearing impairment following aminoglycosides exposure. Previously, we have determined that ferulic acid (FA) protected hair cells against serial concentrations of neomycin-induced ototoxic damage. The aim of the present study is to assess the mechanism and effects of FA on neomycin-induced hair cells loss and impact on mechanosensory-mediated behaviors alteration using transgenic zebrafish (pvalb3b: TagGFP). We first identified the optimal protective condition as pre/co-treatment method in early fish development. Pretreatment of the larvae with FA significantly protected against neomycin-induced hair cells loss through preventing neomycin passed through the cytoplasm of hair cells, and subsequently decreased reactive oxygen species production and TUNEL signals in 4 day post-fertilization (dpf) transgenic zebrafish larvae. Moreover, preservation of functional hair cells correlated directly with rescue of the altered swimming behavior, indicates FA pretreatment protects against neomycin ototoxic damage in 7-dpf transgenic zebrafish larvae. Together, our findings unravel the otoprotective role of FA as an effective agent against neomycin-induced ototoxic effects and offering the theoretical foundation for discovering novel candidates for hearing protection.

NF-κB family of transcription factors are the major contributors to malignant tumor progression, maintenance of cancer stemness, and enhancement of chemoresistance. Fenofibrate, a lipid-lowering drug, has been considered as a candidate for repurposing in the treatment of cancer through various pathways involved in apoptosis, cell cycle, migration, and invasion, including NF-κB. Nevertheless, whether fenofibrate possesses the potential to inhibit cancer stemness remained to be examined.Cytotoxicity of fenofibrate was estimated by MTT assay. The cells expressing stemness marker were detected by flow cytometry using ALDEFLUOR™ Kit. The secondary sphere formation assay was used to assess the self-renewal ability. Transwell system was used to evaluate migration and invasion capacities. NF-κB expression was measured by the immunoblotting system.In the present study, we demonstrated that fenofibrate inhibited cell viability, expression of stemness marker, self-renewal, migration, and invasion capacities in a dose-dependent manner. Of note, fenofibrate targeted cancer stem cells of oral squamous cell carcinoma (OSCC-CSCs) and had minimal effects on normal cells. Moreover, administration of fenofibrate at a lower concentration was sufficient to diminish the expression of NF-κB p50 and p65.This study demonstrated that the inhibitory effects of fenofibrate on OSCC-CSCs properties may be associated with downregulation of NF-κB. These results indicated that administration of fenofibrate may serve as an alternative strategy for OSCC therapy.

Background/Purpose Long non-coding RNA HOXA transcript at the distal tip (HOTTIP) has been reported to contribute to multiple carcinomas, but whether it involves in the progression of precancerous conditions remains to be determined. Oral submucous fibrosis (OSF) has been known as an oral potentially malignant disorder and attributed to the persistent activation of the myofibroblast. Methods The relative expression of HOTTIP in OSF tissues has been employed by RNA-sequencing and RT-PCR analysis. HOTTIP associated myofibroblasts activities and markers in fibrotic buccal mucosal fibroblast (fBMFs) through loss of function approaches have been evaluated. Results In the present study, we found that the expression of HOTTIP was overexpressed in the OSF tissues and positively correlated with several fibrosis markers. To investigate its significance of myofibroblast activation, we first verified the expression level of HOTTIP in the patient-derived fibrotic buccal mucosal fibroblast (fBMFs) was upregulated and conducted the shRNA-mediated knockdown experiment to inhibit its expression followed by numerous examinations. We demonstrated that suppression of HOTTIP downregulated the expression of myofibroblast marker, α-SMA, and type I collagen along with the diminished myofibroblast activities (collagen gel contraction and migration capacities). Furthermore, we showed that silencing HOTTIP lessened the production of various pro-inflammatory cytokines (IL-6 and TNF-α). Conclusion Collectively, our results suggest that HOTTIP plays a crucial role in the persistent activation of myofibroblasts as well as the chronic inflammation and collagen deposition.

Ototoxic hearing loss due to antibiotic medication including aminoglycosides and excess free radical production causes irreversible hair cell injury. Cichoric acid, a naturally occurring phenolic acid, has recently been found to exert anti-oxidative and anti-inflammatory properties through its free radical scavenging capacity. The present study aimed to investigate the protective effects of cichoric acid against neomycin-induced ototoxicity using transgenic zebrafish (pvalb3b: TagGFP). Our results indicated that cichoric acid in concentrations up to 5 μM did not affect zebrafish viability during the 2 h treatment period. Therefore, the otoprotective concentration of cichoric acid was identified as 5 μM under 2 h treatment by counting viable hair cells within the neuromasts of the anterior- and posterior-lateral lines in the study. Pretreatment of transgenic zebrafish with 5 μM of cichoric acid for 2 h significantly protected against neomycin-induced hair cell death. Protection mediated by cichoric acid was, however, lost over time. A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and FM4-64 staining, respectively, provided in situ evidence that cichoric acid ameliorated apoptotic signals and mechanotransduction machinery impairment caused by neomycin. A fish locomotor test (distance move, velocity, and rotation frequency) assessing behavioral alteration after ototoxic damage revealed rescue due to cichoric acid pretreatment before neomycin exposure. These findings suggest that cichoric acid in 5 μM under 2 h treatment has antioxidant effects and can attenuate neomycin-induced hair cell death in neuromasts. Although cichoric acid offered otoprotection, there is only a small difference between pharmacological and toxic concentrations, and hence cichoric acid can be considered a rather prototypical compound for the development of safer otoprotective compounds.

Background/Purpose Gingival overgrowth can occur as a result of poor oral hygiene or a side effect of taking certain medications, such as cyclosporine A (CsA). It has been shown that this immunosuppressant drug induces epithelial-to-mesenchymal transition (EMT) in the gingival epithelium but the associated molecular mechanism remains to be elucidated. Methods We first assessed the relative expression of microRNA-200a (miR-200a) in response to the CsA treatment using qRT-PCR. Next, luciferase reporter assay was applied to examine whether miR-200a was able to regulate ZEB2 and Western blot was utilized to measure the expression of ZEB2 in normal human gingival fibroblasts (HGFs). To confirm the significance of miR-200a and ZEB2 in the CsA-induced gingival overgrowth, miR-200a inhibitor and shRNA mediated knockdown of ZEB2 were used and cell proliferation in HGFs was assessed by MTT assay. Results The expression of miR-200a was dose-dependently downregulated following the CsA treatment. Luciferase reporter assay confirmed that ZEB2 was a direct downstream target regulated by miR-200a and ZEB2 was indeed increased after the administration of CsA. We demonstrated that knockdown of ZEB2 hampered the CsA-induced HGFs proliferation and the elevated cell proliferation due to inhibition of miR-200a was reversed by repression of ZEB2. Conclusion Our results showed that insufficient miR-200a in HGFs caused by CsA administration may lead to gingival enlargement mediated by the upregulation of ZEB2. This finding supported that CsA-induced EMT contributed to the adverse effect of using CsA and miR-200a may serve as an upstream target to prevent the overgrowth of the gingiva.

Five 2-acetoxybriaranes, including two known analogues, excavatolides B (1), E (2), along with three new metabolites, briaviolides V–X (3–5), have been obtained from the octocoral Briareum violaceum. The absolute configurations of 1 and 2 were determined by X-ray analysis for the first time and the structures of 3–5 were established by spectroscopic methods. Bioactivity study showed that briarane 3 decreased the release of elastase from human neutrophils.

Background Surface modification of metallic implants is critical for improving the clinical performance of the dental and orthopedic devices. Bioactive glasses exhibit different levels of cellular function and physicochemical behavior; however, there have been few previous studies on the effect of constituents of the bioactive glasses on the in vitro osteogenic activity and corrosion resistance of apatite-based coatings. Objective The objective of this work was to investigate the effect of SiO2, CaO, Na2O, and P2O5 on plasma-sprayed apatite coatings on Ti alloy substrates for tailoring the properties of implants making them suitable for clinical applications. Methods The corrosion potential and corrosion current of various coatings in simulated body fluid (SBF) were examined. MG63 cell proliferation, differentiation, and mineralization of plasma-sprayed apatite-matrix coatings were evaluated. Results The SiO2 and CaO-containing HA (HSC) coating had a higher corrosion potential than the other three coatings, while SiO2-containing HA (HS) coating displayed the highest corrosion current among all coatings. The effect of the oxides on cell functions followed the order SiO2 > CaO > P2O5 > Na2O in terms of cell attachment, proliferation, differentiation, and mineralization. Conclusions The flexibility in oxide doping may allow for the tunable biological properties and corrosion-resistant ability of the apatite coatings.

(+)-Bornyl p-coumarate is an active substance that is abundant in the Piper betle stem and has been shown to possess bioactivity against bacteria and a strong antioxidative effect. In the current study, we examined the actions of (+)-bornyl p-coumarate against A2058 and A375 melanoma cells. The inhibition effects of (+)-bornyl p-coumarate on these cell lines were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and the underlying mechanisms were identified by immunostaining, flow cytometry and western blotting of proteins associated with apoptosis and autophagy. Our results demonstrated that (+)-bornyl p-coumarate inhibited melanoma cell proliferation and caused loss of mitochondrial membrane potential, demonstrating treatment induced apoptosis. In addition, western blotting revealed that the process is mediated by caspase-dependent pathways, release of cytochrome C, activation of pro-apoptotic proteins (Bax, Bad and caspase-3/-9) and suppression of anti-apoptotic proteins (Bcl-2, Bcl-xl and Mcl-1). Also, the upregulated expressions of p-PERK, p-eIF2&alpha, ATF4 and CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) after treatment indicated that (+)-bornyl p-coumarate caused apoptosis via endoplasmic reticulum (ER) stress. Moreover, increased expressions of beclin-1, Atg3, Atg5, p62, LC3-I and LC3-II proteins and suppression by autophagic inhibitor 3-methyladenine (3-MA), indicated that (+)-bornyl p-coumarate triggered autophagy in the melanoma cells. In conclusion, our findings demonstrated that (+)-bornyl p-coumarate suppressed human melanoma cell growth and should be further investigated with regards to its potential use as a chemotherapy drug for the treatment of human melanoma.

There had been a paradigm shift in tissue engineering studies over the past decades. Of which, part of the hype in such studies was based on exploring for novel biomaterials to enhance regeneration. Strontium ions have been reported by others to have a unique effect on osteogenesis. Both in vitro and in vivo studies had demonstrated that strontium ions were able to promote osteoblast growth, and yet at the same time, inhibit the formation of osteoclasts. Strontium is thus considered an important biomaterial in the field of bone tissue engineering. In this study, we developed a Strontium-calcium silicate scaffold using 3D printing technology and evaluated for its cellular proliferation capabilities by assessing for protein quantification and mineralization of Wharton&rsquo, s Jelly mesenchymal stem cells. In addition, verapamil (an L-type of calcium channel blocker, CCB) was used to determine the mechanism of action of strontium ions. The results found that the relative cell proliferation rate on the scaffold was increased between 20% to 60% within 7 days of culture, while the CCB group only had up to approximately 10% proliferation as compared with the control specimen. Besides, the CCB group had downregulation and down expressions of all downstream cell signaling proteins (ERK and P38) and osteogenic-related protein (Col I, OPN, and OC). Furthermore, CCB was found to have 3&ndash, 4 times lesser calcium deposition and quantification after 7 and 14 days of culture. These results effectively show that the 3D printed strontium-contained scaffold could effectively stimulate stem cells to undergo bone differentiation via activation of L-type calcium channels. Such results showed that strontium-calcium silicate scaffolds have high development potential for bone tissue engineering.

Chemical examination of Briareum excavatum, collected in the waters of Taiwan, led to the isolation of four briarane diterpenoids, including three known compounds, briarenol G (1), briaviolides Y (2), and Z (3), as well as a new metabolite, briarenol L (4). The absolute configuration of 1 was determined by a single-crystal X-ray diffraction analysis for the first time in this study and the structure of 4 was established on the basis of spectroscopic analysis. Briaranes 2 and 3 exerted inhibition effects on COX-2 and iNOS, respectively, release from RAW 264.7 macrophages.

Oral submucous fibrosis (OSF) is a premalignant disorder in the oral cavity, and areca nut chewing habit has been implicated in the persistent activation of myofibroblasts and the subsequent fibrosis. Therefore, it is critical to ameliorate the excessive activities of myofibroblasts prior to the malignant transformation of OSF. In the current study, we evaluated the cytotoxicity of butylidenephthalide (BP), a major phthalide ingredient of Angelica sinensis, in fibrotic buccal mucosal fibroblasts (fBMFs) as well as various myofibroblast hallmarks, including the phenotypical characteristics and fibrosis-related markers. Our results demonstrated that myofibroblast activities, including collagen gel contraction, migration, invasion and wound healing abilities were inhibited in response to BP. The expression levels of myofibroblast marker, α-smooth muscle actin (α-SMA), fibronectin and type 1 collagen A1 were decreased after exposure of BP. Moreover, we found that the EMT-related markers, Twist, Snail and ZEB1 were all downregulated after BP treatment. Most importantly, our findings demonstrated that BP impeded the binding of Snail to the E-box region in the α-SMA promoter, which may lead to inhibition of the arecoline-induced myofibroblast activities. Collectively, our data indicated that BP reduced numerous myofibroblast features in fBMFs and hindered the binding of Snail to α-SMA, thereby may function as an effective and natural antifibrosis compound.

Two new briarane-type diterpenoids, briarenolides K (1) and L (2), were isolated from an octocoral identified as Briareum sp. The structures of new briaranes 1 and 2 were elucidated by spectroscopic methods. In the in vitro anti-inflammatory effects test, briaranes 1 and 2 were found to significantly inhibit the accumulation of the pro-inflammatory iNOS protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.

Six new monocarbocyclic sesterterpenoids, luffalides A–F 1–6, together with four known sesterterpenes 7–10, were isolated from a marine sponge Luffariella sp. The structures of compounds 1–6 were e...

Flaccidoxide-13-acetate, an active compound isolated from cultured-type soft coral Sinularia gibberosa, has been shown to have inhibitory effects against invasion and cell migration of RT4 and T24 human bladder cancer cells. In our study, we used an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation assay, and flow cytometry to determine the mechanisms of the anti-tumor effect of flaccidoxide-13-acetate. The MTT and colony formation assays showed that the cytotoxic effect of flaccidoxide-13-acetate on T24 and RT4 cells was dose-dependent, and the number of colonies formed in the culture was reduced with increasing flaccidoxide-13-acetate concentration. Flow cytometry analysis revealed that flaccidoxide-13-acetate induced late apoptotic events in both cell lines. Additionally, we found that flaccidoxide-13-acetate treatment upregulated the expressions of cleaved caspase 3, cleaved caspase 9, Bax, and Bad, and down-regulated the expressions of Bcl-2, p-Bad, Bcl-x1, and Mcl-1. The results indicated that apoptotic events were mediated by mitochondrial dysfunction via the caspase-dependent pathway. Flaccidoxide-13-acetate also provoked endoplasmic reticulum (ER) stress and led to activation of the PERK-eIF2&alpha, ATF6-CHOP pathway. Moreover, we examined the PI3K/AKT signal pathway, and found that the expressions of phosphorylated PI3K (p-PI3K) and AKT (p-AKT) were decreased with flaccidoxide-13-acetate concentrations. On the other hand, our results showed that the phosphorylated JNK and p38 were obviously activated. The results support the idea that flaccidoxide-13-acetate-induced apoptosis is mediated by mitochondrial dysfunction, ER stress, and activation of both the p38 and JNK pathways, and also relies on inhibition of PI3K/AKT signaling. These findings imply that flaccidoxide-13-acetate has potential in the development of chemotherapeutic agents for human bladder cancer.

Gallic acid is one of the major flavonoids found in plants. It acts as an antioxidant, and seems to have anti-inflammatory, anti-viral, and anti-cancer properties. In this study, we investigated the effects of gallic acid on melanogenesis, including the activation of melanogenesis signaling pathways. Gallic acid significantly inhibited both melanin synthesis and tyrosinase activity in a dose- and time-dependent manner, and decreased the expression of melanogenesis-related proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and dopachrome tautomerase (Dct). In addition, gallic acid also acts by phosphorylating and activating melanogenesis inhibitory proteins such as Akt and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Using inhibitors against PI3K/Akt (LY294002) or MEK/ERK-specific (PD98059), the hypopigmentation effect was suppressed, and the gallic acid-initiated activation of MEK/ERK and PI3K/Akt was also revoked. Gallic acid also increased GSK3β and p-β-catenin expression but down-regulated p-GSK3β. Moreover, GSK3β-specific inhibitor (SB216763) restored gallic acid-induced melanin reduction. These results suggest that activation of the MEK/ERK, PI3K/Akt, and inhibition of Wnt/β-catenin signaling pathways is involved in the melanogenesis signaling cascade, and that activation by gallic acid reduces melanin synthesis via down-regulation of MITF and its downstream signaling pathway. In conclusion, gallic acid may be a potentially agent for the treatment of certain skin conditions.

Psoriasis, an inflammatory skin disease triggered by the immune system, presents keratinocyte hyperproliferation, differentiation and angiogenesis. The role of transglutaminase-2 (TG2) in psoriasis has not been fully established yet. In this retrospective, non-randomized and non-blinded study, skin biopsies were collected from 37 psoriatic patients and immunohistochemical staining was performed. TG2 staining was positive in all 37 samples, among which 32 were strong and five weak. The localization of TG2 staining was present in the epidermis and spreading from basal layer to stratum granulosum in decreasing staining intensity. However, TG2 was also expressed in the basal layer in the non-lesional site of psoriasis and the skin of healthy people. The presence of TG2 was not associated with disease duration, stage of disease and subtype of psoriasis. Overexpression of TG2 seems to be an important role in psoriatic development.

In this study the isolated compound 11-dehydrosinulariolide from soft coral Sinularia leptoclados possessed anti-proliferative, anti-migratory and apoptosis-inducing activities against A2058 melanoma cells. Anti-tumor effects of 11-dehydrosinulariolide were determined by MTT assay, cell migration assay and flow cytometry. Growth and migration of melanoma cells were dose-dependently inhibited by 2–8 μg/mL 11-dehydrosinulariolide. Flow cytometric data indicated that 11-dehydrosinulariolide induces both early and late apoptosis in melanoma cells. It was found that the apoptosis induced by 11-dehydrosinulariolide is relevant to mitochondrial-mediated apoptosis via caspase-dependent pathways, elucidated by loss of mitochondrial membrane potential (∆Ym), release of cytochrome C, activation of caspase-3/-9 and Bax as well as suppression of Bcl-2/Bcl-xL. The cleavage of PARP-1 suggested partial involvement of caspase-independent pathways. Immunoblotting data displayed up-regulations of PERK/eIF2α/ATF4/CHOP and ATF6/CHOP coupling with elevation of ER stress chaperones GRP78, GRP94, calnexin, calreticulin and PDI, implicating the involvement of these factors in ER stress-mediated apoptosis induced by 11-dehydrosinulariolide. The abolishment of apoptotic events after pre-treatment with salubrinal indicated that ER stress-mediated apoptosis is also induced by 11-dehydrosinulariolide against melanoma cells. The data in this study suggest that 11-dehydrosinulariolide potentially induces apoptosis against melanoma cells via mitochondrial dysregulation and ER stress pathways.

The extracts from soft corals have been increasingly investigated for biomedical and therapeutic purposes. The aim of this study is to examine and analyze the anti-tumor effects of the genus Sinularia extract sinularin on A2058 melanoma cells using MTT assay, cell migration assay, wound healing assay, flow cytometric analysis, and proteomic analysis. Sinularin dose-dependently (1-5 μg/mL) inhibited melanoma cell proliferation while the treatment at identical concentrations suppressed cell migration. Sinularin dose-dependently enhanced apoptotic melanoma cells and caused tumor cell accumulation at G2/M phase, indicating that sinularin exerts apoptosis-induced and cell cycle-delayed activities in A2058 melanoma cells. Comparative proteomic analysis was conducted to investigate the effects of sinularin at the molecular level by comparison between the protein profiling of melanoma cells treated with sinularin and without the treatment. Thirty-five differential proteins (13 upregulated and 22 downregulated) concerning the treatment were identified by liquid chromatography-tandem mass spectrometry. Proteomic data and Western blot displayed the levels of several tumor inhibitory or apoptosis-associated proteins including annexin A1, voltage-dependent anion-selective channel protein 1 and prohibitin (upregulated), heat shock protein 60, heat shock protein beta-1, and peroxiredoxin-2 (downregulated) in A2058 melanoma cells exposed to sinularin. Increased expression of p53, cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, p21, and Bax and decreased expression of Bcl-2 in sinularin-treated melanoma cells suggest that the anti-tumor activities of sinularin against melanoma cells are particularly correlated with these pro-apoptotic factors. These data provide important information for the mechanisms of anti-tumor effects of sinularin on melanoma cells and may be helpful for drug development and progression monitoring of human melanoma.

The purpose of this study was to investigate the effect of KCNQ (potassium channel, voltage-gated, KQT-like subfamily) openers in preventing myotonia caused by anthracene-9-carboxylic acid (9-AC, a chloride channel blocker). An animal model of myotonia can be elicited in murine skeletal muscle by 9-AC treatment. KCNQ openers, such as retigabine and flupirtine, can inhibit the increased twitch amplitude (0.1 Hz stimulation) and reduce the tetanic fade (20 Hz stimulations) observed in the presence of 9-AC. Furthermore, the prolonged twitch duration of skeletal muscle was also inhibited by retigabine or flupirtine. Lamotrigine (an anticonvulsant drug) has a lesser effect on the muscle twitch amplitude, tetanic fade, and prolonged twitch duration as compared with KCNQ openers. In experiments using intracellular recordings, retigabine and flupirtine clearly reduced the firing frequencies of repetitive action potentials induced by 9-AC. These data suggested that KCNQ openers prevent the myotonia induced by 9-AC, at least partly through enhancing potassium conductance in skeletal muscle. Taken together, these results indicate that KCNQ openers are potential alternative therapeutic agents for the treatment of myotonia.

Aims The aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d -tubocurarine ( d -TC). Main methods Nerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations. Key findings Amongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d -TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d -TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d -TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose–response inhibition curve for d -TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d -TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors. Significance These data suggest that NF449 was able to compete with the binding of d -TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d -TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.

Metastasis of cancer is the cause of the majority of cancer deaths. Active compound flaccidoxide-13-acetate, isolated from the soft coral Cladiella kashmani, has been found to exhibit anti-tumor activity. In this study, Boyden chamber analysis, Western blotting and gelatin zymography assays indicated that flaccidoxide-13-acetate exerted inhibitory effects on the migration and invasion of RT4 and T24 human bladder cancer cells. The results demonstrated that flaccidoxide-13-acetate, in a concentration-dependent manner, reduced the levels of matrix metalloproteinase-2 (MMP-2), MMP-9, urokinase-type plasminogen activator receptor (uPAR), focal adhesion kinase (FAK), phosphatidylinositide-3 kinases (PI3K), p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, Ras homolog gene family, member A (Rho A), Ras, mitogen-activated protein kinase kinase 7 (MKK7) and mitogen-activated protein kinase kinase kinase 3 (MEKK3), and increased the expressions of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in RT4 and T24 cells. This study revealed that flaccidoxide-13-acetate suppressed cell migration and invasion by reducing the expressions of MMP-2 and MMP-9, regulated by the FAK/PI3K/AKT/mTOR pathway. In conclusion, our study was the first to demonstrate that flaccidoxide-13-acetate could be a potent medical agent for use in controlling the migration and invasion of bladder cancer.

Two eudesmane sesquiterpenoids, verticillatol (1) and 5α-acetoxy-4(14)-eudesmene-1β-ol (2) and two cembrane diterpenoids, (-)-leptodiol acetate (3) and sinulacembranolide A (4) were isolated from the octocoral Sinularia gaweli and compounds 2-4 are new isolates. The structures of new terpenoids 2-4 were elucidated by spectroscopic methods and by comparison the spectral data with those of known analogues. Terpenoid 4 was found to inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 marcophage cells.

Six new monocarbocyclic sesterterpenoids, named luffalides A-F (I), (II), and (III), are isolated from the marine sponge Luffariella sp.

Tumor necrosis factor-alpha (TNF-alpha) can inhibit tumor progression. It can be regulated by its soluble receptors (sTNF-Rs). We examined the expression of TNF-alpha and sTNF-Rs and the TNF-alpha/sTNF-R ratios in betel-quid-chewing patients with oral squamous cell carcinoma (OSCC) to see if these parameters are associated with disease progression according to the treatment stage. Peripheral blood mononuclear cells from 116 OSCC patients at different treatment stages and 19 betel-quid chewers with normal mucosa were assayed with ELISA. Levels of sTNF-RII in the OSCC patients were significantly higher than normal controls, with the recurrence group having the highest levels. After controlling for age and use of alcohol and tobacco, the TNF-alpha/sTNF-RII ratio showed significant differences comparing OSCC patients at each treatment stage with normal controls. Our results suggest that sTNF-RII and TNF-alpha/sTNF-RII ratio may be informative for the diagnosis and prognosis of OSCC.

One new 5alpha,8alpha-epidioxysterol, 3-acetylaxinysterol (1), along with one known sterol, axinysterol (2), were isolated from a Formosan sponge, Axinyssa sp.. The structures of the compounds were determined by spectroscopic methods and the absolute configuration of 2 was further confirmed by single-crystal X-ray diffraction analysis for the first time. Compound 2 exhibited significant cytotoxicity against K562 and Molt 4 cancer cell lines.

The extracts from soft corals have been increasingly investigated for biomedical and therapeutic purposes. The aim of this study is to examine and analyze the anti-tumor effects of the genus Sinularia extract sinularin on A2058 melanoma cells using MTT assay, cell migration assay, wound healing assay, flow cytometric analysis, and proteomic analysis. Sinularin dose-dependently (1-5 μg/mL) inhibited melanoma cell proliferation while the treatment at identical concentrations suppressed cell migration. Sinularin dose-dependently enhanced apoptotic melanoma cells and caused tumor cell accumulation at G2/M phase, indicating that sinularin exerts apoptosis-induced and cell cycle-delayed activities in A2058 melanoma cells. Comparative proteomic analysis was conducted to investigate the effects of sinularin at the molecular level by comparison between the protein profiling of melanoma cells treated with sinularin and without the treatment. Thirty-five differential proteins (13 upregulated and 22 downregulated) concerning the treatment were identified by liquid chromatography-tandem mass spectrometry. Proteomic data and Western blot displayed the levels of several tumor inhibitory or apoptosis-associated proteins including annexin A1, voltage-dependent anion-selective channel protein 1 and prohibitin (upregulated), heat shock protein 60, heat shock protein beta-1, and peroxiredoxin-2 (downregulated) in A2058 melanoma cells exposed to sinularin. Increased expression of p53, cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, p21, and Bax and decreased expression of Bcl-2 in sinularin-treated melanoma cells suggest that the anti-tumor activities of sinularin against melanoma cells are particularly correlated with these pro-apoptotic factors. These data provide important information for the mechanisms of anti-tumor effects of sinularin on melanoma cells and may be helpful for drug development and progression monitoring of human melanoma.

KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopuslaevis oocytes.Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes.Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K(+) current was almost completely blocked by a KCNQ4 blocker, linopirdine (0.25 mmol/L). BIMI (a PKC inhibitor) prevented the effects of PMA (a PKC activator) on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine (2 micromol/L), calyculin A (2 micromol/L) or okadaic acid (1 micromol/L), caused a significant positive shift in V(1/2) and a decrease in the conductance of KCNQ4 channels. The V(1/2) was shifted from -14.6+/-0.5 to -6.4+/-0.4 mV by cyclosporine, -18.8+/-0.5 to -9.2+/-0.4 mV by calyculin A, and -14.1+/-0.5 to -0.7+/-0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors (okadaic acid or calyculin A) on the induction of a positive shift of V(1/2) were augmented by further addition of PMA.These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.

Photolytic cleavage of the nitrogen–nitrogen single bond in benzaldehyde phenylhydrazones produced aminyl (R 2 N ) and iminyl (R 2 CN ) radicals. This photochemical property was utilized in the development of hydrazones as photo-induced DNA-cleaving agents. Irradiation with 350 nm UV light of arylhydrazones bearing substituents of various types in a phosphate buffer solution containing the supercoiled circular φX 174 RFI DNA at pH 6.0 resulted in single-strand cleavage of DNA. Attachment of the electron-donating OMe group to arylhydrazones increased their DNA-cleaving activity. Results from systematic studies indicate that both the aminyl and the iminyl radicals possessed DNA-cleaving ability.

One new 5α,8α-epidioxysterol, 3-acetylaxinysterol (1), along with one known sterol, axinysterol (2), were isolated from a Formosan sponge, Axinyssa sp.. The structures of the compounds were determined by spectroscopic methods and the absolute configuration of 2 was further confirmed by single-crystal X-ray diffraction analysis for the first time. Compound 2 exhibited significant cytotoxicity against K562 and Molt 4 cancer cell lines.

Eight single-stranded oligodeoxyribonucleotides 32P-labeled at the 5′-end were synthesized; they were annealed with the complementary oligodeoxyribonucleotides to form the corresponding double-stranded helices. These duplexes possessed standard Watson–Crick base pairs, locally perturbed sites of a base mismatch, or a bulge. Further, 5′–32P-labeled oligodeoxyribonucleotides with a hairpin loop were also synthesized. Cleavage of these single- and double-stranded oligodeoxyribonucleotides selectively at the deoxyguanosine residue was accomplished by use of 3-(p-tolylamino)-1,5-azulenequinone 1 upon irradiation with 350 nm UV light. The single strands were cleaved more efficiently than the double-helices. For the helices containing a deoxyguanosine residue at a bulge, at a hairpin loop or toward the end, the cleaving efficiency was increased. Computation results indicate that two possibilities exist for agent 1 to form two “Watson–Crick type” hydrogen bonds with guanine in single-stranded oligodeoxyribonucleotides; yet, only one possibility exists in duplexes.