Diindolylmethane Inhibits Cadmium-Induced Autophagic Cell Death via Regulation of Oxidative Stress in HEL299 Human Lung Fibroblasts.

Cadmium (Cd), a harmful heavy metal, can lead to various pulmonary diseases, including chronic obstructive pulmonary disease (COPD), by inducing cytotoxicity and disturbing redox homeostasis. The aim of the present study was to investigate Cd-mediated cytotoxicity using human lung fibroblasts and the therapeutic potential of 3,3'-diindolylmethane (DIM). Cadmium significantly reduced the cell viability of human embryonic lung (HEL299) cells accompanied by enhanced oxidative stress as evidenced by the increased expression of autophagy-related proteins such as LC3B and p62. However, treatment with DIM significantly suppressed autophagic cell death in Cd-induced HEL299 fibroblasts. In addition, DIM induced antioxidant enzyme activity and decreased intracellular reactive oxygen species (ROS) levels in Cd-damaged HEL299 cells. This study suggests that DIM effectively suppressed Cd-induced lung fibroblast cell death through the upregulation of antioxidant systems and represents a potential agent for the prevention of various diseases related to Cd exposure.

In Vitro and In Vivo Protective Effects of Lentil (Lens culinaris) Extract against Oxidative Stress-Induced Hepatotoxicity.

Excessive oxidative stress plays a role in hepatotoxicity and the pathogenesis of hepatic diseases. In our previous study, the phenolic extract of beluga lentil (BLE) showed the most potent in vitro antioxidant activity among extracts of four common varieties of lentils; thus, we hypothesized that BLE might protect liver cells against oxidative stress-induced cytotoxicity. BLE was evaluated for its protective effects against oxidative stress-induced hepatotoxicity in AML12 mouse hepatocytes and BALB/c mice. H2O2 treatment caused a marked decrease in cell viability; however, pretreatment with BLE (25-100 µg/mL) for 24 h significantly preserved the viability of H2O2-treated cells up to about 50% at 100 µg/mL. As expected, BLE dramatically reduced intracellular reactive oxygen species (ROS) levels in a dose-dependent manner in H2O2-treated cells. Further mechanistic studies demonstrated that BLE reduced cellular ROS levels, partly by increasing expression of antioxidant genes. Furthermore, pretreatment with BLE (400 mg/kg) for 2 weeks significantly reduced serum levels of alanine transaminase and triglyceride by about 49% and 40%, respectively, and increased the expression and activity of glutathione peroxidase in CCl4-treated BALB/c mice. These results suggest that BLE protects liver cells against oxidative stress, partly by inducing cellular antioxidant system; thus, it represents a potential source of nutraceuticals with hepatoprotective effects.

Dual targeting of Nur77 and AMPKα by isoalantolactone inhibits adipogenesis in vitro and decreases body fat mass in vivo.

BACKGROUND: Suppression of adipogenesis has been considered as a potential target for the prevention and treatment of obesity and associated metabolic disorders, and the nuclear receptor 4A1 (NR4A1/Nur77) and AMPKα are known to play important roles during early and intermediate stages of adipogenesis. Therefore, we hypothesized that dual targeting Nur77 and AMPKα would show strong inhibitory effect on adipogenesis. METHODS: We screened a herbal medicine-based small molecule library to identify novel natural compounds dual targeting Nur77 and AMPKα, and the antiadipogenic effects and mechanisms of action of a "hit" compound were studied in 3T3-L1 cells. In vivo antiobesity effects of the compound were also investigated in high-fat diet (HFD)-induced obese mice. RESULTS: We identified isoalantolactone (ISO) as a new NR4A1 inactivator that also activates AMPKα in 3T3-L1 cells. ISO, as expected, inhibited adipogenic differentiation of 3T3-L1 preadipocytes, accompanied by reduced mitotic clonal expansion (MCE) which occurs in the early stage of adipogenesis and decreased expression of genes required for MCE and cell cycle markers including cyclin A, cyclin D1. Furthermore, ISO reduced body weight gain and fat mass (epididymal, subcutaneous, perirenal, and inguinal white adipose tissues) in the high-fat diet-fed C57BL/6 N mice. Serum levels of triglycerides, aspartate transaminase, and alanine transaminase and hepatic steatosis were also significantly improved in the ISO-treated group compared to the high-fat diet control group. CONCLUSIONS: These results suggest that ISO dual targeting Nur77 and AMPKα during adipogenesis represents a novel class of mechanism-based antiadipogenic agents for treatment of obesity and associated metabolic disorders, including hyperlipidemia and fatty liver.

Panax notoginseng Inhibits Tumor Growth through Activating Macrophage to M1 Polarization.

Among the herbal ingredients of HangAmDan-B, a medicinal formula that redirects macrophages to become tumoricidal effectors, we found that Panax notoginseng (Burk.) F. H. Chen is the active component responsible for its macrophage-mediated antitumor activity. The water extracted roots of P. notoginseng (PN) did not affect the viability of RAW264.7 murine macrophage-like cells and murine Lewis lung carcinoma (LLC) cells up to a concentration of 100[Formula: see text][Formula: see text]g/mL. However, the transfer of culture media from PN-treated RAW264.7 cells suppressed the growth of LLC cells. The expression of classically activated (M1) markers, such as interleukin (IL)-1[Formula: see text], monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-[Formula: see text], and inducible nitric oxide synthase (iNOS), was increased by PN treatment. The expression of alternatively activated (M2) markers including CD206, IL-10, and [Formula: see text]-[Formula: see text]-acetylhexosaminidases (YM-1) was reduced by PN treatment in the presence of IL-4. Flow cytometry also revealed that PN drives M1 activation of RAW264.7 cells. The transfer of culture media from PN-treated RAW264.7 cells induced the apoptosis of LLC cells as measured by flow cytometry using Annexin-V staining and western blot analysis for caspase cascade-related proteins. In addition, the results from in vivo tumor allograft model demonstrated that PN reduced both tumor volume and weight. The activation of macrophages toward an M1 phenotype was confirmed in the tumor allograft tumor model. These results collectively show that PN can serve as a potent anticancer agent through reeducation of macrophages toward an M1 phenotype.

Water-extracted tubers of Cyperus rotundus L. enhance endometrial receptivity through leukemia inhibitory factor-mediated expression of integrin αVß3 and αVß5.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (CR) has been traditionally used as an herbal medicine in Asian countries to treat diverse gynecological disorders. However, the potential therapeutic effect of CR on endometrial receptivity for successful embryo implantation to treat female infertility has not been fully studied. AIM OF STUDY: The aim of this study was to evaluate the effect of water-extracted CR on endometrial receptivity by investigating the expression of leukemia inhibitory factor (LIF) and integrins, cell adhesion, and embryo implantation using mifepristone (RU486; RU)-induced implantation failure model. MATERIALS AND METHODS: The water extract of CR was prepared and fingerprinted using high-performance liquid chromatography (HPLC). For the expression and regulation of LIF, reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed in CR-stimulated Ishikawa cells. To evaluate LIF-mediated integrin expression, knockdown of LIF by shRNA was performed in Ishikawa cells. The effect of CR on endometrial receptivity was determined by an in vitro adhesion assay between JAr cells and CR-induced Ishikawa cells. In vivo, C57BL/6 female mice (n = 7 per group) orally received CR (31.68mg/kg/day), a similar dose as used clinically. Seven days after CR treatment, all female mice were caged with male mice until pregnancy was verified. On day 4 of pregnancy, RU (4mg/kg) was injected subcutaneously to induce embryo implantation failure. RESULT: CR increased the expression of LIF through the phosphatidylinositol-3-kinase/ protein kinase B (PI-3K/AKT) signaling pathway in Ishikawa cells. In addition, CR enhanced adhesion of JAr cells onto Ishikawa cells by inducing the expression of LIF-dependent integrins αVß3 and αVß5. Furthermore, CR improved the number of implantation sites in pregnant mice despite RU injection. CONCLUSION: CR increased the expression of LIF-mediated integrins αVß3 and αVß5 on the surface of endometrial cells, which is associated with adhesion of trophoblastic cells to endometrial cells for blastocyst implantation. Our findings provide evidence that CR has therapeutic potential against poor endometrial receptivity.

Effect of Sorbus commixta on the invasion and migration of human hepatocellular carcinoma Hep3B cells.

Tumor metastasis is a main cause of cancer-related morbidity and mortality. Thus, a number of medicinal herbs and phytochemicals have been investigated as possible candidates for the inhibition of cancer metastasis. Sorbus commixta Hedl. (SC) is a traditional medicinal plant used in the treatment of inflammatory diseases, as it has antioxidant, anti-inflammatory, anti-atherosclerotic and anti-hepatotoxic activities. In this study, we demonstrate that the water extract of SC exerts inhibitory effect on the invasion and migration of hepatocellular carcinoma Hep3B cells. The activity and expression of matrix metalloproteinase (MMP)-9, which is responsible for the invasion of cancer cells, was decreased by SC treatment. The invasive and migratory potentials of the Hep3B cells were also decreased, as evidence by in vitro assay using the Boyden chamber system. In addition, the expression of the chemokine receptors, C-X-C chemokine receptor type 4 (CXCR)4 and C-X-C chemokine receptor type 6 (CXCR6), were inhibited by SC in Hep3B cells. Furthermore, actin fiber organization was markedly suppressed by SC treatment. Taken together, the findings of this study suggest for the first time, to the best of our knowledge, that SC suppresses the invasion and migration of highly metastatic Hep3B cells.

Apomorphine suppresses TNF-α-induced MMP-9 expression and cell invasion through inhibition of ERK/AP-1 signaling pathway in MCF-7 cells.

Recent studies have shown that dopamine plays an important role in several types of cancer by inhibiting cell growth and invasion via dopamine receptors (DRs), such as dopamine receptor D2. However, the roles of DR agonists in cancer cell growth and invasion remain unclear. In our study, we found that apomorphine (APO), one of the most commonly prescribed DR agonists, inhibited TNF-α-induced matrix metalloprotease-9 (MMP-9) expression and cell invasion in MCF-7 human breast carcinoma cells through DR-independent pathways. Further mechanistic studies demonstrated that APO suppresses TNF-α-induced transcription of MMP-9 by inhibiting activator protein-1 (AP-1), a well-described transcription factor. This is achieved via extracellular signal-regulated kinases 1 and 2 (ERK1/2). Our study has demonstrated that APO targets human MMP-9 in a DR-independent fashion in MCF-7 cells, suggesting that APO is a potential anticancer agent that can suppress the metastatic progression of cancer cells.

Defatted safflower seed extract inhibits adipogenesis in 3T3-L1 preadipocytes and improves lipid profiles in C57BL/6J ob/ob mice fed a high-fat diet.

In the present study, we hypothesized that defatted safflower seed which is known to be rich in polyphenols might influence adipogenesis and obesity-related disorders, and therefore the anti-adipogenic and hypolipidemic effects of ethanol extract from defatted safflower (Cathamus tinctorius L.) seeds (CSE) were investigated both in cultured 3T3-L1 preadipocytes and in C57BL/6J ob/ob mice fed a high-fat diet. CSE inhibited adipocyte differentiation of 3T3-L1 preadipocytes and decreased expression of the adipogenic transcription factors, SREBP1c and PPARγ, and their target genes. Six-week-old obese (ob/ob) mice were fed a high-fat diet and treated with CSE (50 or 100 mg/kg/day) by oral gavage for 6 weeks. Body fat mass (epididymal and perirenal white adipose tissues) in the CSE-treated groups was significantly lower than that in the high-fat diet control (HFD) group, whereas average daily food intake was not significantly different among the groups. Plasma and hepatic triglyceride levels and plasma low-density lipoprotein cholesterol level were also significantly lower in the CSE groups compared to the HFD group. These results suggest that CSE which decreases body fat mass and improves lipid profiles in plasma and liver, represents a potential treatment option for obesity and associated metabolic disorders, including hyperlipidemia.

Water-extracted Perilla frutescens increases endometrial receptivity though leukemia inhibitory factor-dependent expression of integrins.

The leaves and stems of Perilla frutescens var. acuta Kudo (PF) have been used to prevent threatened abortion in traditional medicine in the East Asian countries. Because reduced receptivity of endometrium is a cause of abortion, we analyzed the action of PF on the endometrial receptivity. PF increased the level of leukemia inhibitory factor (LIF), a major cytokine regulating endometrial receptivity, and LIF receptor in human endometrial Ishikawa cells. The PF-induced LIF expression was mediated by c-jun N-terminal kinase (JNK) and p38 pathways. Adhesion between Ishikawa cells and trophoblastic JAr cells stimulated by PF treatment was abolished by knock down of LIF expression or antagonism of LIFR. In addition, the expressions of integrin ß3 and ß5 were increased by PF treatment in Ishikawa cells. The PF-induced expression of integrin ß3 and ß5 was reduced with an LIFR antagonist. Neutralization of both integrins successfully blocked PF-stimulated adhesion of JAr cells and Ishikawa cells. These results suggest that PF enhanced the adhesion between Ishikawa cells and JAr cells by increasing the expression of integrin ß3 and ß5 via an LIF-dependent pathway. Given the importance of endometrial receptivity in successful pregnancy, PF can be a novel and effective candidate for improving pregnancy rate.

Myristica fragrans Suppresses Tumor Growth and Metabolism by Inhibiting Lactate Dehydrogenase A.

Most cancer cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle, i.e., Warburg's effect. In the pathway, the pyruvate produced by glycolysis is converted to lactic acid by lactate dehydrogenase (LDH). Here, we demonstrated that water extracts from the seeds of Myristica fragrans Houtt. (MF) inhibit the in vitro enzymatic activity of LDH. MF effectively suppressed cell growth and the overall Warburg effect in HT29 human colon cancer cells. Although the expression of LDH-A was not changed by MF, both lactate production and LDH activity were decreased in MF-treated cells under both normoxic and hypoxic conditions. In addition, intracellular ATP levels were also decreased by MF treatment, and the uptake of glucose was also reduced by MF treatment. Furthermore, the experiment on tumor growth in the in vivo mice model revealed that MF effectively reduced the growth of allotransplanted Lewis lung carcinoma cells. Taken together, these results suggest that MF effectively inhibits cancer growth and metabolism by inhibiting the activity of LDH, a major enzyme responsible for regulating cancer metabolism. These results implicate MF as a potential candidate for development into a novel drug against cancer through inhibition of LDH activity.

Hepatoprotective effects of fermented field water-dropwort (Oenanthe javanica) extract and its major constituents.

Dropwort (Oenanthe javanica) has been used for many years for the treatment of inflammatory conditions, including hepatitis. We investigated the protective effects of fermented field water-dropwort extract (FDE) on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in HepG2 cells and carbon tetrachloride (CCl4)-induced liver damage in rats. Pretreatment with FDE prior to the t-BHP treatment of HepG2 cells inhibited cell death and lactate dehydrogenase (LDH) leakage in a dose-dependent manner. In addition FDE significantly prevented the increase of hepatic enzyme markers (ALT, AST) in vivo. Moreover, FDE administration for 7 days significantly affected CYP2E1, CYP4A2, and PPARγ gene expressions. CYP2E1 and CYP4A2 gene expression in the liver, increased 2 and 22-fold by CCl4 administration, respectively, was attenuated to normal levels by pretreatment with FDE. PPARγ gene expression, completely blocked by CCl4 treatment, was increased by FDE pretreatment compared to normal control group. Histopathological examination of the livers also revealed that FDE reduced the incidence of liver lesions. Caffeic acid and chlorogenic acid were identified as major constituents of FDE. These results demonstrate the protective effects of FDE against hepatocytotoxicity induced by CCl4 and t-BHP in rats and HepG2 cells, thus indicating the potential of FDE as a therapeutic for acute liver diseases.