Protaetia Brevitarsis-Derived Protein Hydrolysate Reduces Obesity-Related Colitis Induced by High-Fat Diet in Mice through Anti-Inflammatory Pathways.

Ulcerative colitis is an inflammatory bowel disease characterized by inflammation in the mucosal and submucosal layers of the colon. Obesity is closely related to the occurrence and progression of colitis. The most plausible mechanism linking obesity and colitis is an excessive adipogenesis-related inflammatory response, which causes mucosal dysfunction. Obesity and colitis are linked by several etiologic mechanisms, including excessive adipogenesis, lipotoxicity, pro-inflammatory adipokines/cytokines, macrophage polarization, oxidative stress, endoplasmic reticulum (ER) stress, and gut microbiota. These low-grade enteric inflammations cause mucosal layer damage, especially goblet cell dysfunction through mucin 2 (MUC2) misfolding, ultimately leading to colitis. Inhibiting the inflammatory response can be the most effective approach for treating obesity-related colitis. We focused on the anti-inflammatory effects of polyphenols in Protaectia brevitas larvae. The P. brevitas was prepared as a low molecular protein hydrolysate (PHPB) to increase the concentration of anti-inflammatory molecules. In the current study, we investigated the anti-inflammatory effect of PHPB in an obesity-induced colitis mouse model. Compared with the high-fat diet (HFD) group, the group treated with PHPB exhibited reduced body/organ/fat weight, appetite/food intake inhibition, hypolipidemic effect on ectopic fat, and anti-adipogenic mechanism through the AMPK signaling pathway. Furthermore, we observed attenuated expression of PPARγ and C/EBPα, inhibition of pro-inflammatory molecules, stimulation of anti-inflammatory molecules, probiotic-like effect against obesogenic gut microbiota, inhibition of macrophage polarization into M1, suppression of oxidative/ER stress, and reduction of Muc2 protein misfolding in colon. These diverse anti-inflammatory responses caused histological and functional recovery of goblet cells, eventually improving colitis. Therefore, our findings suggest that the protein hydrolysate of Protaetia brevitarsis can improve obesity-related colitis through its anti-inflammatory activities.

Nrf2-mediated protective effect of protein hydrolysates from Protaetia brevitarsis larvae against oxidative stress-induced hepatotoxicity.

In this study, we hypothesized that Protaetia brevitarsis larvae (PBL) protein hydrolysates, which have been previously reported to exhibit strong antioxidant activity, might protect liver cells against oxidative stress-induced injury. Thus, the cytoprotective effects and related mechanisms of PBL hydrolysates were investigated in AML12 liver cells. Among PBL hydrolysates prepared by three different proteases, the PBL flavouryzme hydrolysate showed significantly higher protective effect against H2O2-induced cytotoxicity than other hydrolysates in AML12 cells. Further mechanistic studies showed that pretreatment with PFH reduces cellular level of reactive oxygen species through induction of Nrf2-mediated antioxidant enzymes such as catalase and heme oxygenase-1. In addition, the free amino acid analysis revealed that PFH was rich in branched-chain amino acids, aromatic amino acids, and hydrophobic amino acids as compared to other hydrolysates, which could contribute to its hepatoprotective effect. These findings suggest that PFH represents a potential source of nutraceuticals that supports liver functions. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01279-0.

Flavone and Hydroxyflavones Are Ligands That Bind the Orphan Nuclear Receptor 4A1 (NR4A1).

It was recently reported that the hydroxyflavones quercetin and kaempferol bind the orphan nuclear receptor 4A1 (NR4A1, Nur77) and act as antagonists in cancer cells and tumors, and they inhibit pro-oncogenic NR4A1-regulated genes and pathways. In this study, we investigated the interactions of flavone, six hydroxyflavones, seven dihydroxyflavones, three trihydroxyflavones, two tetrahydroxyflavones, and one pentahydroxyflavone with the ligand-binding domain (LBD) of NR4A1 using direct-binding fluorescence and an isothermal titration calorimetry (ITC) assays. Flavone and the hydroxyflavones bound NR4A1, and their KD values ranged from 0.36 µM for 3,5,7-trihydroxyflavone (galangin) to 45.8 µM for 3'-hydroxyflavone. KD values determined using ITC and KD values for most (15/20) of the hydroxyflavones were decreased compared to those obtained using the fluorescence assay. The results of binding, transactivation and receptor-ligand modeling assays showed that KD values, transactivation data and docking scores for these compounds are highly variable with respect to the number and position of the hydroxyl groups on the flavone backbone structure, suggesting that hydroxyflavones are selective NR4A1 modulators. Nevertheless, the data show that hydroxyflavone-based neutraceuticals are NR4A1 ligands and that some of these compounds can now be repurposed and used to target sub-populations of patients that overexpress NR4A1.

Plant Alkaloid Tetrandrine Is a Nuclear Receptor 4A1 Antagonist and Inhibits Panc-1 Cell Growth In Vitro and In Vivo.

The orphan nuclear receptor 4A1 (NR4A1) is highly expressed in human pancreatic cancer cells and exerts pro-oncogenic activity. In a previous study, we demonstrated that fangchinoline (FCN), a natural inhibitor of nuclear NR4A1, induces NR4A1-dependent apoptosis in human pancreatic cancer cells. In this study, we evaluated FCN and its structural analogs (berbamine, isotetrandrine, tetrandrine, and tubocurarine) for their inhibitory effects on NR4A1 transactivity, and confirmed that tetrandrine (TTD) showed the highest inhibitory effect in pancreatic cancer cells. Moreover, in a tryptophan fluorescence quenching assay, TTD directly bound to the ligand binding domain (LBD) of NR4A1 with a KD value of 10.60 µM. Treatment with TTD decreased proliferation and induced apoptosis in Panc-1 human pancreatic cancer cells in part through the reduced expression of the Sp1-dependent anti-apoptotic gene survivin and induction of ROS-mediated endoplasmic reticulum stress, which are the well-known NR4A1-dependent proapoptotic pathways. Furthermore, at a dose of 25 mg/kg/day, TTD reduced tumor growth in an athymic nude mouse xenograft model bearing Panc-1 cells. These data show that TTD is an NR4A1 antagonist and that modulation of the NR4A1-mediated pro-survival pathways is involved in the antitumor effects of TTD.

Antiobesity and Hepatoprotective Effects of Protein Hydrolysates Derived from Protaetia brevitarsis in an Obese Mouse Model.

Background: Obesity induced by excessive nutrients can cause fatty liver and metabolic dysfunction, which leads to hepatic dysfunction and local/systemic inflammatory responses. Previously, we analyzed the antioxidant, antilipotoxicity, and anti-inflammatory effects of protein hydrolysates in vitro. The aim of the present study is to investigate the antiobesity and hepatoprotective effects of protein hydrolysates derived from Protaectia brevitas (PHPB) in an obese mouse model. Methods: For this in vivo study, 40 mice were included and divided into four groups: (1) normal diet group, (2) high-fat-diet (ctrl(-)) group, (3) high-fat-diet and silymarin-treated (ctrl(+)) group, and (4) high-fat-diet and PHPB-treated group. After 6 weeks of treatment, body weight and the amount of daily food intake were observed. Moreover, the major organs and blood of animals were collected for the analysis of serum chemistry, histopathological examination, and obesity- and inflammation-related gene expressions. Results: The body weight and the amount of daily food intake significantly decreased in the PHPB-treated group compared with those in the ctrl(-) group. The levels of serum ALT, AST, ALP, creatinine, blood urea nitrogen, glucose, bilirubin, total cholesterol, TG, low-density lipoprotein, IL-6, TNF-α, and IGF-1 significantly reduced in the PHPB-treated group, whereas the serum free fatty acid, albumin, high-density lipoprotein, and adiponectin concentrations increased. In the analysis of weight of the liver, kidney, lungs, spleen, and fat tissues (from epididymal, perirenal, and mesentery tissues), the PHPB-treated group showed decreased values compared with the ctrl(-) group. In the histopathological analysis, the PHPB-treated group showed significantly reduced macrovesicular fatty change and inflammatory cell infiltration in the liver, and the size of the adipocyte in the epididymis also significantly decreased. The obesity- and inflammation-related gene (IL-6, TNF-α, IGF-1, leptin, AP2/FABP4, AMPK-α2, ß3AR, and PPAR-γ) expressions in the liver and epididymal adipose tissue were reduced in the PHPB-treated group. Conclusions: Overall, the results of this study suggest that the protein hydrolysates that derived from Protaectia brevitas produce antiobesity and hepatoprotective effects via anti-inflammatory activities.

Flavonoids kaempferol and quercetin are nuclear receptor 4A1 (NR4A1, Nur77) ligands and inhibit rhabdomyosarcoma cell and tumor growth.

BACKGROUND: Flavonoids exhibit both chemopreventive and chemotherapeutic activity for multiple tumor types, however, their mechanisms of action are not well defined. Based on some of their functional and gene modifying activities as anticancer agents, we hypothesized that kaempferol and quercetin were nuclear receptor 4A1 (NR4A1, Nur77) ligands and confirmed that both compounds directly bound NR4A1 with KD values of 3.1 and 0.93 µM, respectively. METHODS: The activities of kaempferol and quercetin were determined in direct binding to NR4A1 protein and in NR4A1-dependent transactivation assays in Rh30 and Rh41 rhabdomyosarcoma (RMS) cells. Flavonoid-dependent effects as inhibitors of cell growth, survival and invasion were determined in XTT and Boyden chamber assays respectively and changes in protein levels were determined by western blots. Tumor growth inhibition studies were carried out in athymic nude mice bearing Rh30 cells as xenografts. RESULTS: Kaempferol and quercetin bind NR4A1 protein and inhibit NR4A1-dependent transactivation in RMS cells. NR4A1 also regulates RMS cell growth, survival, mTOR signaling and invasion. The pro-oncogenic PAX3-FOXO1 and G9a genes are also regulated by NR4A1 and, these pathways and genes are all inhibited by kaempferol and quercetin. Moreover, at a dose of 50 mg/kg/d kaempferol and quercetin inhibited tumor growth in an athymic nude mouse xenograft model bearing Rh30 cells. CONCLUSION: These results demonstrate the clinical potential for repurposing kaempferol and quercetin for clinical applications as precision medicine for treating RMS patients that express NR4A1 in order to increase the efficacy and decrease dosages of currently used cytotoxic drugs.

Broussochalcone A Is a Novel Inhibitor of the Orphan Nuclear Receptor NR4A1 and Induces Apoptosis in Pancreatic Cancer Cells.

The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity, and NR4A1 silencing and treatment with its inactivators has been shown to inhibit pancreatic cancer cells and tumor growth. In this study, we identified broussochalcone A (BCA) as a new NR4A1 inhibitor and demonstrated that BCA inhibits cell growth partly by inducing NR4A1-mediated apoptotic pathways in human pancreatic cancer cells. BCA downregulated specificity protein 1 (Sp1)-mediated expression of an anti-apoptotic protein, survivin, and activated the endoplasmic reticulum (ER) stress-mediated apoptotic pathway. These results suggest that NR4A1 inactivation contributes to the anticancer effects of BCA, and that BCA represents a potential anticancer agent targeting NR4A1 that is overexpressed in many types of human cancers.

Caesalpinia sappan induces apoptotic cell death in ectopic endometrial 12Z cells through suppressing pyruvate dehydrogenase kinase 1 expression.

Endometriosis is a common gynecological disease defined as the growth of endometrial tissues outside the uterus. Although the mechanism underlying the progression of endometriosis has not been fully elucidated, cancer-like aerobic glycolysis is considered to mediate the elevated growth and resistance to apoptosis of endometriotic cells. The heartwood of Caesalpinia sappan L. (family Leguminosae) is a herbal medicinal product used to treat gynecological symptoms, including algomenorrhea and amenorrhea. The results of the present study revealed that endometriotic 12Z cells exhibited more rapid growth than normal endometrial cells (THES). The expression levels of pyruvate dehydrogenase kinase (PDK)1 and 3 and lactate production were higher in 12Z cells than in THES cells. In addition, the 12Z cells were more sensitive to the cytotoxicity of the aqueous extract of C. sappan heartwood (CS) than the THES cells. CS inhibited lactate production and phosphorylation of pyruvate dehydrogenase A by reducing the expression of PDK1. CS also increased mitochondrial reactive oxygen species (ROS) levels, decreased mitochondrial membrane potential and consequently stimulated the apoptosis of 12Z cells. CS-induced cell death was substantially inhibited by exogenous PDK1 expression. In conclusion, CS may be a novel drug candidate for treating endometriosis by inhibiting aerobic glycolysis and inducing ROS-mitochondria-mediated apoptotic cell death.

Renal Protective Effect of Beluga Lentil Pretreatment for Ischemia-Reperfusion Injury.

MATERIALS AND METHODS: Mice were divided into four groups: normal, untreated, low- (2 mg), and high-dose (8 mg) beluga lentil treatment groups. Beluga lentil was orally administered for 2 weeks, followed by bilateral renal ischemia for 20 min and reperfusion for 30 min. Blood samples and kidney tissues were collected and analyzed to investigate renal function, histopathology, epithelial and endothelial cell damage, apoptosis, oxidative stress, and inflammatory responses. RESULTS: The pretreated groups maintained renal function, with significantly lower blood urea nitrogen (BUN) and creatinine levels, compared with the other groups. The histopathological analysis showed reduced proximal tubule injury and decreased injury-related molecule (kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)) secretion in the pretreated groups compared with the other groups. Terminal deoxynucleotidyl transferase dUTP nick-end labeling- (TUNEL-) positive cells and the secretion of apoptosis-related molecules (Fas and caspase 3) were significantly reduced in the pretreated groups compared with the other groups. The pretreated groups showed positive microvessel-associated gene (cluster of differentiation (CD31)) expression and negative adhesion molecule (intracellular adhesion molecule 1 (ICAM-1)) expression. An antioxidant effect was observed in the pretreatment groups, with reduced malonaldehyde (MDA) expression and increased antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx)) secretion. In the pretreated groups, F4/80+ macrophages and CD4+ T cell infiltration were inhibited and proinflammatory cytokine (interleukin- (IL-) 1ß, IL-6, and tumor necrosis factor- (TNF-) α) levels decreased; however, the levels of anti-inflammatory cytokines (transforming growth factor- (TGF-) ß, IL-10, and IL-22) increased. CONCLUSIONS: Beluga lentil pretreatment demonstrated protective effects against I/R-induced renal damage, via antiapoptotic, anti-inflammatory, and antioxidant activities.

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.

Novel hepatoprotective peptides derived from protein hydrolysates of mealworm (Tenebrio molitor).

In the present study, we hypothesized that protein hydrolysates of mealworm (Tenebrio molitor) which is known to exert significant scavenging activity toward reactive oxygen species (ROS) might protect liver cells against ROS-induced cytotoxicity. Therefore, hepatoprotective effects of protein hydrolysates of mealworm and their underlying mechanisms were investigated in AML12 mouse liver cells and the responsible peptides were further identified. Pretreatment with the mealworm alcalase hydrolysate (MAH; <1 kDa) showed the highest protective effect against H2O2-induced cytotoxicity in AML12 cells among three mealworm hydrolysates produced by different proteases (alcalase, flavourzyme, and neutrase). Further mechanistic studies demonstrated that MAH reduces ROS levels through increasing NF-E2-related factor 2-mediated expression of catalase, heme oxygenase-1, and genes involved in glutathione synthesis. Moreover, two novel hepatoprotective peptides, Ala-Lys-Lys-His-Lys-Glu and Leu-Glu, which shared similar mechanisms of action with MAH were identified. These results suggest that MAH and the two peptides represent potential sources of natural hepatoprotective agents.

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.

Water-extracted branch of Cinnamomum cassia promotes lung cancer cell apoptosis by inhibiting pyruvate dehydrogenase kinase activity.

Cinnamomum cassia Blume has been widely reported as the anti-tumor agent. However, the precise mechanism underlying its pro-apoptotic action is still not clear. Restraining aerobic glycolysis through suppression of pyruvate dehydrogenase kinase (PDHK) is a promising strategy for cancer inhibition. In this study, we performed to investigate the anti-tumor action of C. cassia is mediated by PDHK inhibition. The inhibition of water-extracted branch of C. cassia (WBCC) on the activity of PDHK using both in vitro and cell-based kinase assay were examined in several lung cancer cells. WBCC reduced viabilities of several lung cancer cells with minimal cytotoxicity on normal bronchial epithelial cells. WBCC decreased lactate production through inhibiting activity of PDHK. In consequence of PDHK inhibition, WBCC increased ROS production, which damage mitochondria membrane stability. In addition, WBCC induced ROS- and mitochondria-dependent apoptotic cell death. Among the components of WBCC, cinnamic acid was founded as a major inhibitor on PDHK activity. This is first report that WBCC induces apoptosis of lung cancer cells through inhibiting PDHK activity. Our findings suggest that WBCC and cinnamic acid can be potential candidates for developing novel anti-cancer drugs through glycolysis metabolism.

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.

Sorbus commixta water extract induces apoptotic cell death via a ROS-dependent pathway.

The stembark of Sorbus commixta Hedl. has been used for treating asthma, bronchitis, gastritis and edema. However, the anticancer and proapoptotic effects of the water extract of the stembark of S. commixta (SCE) remain unknown. In the present study, it was shown that SCE inhibited the cell viability of the hepatocellular carcinoma cell lines Hep3B and HepG2, and of the colon carcinoma cell line HCT116. DNA content analysis indicated that SCE increased the sub-G1 population of HCT116 cells. In addition, degradation of nuclear DNA and levels of proapoptotic cascade components, including caspase-9, caspase-3 and poly ADP-ribose polymerase, were augmented by SCE treatment. Mitochondrial membrane potential and the ratio of B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax) were also reduced. Furthermore, SCE increased the expression of proapoptotic proteins, including p21, p27 and p53. Mouse double minute 2 homology, a negative regulator of p53, was cleaved by SCE treatment. Intracellular reactive oxygen species (ROS) production was also increased by SCE treatment. However, the SCE-induced cytotoxic effects and the increased expression of proapoptotic proteins, including p53 and p21, and reduced Bcl-2/Bax ratio, could be attenuated by N-acetyl cysteine, an ROS inhibitor. Taken together, these results indicate that SCE is a potent proapoptotic herbal medicine, which exerts its effects via the ROS-mediated mitochondrial pathway.

Inactivation of the orphan nuclear receptor NR4A1 contributes to apoptosis induction by fangchinoline in pancreatic cancer cells.

Previous studies have demonstrated that the orphan nuclear receptor NR4A1 is overexpressed in human pancreatic cancer and antagonizing this receptor promotes apoptosis and inhibits pancreatic cancer cells and tumor growth. In the present study, we identified fangchinoline, a bisbenzyltetrahydroisoquinoline alkaloid from Stephania tetrandra, as a new inactivator of nuclear NR4A1 and demonstrated that fangchinoline inhibits cell proliferation and induces apoptosis, in part, via the NR4A1-dependent pro-apoptotic pathways in human pancreatic cancer cells. It decreased expression of the antiapoptotic protein survivin by inhibiting Sp1-mediated transcription and induced oxidative stress-mediated endoplasmic reticulum (ER) stress in pancreatic cancer cells. These results suggest that inhibition of NR4A1-mediated transcriptional activity was involved in the anticancer effects of fangchinoline, and fangchinoline represents a novel class of mechanism-based anticancer agents targeting NR4A1 that is overexpressed in pancreatic cancer.

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.