Cytotoxic and Apoptosis-Inducing Activity of Triterpene Glycosides from Holothuria scabra and Cucumaria frondosa against HepG2 Cells

The cytotoxic effects of thirteen triterpene glycosides from Holothuria scabra Jaeger and Cucumaria frondosa Gunnerus (Holothuroidea) against four human cell lines were detected and their cytotoxicity-structure relationships were established. The apoptosis-inducing activity of a more potent glycoside echinoside A (1) in HepG2 cells was further investigated by determining its effect on the morphology, mitochondrial transmembrane potential (Δψ_m) and mRNA expression levels of the apoptosis-related genes. The results showed that the number of glycosyl residues in sugar chains and the side chain of aglycone could affect their cytotoxicity towards tumor cells and selective cytotoxicity. 1 significantly inhibited cell viability and induced apoptosis in HepG2 cells. 1 also markedly decreased the Δψ_m and Bcl-2/Bax mRNA express ratio, and up-regulated the mRNA expression levels of Caspase-3, Caspase-8 and Caspase-9 in HepG2 cells. Therefore, 1 induced apoptosis in HepG2 cells through both intrinsic and extrinsic pathway. These findings could potentially promote the usage of these glycosides as leading compounds for developing new antitumor drugs.     

Marine Bromophenol Derivative 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-isopropoxymethyl benzyl)benzene-1,2-diol Protects Hepatocytes from Lipid-Induced Cell Damage and Insulin Resistance via PTP1B Inhibition

3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-isopropoxymethyl benzyl)benzene-1,2-diol (HPN) is a bromophenol derivative from the marine red alga Rhodomela confervoides. We have previously found that HPN exerted an anti-hyperglycemic property in db/db mouse model. In the present study, we found that HPN could protect HepG2 cells against palmitate (PA)-induced cell death. Data also showed that HPN inhibited cell death mainly by blocking the cell apoptosis. Further studies demonstrated that HPN (especially at 1.0 μM) significantly restored insulin-stimulated tyrosine phosphorylation of IR and IRS1/2, and inhibited the PTP1B expression level in HepG2 cells. Furthermore, the expression of Akt was activated by HPN, and glucose uptake was significantly increased in PA-treated HepG2 cells. Our results suggest that HPN could protect hepatocytes from lipid-induced cell damage and insulin resistance via PTP1B inhibition. Thus, HPN can be considered to have potential for the development of anti-diabetic agent that could protect both hepatic cell mass and function.     

Consumption of Syzygium gratum Promotes the Antioxidant Defense System in Mice

Several vegetables have been shown to possess cytoprotective and antioxidant effects with various mechanisms of action. The aim of this study was to determine the antioxidant effects and mechanism underlying of Syzygium gratum, a dietary and herbal plant commonly found in the Southeast Asia. Additionally, its effects on the induction of endogenous antioxidant defensive system were also investigated. Results showed that the leaf extract possessed an exceptionally strong antioxidant and intracellular oxygen radical scavenging activity in both aqueous and ethanolic extracts. The plant aqueous extract was further studied in C57BL/6J mice to evaluate its effects in vivo. The extract was well tolerated by the animals throughout the 30 days of study. The cytoprotective enzyme, heme oxygenase (HO-1) activity was significantly increased in the high dose-treated animals (1 g/kg/day). Consistent with the enzymatic activity, the expression of HO-1 mRNA tended to increase in those mice. There was no significant increase in hepatic γ-glutamylcysteine ligase (γ-GCL) activity, glutathione levels and GCL mRNA expression. Taken together, this study provides evidence that S. gratum exhibits potent direct antioxidant properties and can induce cytoprotective enzyme in vivo. Consumption of S. gratum may provide a health benefit against oxidative stress and other related disorders.     

Calcitriol Treatment Attenuates Uric Acid-Induced Kidney Injury via Super Oxide Dismutase-1 (SOD-1) Upregulation and Fibrosis Reduction

Background:Hyperuricemia induces nephropathy through the mediation of oxidative stress, tubular injury, inflammation, and fibrosis. The high uric acid level is associated with the reduction of vitamin D levels. However, the reno-protective effects of this vitamin in hyperuricemia condition remain unknown. This study aimed to elucidate calcitriol treatment in a uric acid-induced hyperuricemia mice model. Methods:Uric acid (125 mg/kg BW) was administered intraperitoneally for 7 (UA7) and 14 (UA14) days. Calcitriol (0.5 g/kg BW) was intraperitoneally injected for the following seven days, after 14 days of uric acid induction (UA14VD7 group). The control group received NaCl 0.9%, by the same route. Serum creatinine was measured using calorimetric method, and uric acid levels were assessed using enzymatic calorimetric assay. Tubular injury and fibrosis were assessed using PAS and Sirius red staining. RT-PCR and qRT-PCR were carried out for the analyses of SOD-1, Collagen-1, and TGF-1 mRNA expression in the kidney. Immunostaining of SOD-1 was performed to detect its expression in the kidney. Results:Uric acid and creatinine levels markedly increased in UA14 groups, followed by an exacerbation of tubular injury. RT-PCR revealed the upregulation of Collagen-1 and TGF-1, along with the downregulation of SOD-1. Calcitriol treatment attenuated the injury with reducing uric acid and creatinine levels, as well as tubular injury. This was associated with lower Collagen-1 and TGF-1 mRNA expression compared to the UA7 and UA14 groups. SOD-1 was upregulated in epithelial cells in the UA14VD7 group. Conclusion:Calcitriol treatment after uric acid induction may attenuate kidney injury through upregulation of SOD-1 and downregulation of Collagen-1 and TGF-1 gene expression. Key Words: Fibrosis, Hyperuricemia, Kidney injury, Superoxide dismutase-1, Vitamin D     

The Anticancer Effect of Fucoidan in PC-3 Prostate Cancer Cells

Fucoidan, a sulfated polysaccharide, has a variety of biological activities, such as anti-cancer, anti-angiogenic and anti-inflammatory. However, the mechanisms of action of fucoidan as an anti-cancer agent have not been fully elucidated. The present study examined the anti-cancer effect of fucoidan obtained from Undaria pinnatifida in PC-3 cells, human prostate cancer cells. Fucoidan induced the apoptosis of PC-3 cells by activating both intrinsic and extrinsic pathways. The induction of apoptosis was accompanied by the activation of extracellular signal-regulated kinase mitogen-activated protein kinase (ERK1/2 MAPK) and the inactivation of p38 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt. In addition, fucoidan also induced the up-regulation of p21^Cip1/Waf and down-regulation of E2F-1 cell-cycle-related proteins. Furthermore, in the Wnt/β-catenin pathway, fucoidan activated GSK-3β that resulted in the decrease of β-catenin level, followed by the decrease of c-myc and cyclin D1 expressions, target genes of β-catenin in PC-3 cells. These results suggested that fucoidan treatment could induce intrinsic and extrinsic apoptosis pathways via the activation of ERK1/2 MAPK, the inactivation of p38 MAPK and PI3K/Akt signaling pathway, and the down-regulation of Wnt/β-catenin signaling pathway in PC-3 prostate cancer cells. These data support that fucoidan might have potential for the treatment of prostate cancer.     

EGCG抑制Nicotine诱导肺腺癌H1299细胞JAK2/STAT3 mRNA的表达研究

为探索EGCG对Nicotine诱导肺癌细胞增殖的抑制作用,本研究通过MTT实验筛选出EGCG、Nicotine对肺腺癌细胞H1299的最佳作用浓度,利用实时荧光定量PCR技术检测EGCG、Nicotine对H1299细胞中Bax、Bcl-2、Jak2和Stat3基因mRNA相对表达量的变化。实验结果表明,EGCG对H1299细胞的半抑制浓度IC₅₀值约为32βμmol·L^-1（24βh）、15βμmol·L^-1（48βh）;1βμmol·L^-1的Nicotine对H1299细胞促增殖作用明显;以15βμmol·L^-1的EGCG预处理H1299细胞24βh可显著下调1βμmol·L^-1 Nicotine的促增殖作用（P<0.05）。1βμmol·L^-1的Nicotine处理H1299细胞可明显降低JAK2/STAT3信号通路中Bax基因mRNA的表达,增加Bcl-2、Jak2、Stat3基因的mRNA表达;15βμmol·L^-1 EGCG预处理H1299细胞可反向调控Nicotine诱导所致JAK2/STAT3信号通路中Jak2、Stat3和Bax、Bcl-2基因表达量的变化,结果具有显著性差异（P<0.05）。由此可知,EGCG对Nicotine诱导的肺腺癌H1299细胞增殖及JAK2/STAT3信号通路中促增殖基因mRNA的表达起抑制作用。     

黑茶对胎牛血清诱导脂肪变性的HepG2细胞的影响

利用含有50%的胎牛血清的培养液处理HepG2细胞48 h,建立非酒精性脂肪变性细胞模型,分别从噻唑兰染色吸光度法(MTT值)、甘油三酯(TG)、胆固醇(TC)3个方面评价茯砖提取物(FZ)、六堡茶提取物(LB)、青砖茶提取物(QZ)、千两茶提取物(QL)清除肝细胞脂肪堆积的作用。结果表明,与模型组相比,加药处理组细胞的脂质堆积有缓解趋势,茯砖茶、六堡茶、千两茶、青砖茶提取物都可显著降低细胞内TG累积(P<0.01);而茯砖、青砖茶提取物对TC的清除效果较好(P<0.01),千两茶与六堡茶效果不明显。以上结果表明,黑茶提取物通过降低细胞内的脂类堆积,可能起到缓解脂肪变性的作用。     

Antifungal activity of Vitex agnus-castus extract against Pythium ultimum in tomato

Vitex agnus-castus methanolic extract showed strong antifungal activity against Pythium ultimum in tomato under both in vitro and in vivo conditions. The 0.2% extract delayed the mycelial growth of the fungus and showed significant antifungal activity against P. ultimum on tomato seedlings with an efficacy comparable to that of the synthetic fungicide. To determine the involvement both of plant extract and pathogenic fungus in PR gene induction, tomato plants were treated with V. agnus-castus extract and/or inoculated with P. ultimum. The expression of four PR genes (PR-1, PR-2, PR-5, PR-6) was monitored at five time points within 48 h of the extract treatment and fungal inoculation. The PR-1 and PR-4 genes were activated directly by V. agnus-castus extract up to 12 h after treatments; at 24 h, the direct activation by plant extract disappeared and a synergistic inducing effect of extract and pathogen applied simultaneously on the plant was observed. The PR-6 gene was not activated directly by the V. agnus-castus extract but only when applied together with the pathogen; activation of the PR-6 gene occurred 24 h after treatments and the gene expression increased at 48 h. There was no activation of PR-5 gene by the plant extract. The ability of V. agnus-castus extract to enhance plant defence responses upon pathogen inoculation might be further investigated. The activation of various PR genes suggests that induction of defence responses by V. agnus-castus extract in tomato may be regulated by more than one signalling pathway.     

Polyphenol-Rich Fraction of Ecklonia cava Improves Nonalcoholic Fatty Liver Disease in High Fat Diet-Fed Mice

Ecklonia cava (E. cava; CA) is an edible brown alga with beneficial effects in diabetes via regulation of various metabolic processes such as lipogenesis, lipolysis, inflammation, and the antioxidant defense system in liver and adipose tissue. We investigated the effect of the polyphenol-rich fraction of E. cava produced from Gijang (G-CA) on nonalcoholic fatty liver disease (NAFLD) in high-fat diet (HFD)-fed mice. C57BL6 mice were fed a HFD for six weeks and then the HFD group was administered 300 mg/kg of G-CA extracts by oral intubation for 10 weeks. Body weight, fat mass, and serum biochemical parameters were reduced by G-CA extract treatment. MRI/MRS analysis showed that liver fat and liver volume in HFD-induced obese mice were reduced by G-CA extract treatment. Further, we analyzed hepatic gene expression related to inflammation and lipid metabolism. The mRNA expression levels of inflammatory cytokines and hepatic lipogenesis-related genes were decreased in G-CA-treated HFD mice. The mRNA expression levels of cholesterol 7 alpha-hydroxylase 1 (CYP7A1), the key enzyme in bile acid synthesis, were dramatically increased by G-CA treatment in HFD mice. We suggest that G-CA treatment ameliorated hepatic steatosis by inhibiting inflammation and improving lipid metabolism.     

Antioxidant Activity of Gracilaria lemaneiformis Polysaccharide Degradation Based on Nrf-2/Keap-1 Signaling Pathway in HepG2 Cells with Oxidative Stress Induced by H2O2

The objective of this research was to investigate the antioxidant activity of Gracilaria lemaneiformis polysaccharide degradation and its underlying mechanism involved in the Nrf-2/Keap-1 signaling pathway in HepG2 cells with oxidative stress induced by H₂O₂. The result of the scavenging ability of free radicals showed that GLP-HV (polysaccharide degraded by H₂O₂–vitamin C (Vc)) performed a better scavenging ability than GLP (G. lemaneiformis polysaccharide). Moreover, the scavenging ability of polysaccharide to these free radicals from strong to weak was as follows: superoxide radical, ferric ion, ABTS⁺, and DPPH radical, and their IC₅₀ values were 3.56 ± 0.0028, 4.97 ± 0.18, 9.62 ± 0.35, and 23.85 ± 1.78 mg/mL, respectively. Furthermore, GLP-HV obviously relieved oxidative stress in HepG2 cells, which strengthened the activity of T-AOC, CAT, GSH-PX, and SOD, and diminished the intensity of MDA, intracellular ROS, and calcium ion based on the Nrf-2/Keap-1 signaling pathway. The PCR result revealed that polysaccharide upregulated the expression of the genes Nrf-2, HO-1, NQO-1, and ZO-1 and downregulated Keap-1. The correlation between chemical properties and antioxidant mechanism of GLP-HV was evaluated via a heat map. The results illustrated that reducing sugar and active groups presented a positive correlation, and molecular weight and viscosity exhibited a negative relation with antioxidant activity.     

SD118-xanthocillin X (1), a novel marine agent extracted from Penicillium commune, induces autophagy through the inhibition of the MEK/ERK pathway

A compound named SD118-xanthocillin X (1) (C(18)H(12)N(2)O(2)), isolated from Penicillium commune in a deep-sea sediment sample, has been shown to inhibit the growth of several cancer cell lines in vitro. In the present study, we employed a growth inhibition assay and apoptotic analysis to identify the biological effect and detailed mechanism of SD118-xanthocillin X (1) in human hepatocellular carcinoma (HepG2) cells. SD118-xanthocillin X (1) demonstrated a concentration-dependent inhibitory effect on the growth of HepG2 cells and caused slight cellular apoptosis and significantly induced autophagy. Autophagy was detected as early as 12 h by the conversion of microtubule-associated protein 1 light chain 3 (LC3-I) to LC3-II, following cleavage and lipid addition to LC3-I. The pharmacological autophagy inhibitor 3-methyladenine largely attenuates the growth inhibition and autophagic effect of SD118-xanthocillin X (1) in HepG2 cells. Our data also indicated that the autophagic effect of SD118-xanthocillin X (1) occurs via the down-regulation of the MEK/ERK signaling pathway and the up-regulated class III PI3K/Beclin 1 signaling pathway.     

Citromycin Isolated from the Antarctic Marine-Derived Fungi,Sporothrix sp., Inhibits Ovarian Cancer Cell Invasion via Suppression of ERK Signaling

Recently, microorganisms and their metabolites in the Antarctic marine environment have attracted attention as useful sources for novel therapeutics, including anticancer drugs. Here, we investigated the effects of citromycin, isolated from the Antarctic marine-derived fungus, Sporothrix sp., on human ovarian cancer cells. Citromycin inhibited the migration and invasion of human ovarian cancer SKOV3 and A2780 cells, but had no cytotoxic activity against them. Additionally, it inhibited the expression of epithelial–mesenchymal transition (EMT) markers and the activation of matrix metalloproteinase (MMP)-2 and MMP9. Moreover, extracellular signal-regulated kinase (ERK)-1/2 signaling was inhibited after citromycin treatment, and the ectopic expression of ERK negated the anti-invasive activity of citromycin. Our findings suggest that citromycin inhibits the migration and invasion of human ovarian cancer cells by downregulating the expression levels of EMT markers and MMP-2/9 via inhibition of the ERK1/2 pathway.     

Antiproliferation and Apoptosis on RKO Colon Cancer by <Emphasis Type="Italic">Millingtonia hortensis</Emphasis>

Millingtonia hortensis is a medicinal plant widely used in many Asian countries. An aqueous crude extract of this plant has been shown the apoptosis induction on RKO colon cancer cells. However, its mechanism remains unknown. To learn more about this plant extract, we partially purified the crude extract using Sephadex LH-20 and three aqueous fractions were collected. Each fraction was investigated for cytotoxicity using MTT assay. Fraction 1 showed antiproliferative effect on RKO cells with dose-dependent manner, while fraction 2 and 3 had no effect. Induction of apoptosis was determined using flow cytometry and DNA fragmentation method. Apoptotic cell numbers and the appearance of fragmented DNA increased with dose-dependent manner after treatment with fraction 1 for 48 h. We further investigated the expression of apoptotic protein by western blot analysis. Fraction 1 decreased the expression of anti-apoptotic protein, Bcl-x_L and p-Bad, while pro-apoptotic protein Bad, was not changed. Fraction 1 also decreased the expression of p-Akt and slightly increased the level of total Akt. These results indicated that fraction 1 is able to inhibit cell proliferation and induce apoptosis on RKO cells by decreasing the expression of Bcl-x_L, p-Bad and p-Akt which are involving in survival of cancer cells.     

Ameliorative Effects of Oyster Protein Hydrolysates on Cadmium-Induced Hepatic Injury in Mice

Cadmium (Cd) is a widespread environmental toxicant that can cause severe hepatic injury. Oyster protein hydrolysates (OPs) have potential effects on preventing liver disease. In this study, thirty mice were randomly divided into five groups: the control, Cd, Cd + ethylenediaminetetraacetic acid (EDTA, 100 mg/kg), and low/high dose of OPs-treatment groups (100 mg/kg or 300 mg/kg). After continuous administration for 7 days, the ameliorative effect of OPs on Cd-induced acute hepatic injury in Cd-exposed mice was assessed. The results showed that OPs significantly improved the liver function profiles (serum ALT, AST, LDH, and ALP) in Cd-exposed mice. Histopathological analysis showed that OPs decreased apoptotic bodies, hemorrhage, lymphocyte accumulation, and inflammatory cell infiltration around central veins. OPs significantly retained the activities of SOD, CAT, and GSH-Px, and decreased the elevated hepatic MDA content in Cd-exposed mice. In addition, OPs exhibited a reductive effect on the inflammatory responses (IL-1β, IL-6, and TNF-α) and inhibitory effects on the expression of inflammation-related proteins (MIP-2 and COX-2) and the ERK/NF-κB signaling pathway. OPs suppressed the development of hepatocyte apoptosis (Bax, caspase-3, and Blc-2) and the activation of the PI3K/AKT signaling pathway in Cd-exposed mice. In conclusion, OPs ameliorated the Cd-induced hepatic injury by inhibiting oxidative damage and inflammatory responses, as well as the development of hepatocyte apoptosis via regulating the ERK/NF-κB and PI3K/AKT-related signaling pathways.     

Hypocholesterolemic Activity from the Leaf Extracts of Cnidoscolus chayamansa

The aim of this study was to determine the hypocholesterolemic activity of Cnidoscolus chayamansa. In an in vivo model, high-cholesterol diet administered to mice Balb/c induced hypercholesterolemia. Three extracts from Cnidoscolus chayamansa (ethanol, methanol and an aqueous extract) were tested on hypercholesterolemic mice. Active extracts were assessed against the in vitro inhibitory activity of the same three extracts on the HMG-CoA reductase enzyme by using Vero cells. The specific chemical groups present in the phytochemical extracts were also determined. Only the aqueous extract (at either doses employed) showed a significant cholesterol reduction (27.9 and 31.1%, for 50 and 100 mg kg⁻¹, respectively P < 0.01). The extract did not inhibit the HMG-CoA reductase enzyme, suggesting that its compounds act at another level in cholesterol metabolism. Reactions to secondary metabolites indicate the presence of alkaloids in the aqueous and ethanol extracts and phenol hydroxyls in the ethanol and methanol extracts.     

Chemopreventive Effects of Free and Bound Phenolics Associated to Steep Waters (Nejayote) Obtained After Nixtamalization of Different Maize Types

Free and bound phenolics extracts from nejayote solids were obtained after optimally lime-cooking blue, normal white, red, normal yellow, high-carotenoid and quality protein maize types. The extraction yield ranged from 4.47 to 10.05%. Bound phenolics extracts had higher content of total phenolics, antioxidant activity and ferulic acid compared to the free phenolics extracts. In general, free phenolics extracts were less cytotoxic than the bound phenolics counterparts. Bound phenolics extracts had higher induction of quinone reductase (QR) and particularly the normal yellow nejayote exerted the highest chemopreventive index tested in Hepa1c1c7 cells. When tested for monofunctional phase 2 induction capacity in BPrc1 cells, the bound phenolics extracts of blue, normal white and quality protein nejayotes were better inducers than the normal yellow counterpart. Particularly, the free phenolics extract of the white maize nejayote induced BPrc1 cells QR and exerted a higher chemopreventive index compared to the bound phenolics extract. Therefore, the nejayote of the normal white maize was the best source of monofunctional phase 2 enzyme inducers.