Anti-Obesity Effects of Hovenia Dulcis Branches in Mouse Adipocytes, 3t3-l1 Cells, and High-Fat Diet Obese Mice.

Hovenia dulcis has been reported to have various pharmacological activities, but most studies were done with its fruits. However, from an economic point of view, the use of discarded leaves and branches as by-products is very valuable. In this study, thein vitro andin vivo anti-obesity activities of Hovenia dulcis branch extract (HDB) were investigated to evaluate the applicability of HDB as an anti-obesity agent. In differentiated 3T3-L1 cells, HDB inhibited lipid droplet accumulation. And HDB downregulated CEBPα, PPARγ, and perilipin-1, and upregulated ATGL, p-HSL, HSL, p-AMPK, UCP-1, PGC-1α, PRDM16, LC3-II, and p62/SQSTM1. In addition, HDB increased free glycerol content. In HFD-induced obese mice, HDB reduced body weight and total fat weight. In addition, HDB decreased blood LDL-cholesterol, blood total cholesterol, and blood triglyceride. These results indicate that HDB has anti-obesity activity and HDB can be used as a healthy functional food agent for weight reduction.

Anti-inflammatory activity of the extracts from Rodgersia podophylla leaves through activation of Nrf2/HO-1 pathway, and inhibition of NF-κB and MAPKs pathway in mouse macrophage cells.

OBJECTIVE: Recently, Rodgersia podophylla has been reported to exhibit anti-inflammatory activity. However, little is known about the potential mechanisms about its anti-inflammatory activity. We elucidated the anti-inflammatory mechanisms of leaves extracts from Rodgersia podophylla (RP-L) in RAW264.7 cells. MATERIALS AND METHODS: LPS-induced NO was measured by Griess and mRNA of pro-inflammatory mediators was analyzed by RT-PCR. Cell viability was measured using MTT assay. The protein level was analyzed by Western blot. RESULTS: RP-L significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, IL-1ß and IL-6 in LPS-stimulated RAW264.7 cells. RP-L increased HO-1 expression in RAW264.7 cells, and the inhibition of HO-1 by ZnPP reduced the inhibitory effect of RP-L against LPS-induced NO production in RAW264.7 cells. Inhibition of p38, ROS and GSK3ß attenuated RP-L-mediated HO-1 expression. Inhibition of ROS inhibited p38 phosphorylation and GSK3ß expression induced by RP-L. In addition, inhibition of GSK3ß blocked RP-L-mediated p38 phosphorylation. RP-L induced nuclear accumulation of Nrf2, and inhibition of p38, ROS and GSK3ß abolished RP-L-mediated nuclear accumulation of Nrf2. Furthermore, RP-L blocked LPS-induced degradation of IκB-α and nuclear accumulation of p65. RP-L also attenuated LPS-induced phosphorylation of ERK1/2 and p38. In GC/MS analysis of RP-L, pyrogallol was detected as bioactive compound for anti-inflammatory activity of RP-L. Pyrogallol was observed to activate HO-1 expression through ROS/GSK3ß/p38/Nrf2/HO-1 signaling. CONCLUSIONS: Our results suggest that RP-L exerts potential anti-inflammatory activity by activating ROS/GSK3ß/p38/Nrf2/HO-1 signaling and inhibiting NF-κB and MAPK signaling in RAW264.7 cells. These findings suggest that RP-L may have great potential for the development of anti-inflammatory drug.

Heracleum moellendorffii roots inhibit the production of pro-inflammatory mediators through the inhibition of NF-κB and MAPK signaling, and activation of ROS/Nrf2/HO-1 signaling in LPS-stimulated RAW264.7 cells.

BACKGROUND: Heracleum moellendorffii roots (HM-R) have been long treated for inflammatory diseases such as arthritis, backache and fever. However, an anti-inflammatory effect and the specific mechanism of HM-R were not yet clear. In this study, we for the first time explored the anti-inflammatory of HM-R. METHODS: The cytotoxicity of HM-R against RAW264.7 cells was evaluated using MTT assay. The inhibition of NO and PGE2 production by HM-R was evaluated using Griess reagent and Prostaglandin E2 ELISA Kit, respectively. The changes in mRNA or protein level following HM-R treatment were assessed by RT-PCR and Western blot analysis, respectively. RESULTS: HM-R dose-dependently blocked LPS-induced NO and PGE2 production. In addition, HM-R inhibited LPS-induced overexpression of iNOS, COX-2, IL-1ß and IL-6 in RAW264.7 cells. HM-R inhibited LPS-induced NF-κB signaling activation through blocking IκB-α degradation and p65 nuclear accumulation. Furthermore, HM-R inhibited MAPK signaling activation by attenuating the phosphorylation of ERK1/2, p38 and JNK. HM-R increased nuclear accumulation of Nrf2 and HO-1 expression. However, NAC reduced the increased nuclear accumulation of Nrf2 and HO-1 expression by HM-R. In HPLC analysis, falcarinol was detected from HM-R as an anti-inflammatory compound. CONCLUSIONS: These results indicate that HM-R may exert anti-inflammatory activity by inhibiting NF-κB and MAPK signaling, and activating ROS/Nrf2/HO-1 signaling. These findings suggest that HM-R has a potential as a natural material for the development of anti-inflammatory drugs.

Anti-inflammatory effect of Vaccinium oldhamii stems through inhibition of NF-κB and MAPK/ATF2 signaling activation in LPS-stimulated RAW264.7 cells.

BACKGROUND: Vaccinium oldhamii (V. oldhamii) has been reported to exert a variety of the pharmacological properties such as anti-oxidant activity, anti-cancer activity, and inhibitory activity of α-amylase and acetylcholinesterase. However, the anti-inflammatory activity of V. oldhamii has not been studied. In this study, we aimed to investigate anti-inflammatory activity of the stem extracts from V. oldhamii, and to elucidate the potential mechanisms in LPS-stimulated RAW264.7 cells. METHODS: Cell viability was evaluated by MTT assay. The determination of NO and PGE2 production was performed using Griess reagent and Prostaglandin E2 ELISA Kit, respectively. The change of mRNA or protein level was evaluated by RT-PCR and Western blot. RESULTS: Among VOS, VOL and VOF, the inhibitory effect of NO and PGE2 production induced by LPS was highest in VOS treatment. Thus, VOS was selected for the further study. VOS dose-dependently blocked LPS-induced NO and PGE2 production by inhibiting iNOS and COX-2 expression, respectively. VOS inhibited the expression of pro-inflammatory cytokines such as IL-1ß, IL-6 and TNF-α. In addition, VOS suppressed TRAP activity and attenuated the expression of the osteoclast-specific genes such as NFATc1, c-FOS, TRAP, MMP-9, cathepsin K, CA2, OSCAR and ATPv06d2. VOS inhibited LPS-induced NF-κB signaling activation through blocking IκB-α degradation and p65 nuclear accumulation. VOS inhibited MAPK signaling activation by attenuating the phosphorylation of ERK1/2, p38 and JNK. Furthermore, VOS inhibited ATF2 phosphorylation and blocked ATF2 nuclear accumulation. CONCLUSIONS: These results indicate that VOS may exert anti-inflammatory activity by inhibiting NF-κB and MAPK/ATF2 signaling. From these findings, VOS has potential to be a candidate for the development of chemopreventive or therapeutic agents for the inflammatory diseases.

Extracts from Sageretia thea reduce cell viability through inducing cyclin D1 proteasomal degradation and HO-1 expression in human colorectal cancer cells.

BACKGROUND: Sageretia thea (S. thea) has been used as the medicinal plant for treating hepatitis and fevers in Korea and China. Recently, anticancer activity of S. thea has been reported, but the potential mechanism for the anti-cancer property of S. thea is still insufficient. Thus, we evaluated whether extracts from the leaves (STL) and branches (STB) of S. thea exert anticancer activity and elucidated its potential mechanism in SW480 cells. METHODS: MTT assay was performed for measuring cell viability. Western blot and RT-PCR were used for analyzing the level of protein and mRNA, respectively. RESULTS: Treatment of STL or STB decreased the cell viability and induced apoptosis in SW480 cells. Decreased level of cyclin D1 protein was observed in SW480 cells treated with STL or STB, but no change in cyclin D1 mRNA level was observed with the treatment of STL or STB. MG132 blocked downregulation of cyclin D1 protein by STL or STB. Thr286 phosphorylation of cyclin D1 by STL or STB occurred faster than downregulation of cyclin D1 protein in SW480 cells. When SW480 cells were transfected with T286A-cyclin D1, cyclin D1 degradation by STL or STB did not occur. Inhibition of GSK3ß and cyclin D1 nuclear export attenuated STL or STB-mediated cyclin D1 degradation. In addition, STL or STB increased HO-1 expression, and the inhibition of HO-1 attenuated the induction of apoptosis by STL or STB. HO-1 expression by STL or STB resulted from Nrf2 activation through ROS-dependent p38 activation. CONCLUSIONS: These results indicate that STL or STB may induce GSK3ß-dependent cyclin D1 degradation, and increase HO-1 expression through activating Nrf2 via ROS-dependent p38 activation, which resulted in the decrease of the viability in SW480 cells. These findings suggest that STL or STB may have great potential for the development of anti-cancer drug.

Cytotoxic activity of the twigs of Cinnamomum cassia through the suppression of cell proliferation and the induction of apoptosis in human colorectal cancer cells.

BACKGROUND: Because twigs of Cinnamomum cassia (TC) have been reported to exert anti-cancer activity, the mechanistic study for TC's anti-cancer activity is required. Thus, we elucidated the potential molecular mechanism of TC's anti-proliferative effect and the induction of apoptosis in human colorectal cancer cells. METHODS: How water extracts form TC (TC-HW) was used in this study. Anti-cell proliferative effect of TC-HW was evaluated by MTT assay. The change of protein or mRNA level by TC-HW was evaluated by Western blot and RT-RCR, respectively. The promoter construct for ATF3, NF-κB, TOP-FLASH or FOP-FLASH was used for the investigation of the transcriptional activity for ATF3, NF-κB or Wnt. siRNA for ATF3 or p65 was used for the knockdown of ATF3 and p65. RESULTS: TC-HW reduced the cell viability in human colorectal cancer cells. TC-HW decreased cyclin D1 protein level through cyclin D1 degradation via GSK3ß-dependent threonine-286 (T286) phosphorylation of cyclin D1, indicating that cyclin D1 degradation may contribute to TC-HW-mediated decrease of cyclin D1 protein level. TC-HW downregulated the expression of cyclin D1 mRNA level and inhibited Wnt activation through the downregulation of ß-catenin and TCF4 expression, indicating that inhibition of cyclin D1 transcription may also result in TC-HW-mediated decrease of cyclin D1 protein level. In addition, TC-HW was observed to induce apoptosis through ROS-dependent DNA damage. TC-HW-induced ROS increased NF-κB and ATF3 activation, and inhibition of NF-κB and ATF3 activation attenuated TC-HW-mediated apoptosis. CONCLUSIONS: Our results suggest that TC-HW may suppress cell proliferation through the downregulation of cyclin D1 via proteasomal degradation and transcriptional inhibition, and may induce apoptosis through ROS-dependent NF-κB and ATF3 activation. These effects of TC-HW may contribute to the reduction of cell viability in human colorectal cancer cells. From these findings, TC-HW has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Ethanol extracts from the branch of Taxillus yadoriki parasitic to Neolitsea sericea induces cyclin D1 proteasomal degradation through cyclin D1 nuclear export.

BACKGROUND: Although the inhibitory effect of mistletoe on cancer cell growth has been reported, the underlying mechanisms to explain its anti-proliferative activity are not fully studied. Thus, we elucidated the potential molecular mechanism of the branch from Taxillus yadoriki (TY) parasitic to Neolitsea sericea (NS) (TY-NS-B) for the anti-proliferative effect. METHODS: Anti-cell proliferative effect was evaluated by MTT assay. The change of cyclin D1 protein or mRNA level was evaluated by Western blot and RT-RCR, respectively. RESULTS: In comparison of anti-proliferative effect of TY from the host trees such as Cryptomeria japonica (CJ), Neolitsea sericea (NS), Prunus serrulata (PS), Cinnamomum camphora (CC) and Quercus acutissima (QA), TY-NS showed higher anti-cell proliferative effect than TY-CJ, TY-PS, TY-CC or TY-QA. In addition, the anti-proliferative effect of branch from TY from all host trees was better than leaves. Thus, we selected the branch from Taxillus yadoriki parasitic to Neolitsea sericea (TY-NS-B) for the further study. TY-NS-B inhibited the cell proliferation in the various cancer cells and downregulated cyclin D1 protein level. MG132 treatment attenuated cyclin D1 downregulation of cyclin D1 protein level by TY-NS-B. In addition, TY-NS-B increased threonine-286 (T286) phosphorylation of cyclin D1, and the mutation of T286 to alanine (T286A) blocked cyclin D1 proteasomal degradation by TY-NS-B. But the upstream factors related to cyclin D1 degradation such as ERK1/2, p38, JNK, GSK3ß, PI3K, IκK or ROS did not affect cyclin D1 degradation by TY-NS-B. However, LMB treatment was observed to inhibit cyclin D1 degradation by TY-NS-B, and T286A blocked cyclin D1 degradation through suppressing cyclin D1 redistribution from nucleus to cytoplasm by TY-NS-B. In addition, TY-NS-B activated CRM1 expression. CONCLUSIONS: Our results suggest that TY-NS-B may suppress cell proliferation by downregulating cyclin D1 protein level through proteasomal degradation via T286 phosphorylation-dependent cyclin D1 nuclear export. These findings will provide the evidence that TY-NS-B has potential to be a candidate for the development of chemoprevention or therapeutic agents for human cancer.

Anticancer activity of calyx of Diospyros kaki Thunb. through downregulation of cyclin D1 via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells.

BACKGROUND: Although it has been reported to contain high polyphenols, the pharmacological studies of the calyx of Diospyros kaki Thunb (DKC) have not been elucidated in detail. In this study, we elucidated anti-cancer activity and potential molecular mechanism of DKC against human colorectal cancer cells. METHODS: Anti-cell proliferative effect of 70% ethanol extracts from the calyx of Diospyros kaki (DKC-E70) was evaluated by MTT assay. The effect of DKC-E70 on the expression of cyclin D1 in the protein and mRNA level was evaluated by Western blot and RT-PCR, respectively. RESULTS: DKC-E70 suppressed the proliferation of human colorectal cancer cell lines such as HCT116, SW480, LoVo and HT-29. Although DKC-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by DKC-E70 occurred at the earlier time than that of cyclin D1 mRNA, which indicates that DKC-E70-mediated downregulation of cyclin D1 protein may be a consequence of the induction of degradation and transcriptional inhibition of cyclin D1. In cyclin D1 degradation, we found that cyclin D1 downregulation by DKC-E70 was attenuated in presence of MG132. In addition, DKC-E70 phosphorylated threonine-286 (T286) of cyclin D1 and T286A abolished cyclin D1 downregulation by DKC-E70. We also observed that DKC-E70-mediated T286 phosphorylation and subsequent cyclin D1 degradation was blocked in presence of the inhibitors of ERK1/2, p38 or GSK3ß. In cyclin D1 transcriptional inhibition, DKC-E70 inhibited the expression of ß-catenin and TCF4, and ß-catenin/TCF-dependent luciferase activity. CONCLUSIONS: Our results suggest that DKC-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through cyclin D1 degradation by T286 phosphorylation dependent on ERK1/2, p38 or GSK3ß, and cyclin D1 transcriptional inhibition through Wnt signaling. From these findings, DKC-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Anti-cancer activity of Bacillus amyloliquefaciens AK-0 through cyclin D1 proteasomal degradation via GSK3ß-dependent phosphorylation of threonine-286.

Microorganisms have been regarded as important sources of novel bioactive natural products. In this study, we evaluated the anti-cancer activity and the potential mechanism of Bacillus amyloliquefaciens AK-0 newly isolated from the rhizosphere soil of Korean ginseng. The ethyl acetate fraction from the culture medium of B. amyloliquefaciens AK-0 (EA-AK0) inhibited markedly the proliferation of human colorectal cancer cells such as HCT116, SW480, LoVo and HT-29. EA-AK0 effectively decreased cyclin D1 protein level in human colorectal cancer cells, while cyclin D1 mRNA level was not changed by EA-AK0 treatment. Inhibition of proteasomal degradation by MG132 blocked EA-AK0-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with MRB. In addition, EA-AK0 increased threonine-286 (T286) phosphorylation of cyclin D1, and a point mutation of T286 to alanine attenuated cyclin D1 degradation by EA-AK0. Inhibition of GSK3ß by LiCl suppressed cyclin D1 phosphorylation and downregulation by EA-AKO. From these results, EA-AK0 may suppress the proliferation of human colorectal cancer cells by inducing cyclin D1 proteasomal degradation through GSK3ß-dependent T286 phosphorylation. These results indicate that EA-AK0 could be used for treating colorectal cancer and serve as a potential candidate for anticancer drug development. In addition, these findings will be helpful for expanding the knowledge on the molecular anti-cancer mechanisms of EA-AK0.

In Vitro Anticancer Activity of Phlorofucofuroeckol A via Upregulation of Activating Transcription Factor 3 against Human Colorectal Cancer Cells.

Phlorofucofuroeckol A (PFF-A), one of the phlorotannins found in brown algae, has been reported to exert anti-cancer property. However, the molecular mechanism for the anti-cancer effect of PFF-A has not been known. Activating transcription factor 3 (ATF3) has been reported to be associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which PFF-A stimulates ATF3 expression and apoptosis in human colorectal cancer cells. PFF-A decreased cell viability through apoptosis of human colorectal cancer cells. PFF-A increased ATF3 expression through regulating transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by PFF-A was cAMP response element binding protein (CREB), located between positions -147 and -85 of the ATF3 promoter. Inhibition of p38, c-Jun N-terminal kinases (JNK), glycogen synthase kinase (GSK) 3ß, and IκB kinase (IKK)-α blocked PFF-A-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of poly (ADP-ribose) polymerase (PARP) by PFF-A, while ATF3 overexpression increased PFF-A-mediated cleaved PARP. These results suggest that PFF-A may exert anti-cancer property through inducing apoptosis via the ATF3-mediated pathway in human colorectal cancer cells.

Anti-cancer activity of Psoralea fructus through the downregulation of cyclin D1 and CDK4 in human colorectal cancer cells.

BACKGROUND: Psoralea Fructus (PF), the dried and ripe fruit of Psoralea corylifolia exhibits an anti-cancer activity. However, the molecular mechanisms by which PF inhibits the proliferation of cancer cells have not been elucidated in detail. Cyclin D1 and CDK4 are important regulatory proteins in cell growth and are overexpressed in many cancer cells. In this study, we investigated the molecular mechanism of PF on the downregulation of cyclin D1 and CDK4 level. METHODS: Cell growth was evaluated by MTT assay. The effect of PF on cyclin D1 and CDK4 expression was evaluated by Western blot or RT-PCR. RESULTS: PF suppressed the proliferation of human colorectal cancer cell lines such as HCT116 (IC50: 45.3 ± 1.2 µg/ml), SW480 (IC50: 37.9 ± 1.6 µg/ml), LoVo (IC50: 23.3 ± 1.9 µg/ml µg/ml) HT-29 (IC50 value: 40.7 ± 1.5 µg/ml). PF induced decrease in the protein expression of cyclin D1 and CDK4. However, the mRNA expression of cyclin D1 and CDK4 did not be changed by PF; rather it suppressed the expression of cyclin D1 and CDK4 via the proteasomal degradation. In cyclin D1 degradation, we found that T286 of cyclin D1 play a pivotal role in PF-mediated cyclin D1 degradation. Subsequent experiments with several kinase inhibitors suggest that PF-mediated degradation of cyclin D1 and CDK4 is dependent on ERK1/2 and/or GSK3ß. CONCLUSIONS: Our results suggest that PF has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

TCF4 Is a Molecular Target of Resveratrol in the Prevention of Colorectal Cancer.

The Wnt/ß-catenin pathway plays an essential role in the tumorigenesis of colorectal cancer. T-cell factor-4 (TCF4) is a member of the TCF/LEF (lymphoid enhancer factor) family of transcription factors, and dysregulation of ß-catenin is decisive for the initiation and progression of colorectal cancer. However, the role of TCF4 in the transcriptional regulation of its target gene remained poorly understood. Resveratrol is a dietary phytoalexin and present in many plants, including grape skin, nuts and fruits. Although resveratrol has been widely implicated in anti-tumorigenic and pro-apoptotic properties in several cancer models, the underlying cellular mechanisms are only partially understood. The current study was performed to elucidate the molecular mechanism of the anti-cancer activity of resveratrol in human colorectal cancer cells. The treatment of resveratrol and other phytochemicals decreased the expression of TCF4. Resveratrol decreases cellular accumulation of exogenously-introduced TCF4 protein, but did not change the TCF4 transcription. The inhibition of proteasomal degradation using MG132 (carbobenzoxy-Leu-Leu-leucinal) and lactacystin ameliorates resveratrol-stimulated down-regulation of TCF4. The half-life of TCF4 was decreased in the cells exposed to resveratrol. Resveratrol increased phosphorylation of TCF4 at serine/threonine residues through ERK (extracellular signal-regulated kinases) and p38-dependent pathways. The TCF4 knockdown decreased TCF/ß-catenin-mediated transcriptional activity and sensitized resveratrol-induced apoptosis. The current study provides a new mechanistic link between resveratrol and TCF4 down-regulation and significant benefits for further preclinical and clinical practice.

Anti-inflammatory activity of mushroom-derived hispidin through blocking of NF-κB activation.

BACKGROUND: Hispidin, a polyphenol compound mainly derived from the valuable medicinal mushroom Phellinus species, has been found to possess distinct biological effects. However, the anti-inflammatory potential of hispidin still remains uncharacterized. RESULTS: In this study, the effects of hispidin on activation of nuclear factor kappa B (NF-κB) and the subsequent production of inducible nitric oxide synthase (iNOS) were determined in the lipopolysaccharide (LPS)-induced macrophage RAW 264.7 cells. Our data indicated that hispidin inhibits transcriptional activity of NF-κB in a dose-dependent manner. Hispidin also attenuated LPS-induced NF-κB nuclear translocation and associated inhibitor of kappa B (IκB-α) degradation. Furthermore, hispidin deceased iNOS protein expression and the generation of reactive oxygen species (ROS) in the LPS-induced cells, but did not affect phosphorylation of mitogen-activated protein kinases. CONCLUSION: These findings suggest that hispidin exhibits anti-inflammatory activity through suppressing ROS mediated NF-κB pathway in mouse macrophage cells.

The contribution of activating transcription factor 3 to apoptosis of human colorectal cancer cells by protocatechualdehyde, a naturally occurring phenolic compound.

Protocatechualdehyde (PCA) is one of the important compounds found in barley, green cavendish bananas and grapevine leaves. PCA shows anti-cancer activities in breast, leukemia and colorectal cancer cells. Previous study reported that PCA exerts anti-cancer activity through down-regulating cyclin D1 and HDAC2 in human colorectal cancer cells. However, the underlying mechanisms for the expression of activating transcription factor 3 (ATF3) by PCA has not been studied. Thus, we performed in vitro study to investigate if treatment of PCA affects ATF3 expression and ATF3-mediated apoptosis in human colorectal cancer cells. PCA decreased cell viability in a dose-dependent manner in HCT116 and SW480 cells. In addition, PCA reduced cell viability in MCF-7, MDA-MB-231 and HepG-2 cells. Exposure of PCA activated the levels of ATF3 protein and mRNA in HCT116 and SW480 cells. Inhibition of ERK1/2/ by PD98059 and p38 by SB203580 inhibited PCA-induced ATF3 expression and transcriptional activation. ATF3-knockdown inhibited PCA-induced apoptosis and cell viability. In addition, ATF3 overexpression enhanced PCA-mediated cleavage of PARP. These findings suggest that inhibition of cell viability and apoptosis by PCA may be result of ATF3 expression through ERK1/2 and p38-mediated transcriptional activation.

Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark.

BACKGROUND: Root bark of mulberry (Morus alba L.) has been used in herbal medicine as anti-phlogistic, liver protective, kidney protective, hypotensive, diuretic, anti-cough and analgesic agent. However, the anti-cancer activity and the potential anti-cancer mechanisms of mulberry root bark have not been elucidated. We performed in vitro study to investigate whether mulberry root bark extract (MRBE) shows anti-inflammatory and anti-cancer activity. METHODS: In anti-inflammatory activity, NO was measured using the griess method. iNOS and proteins regulating NF-κB and ERK1/2 signaling were analyzed by Western blot. In anti-cancer activity, cell growth was measured by MTT assay. Cleaved PARP, ATF3 and cyclin D1 were analyzed by Western blot. RESULTS: In anti-inflammatory effect, MRBE blocked NO production via suppressing iNOS over-expression in LPS-stimulated RAW264.7 cells. In addition, MRBE inhibited NF-κB activation through p65 nuclear translocation via blocking IκB-α degradation and ERK1/2 activation via its hyper-phosphorylation. In anti-cancer activity, MRBE deos-dependently induced cell growth arrest and apoptosis in human colorectal cancer cells, SW480. MRBE treatment to SW480 cells activated ATF3 expression and down-regulated cyclin D1 level. We also observed that MRBE-induced ATF3 expression was dependent on ROS and GSK3ß. Moreover, MRBE-induced cyclin D1 down-regulation was mediated from cyclin D1 proteasomal degradation, which was dependent on ROS. CONCLUSIONS: These findings suggest that mulberry root bark exerts anti-inflammatory and anti-cancer activity.

Anti-cancer activity of Ginger (Zingiber officinale) leaf through the expression of activating transcription factor 3 in human colorectal cancer cells.

BACKGROUND: Ginger leaf (GL) has long been used as a vegetable, tea and herbal medicine. However, its pharmacological properties are still poorly understood. Thus, we performed in vitro studies to evaluate anti-cancer properties of ginger leaf and then elucidate the potential mechanisms involved. METHODS: Cell viability was measured by MTT assay. ATF3 expression level was evaluated by Western blot or RT-PCR and ATF3 transcriptional activity was determined using a dual-luciferase assay kit after the transfection of ATF3 promoter constructs. In addition, ATF3-dependent apoptosis was evaluated by Western blot after ATF3 knockdown using ATF3 siRNA. RESULTS: Exposure of GL to human colorectal cancer cells (HCT116, SW480 and LoVo cells) reduced the cell viability and induced apoptosis in a dose-dependent manner. In addition, GL reduced cell viability in MCF-7, MDA-MB-231 and HepG-2 cells. ATF3 knockdown attenuated GL-mediated apoptosis. GL increased activating transcription factor 3 (ATF3) expressions in both protein and mRNA level and activated ATF3 promoter activity, indicating transcriptional activation of ATF3 gene by GL. In addition, our data showed that GL-responsible sites might be between -318 and -85 region of the ATF3 promoter. We also observed that ERK1/2 inhibition by PD98059 attenuated GL-mediated ATF3 expression but not p38 inhibition by SB203580, indicating ERK1/2 pathway implicated in GL-induced ATF3 activation. CONCLUSIONS: These findings suggest that the reduction of cell viability and apoptosis by GL may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression through ERK1/2 activation in human colorectal cancer cells.

The induction of activating transcription factor 3 (ATF3) contributes to anti-cancer activity of Abeliophyllum distichum Nakai in human colorectal cancer cells.

BACKGROUND: Recently, Abeliophyllum distichum Nakai (A. distichum) has been reported to exert the inhibitory effect on angiotensin converting enzyme. However, no specific pharmacological effects from A. distichum have been described. We performed in vitro study to evaluate anti-cancer properties of A. distichum and then elucidate the potential mechanisms. METHODS: Cell viability was measured by MTT assay. ATF3 expression level was evaluated by Western blot or RT-PCR and ATF3 transcriptional activity was determined using a dual-luciferase assay kit after the transfection of ATF3 promoter constructs. In addition, ATF3-dependent apoptosis was evaluated by Western blot after ATF3 knockdown using ATF3 siRNA. RESULTS: Exposure of ethyl acetate fraction from the parts of A. distichum including flower, leaf and branch to human colorectal cancer cells, breast cancer cells and hepatocellular carcinoma reduced the cell viability. The branch extracts from A. distichum (EAFAD-B) increased the expression of activating transcription factor 3 (ATF3) and promoter activity, indicating transcriptional activation of ATF3 gene by EAFAD-B. In addition, our data showed that EAFAD-B-responsible sites might be between -147 and -85 region of the ATF3 promoter. EAFAD-B-induced ATF3 promoter activity was significantly decreased when the CREB site was deleted. However, the deletion of Ftz sites did not affect ATF3 promoter activity by EAFAD-B. We also observed that inhibition of p38MAPK and GSK3ß attenuated EAFAD-B-mediated ATF3 promoter activation. Also, EAFAD-B contributes at least in part to increase of ATF3 accumulation. CONCLUSION: These findings suggest that the anti-cancer activity of EAFAD-B may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression.

Inhibitory Activity of Sparassis latifolia on the Lipid Accumulation through Suppressing Adipogenesis and Activating Lipolysis in 3T3-L1 Cells.

Sparassis latifolia (SL) has been reported to exhibit anti-obesity effects in high-fat diet animal models, yet research into its mechanisms of action remains limited. Therefore, this study aimed to elucidate the mechanisms behind the anti-obesity activity of SL's 30% ethanol extract (SL30E) using 3T3-L1 cells in an in vitro setting. SL30E effectively mitigated the accumulation of lipid droplets and triacylglycerol. SL30E downregulated PPARγ and CEBPα protein levels. The diminishment of PPARγ and C/EBPα, facilitated by SL30E, was impeded by the knockdown of ß-catenin using ß-cateninspecific siRNA. Furthermore, SL30E was observed to increase the protein levels of ATGL and p-HSL, while it concurrently decreased the protein levels of perilipin-1. SL30E downregulated p62/SQSTM1 protein level and upregulated LC3-II protein level. Moreover, SL30E was demonstrated to elevate the protein levels of p-AMPK and PGC-1α. The results indicate that SL30E inhibits lipid accumulation by suppressing adipogenesis and inducing lipolysis, lipophagy, and thermogenesis in 3T3-L1 cells. These observations provide potential insights into the mechanisms underlying the anti-obesity effects of SL, contributing valuable information to the existing body of knowledge.

Anti­obesity and immunostimulatory activity of Chrysosplenium flagelliferum in mouse preadipocytes 3T3­L1 cells and mouse macrophage RAW264.7 cells.

Chrysosplenium flagelliferum (CF) is known for its anti-inflammatory, antioxidant and antibacterial activities. However, there is a lack of research on its other pharmacological properties. In the present study, the bifunctional roles of CF in 3T3-L1 and RAW264.7 cells were investigated, focusing on its anti-obesity and immunostimulatory effects. In 3T3-L1 cells, CF effectively mitigated the accumulation of lipid droplets and triacylglycerol. Additionally, CF downregulated the peroxisome proliferator-activated receptor (PPAR)-γ and CCAAT/enhancer-binding protein α protein levels; however, this effect was impeded by the knockdown of ß-catenin using ß-catenin-specific small interfering RNA. Consequently, CF-mediated inhibition of lipid accumulation was also decreased. CF increased the protein levels of adipose triglyceride lipase and phosphorylated hormone-sensitive lipase, while decreasing those of perilipin-1. Moreover, CF elevated the protein levels of phosphorylated AMP-activated protein kinase and PPARγ coactivator 1-α. In RAW264.7 cells, CF enhanced the production of pro-inflammatory mediators, such as nitric oxide (NO), inducible NO synthase, interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α, and increased their phagocytic capacities. Inhibition of Toll-like receptor (TLR)-4 significantly reduced the effects of CF on the production of pro-inflammatory mediators and phagocytosis, indicating its crucial role in facilitating these effects. CF-induced increase in the production of pro-inflammatory mediators was controlled by the activation of c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-κB pathways, and TLR4 inhibition attenuated the phosphorylation of these kinases. The results of the pesent study suggested that CF inhibits lipid accumulation by suppressing adipogenesis and inducing lipolysis and thermogenesis in 3T3-L1 cells, while stimulating macrophage activation via the activation of JNK and NF-κB signaling pathways mediated by TLR4 in RAW264.7 cells. Therefore, CF simultaneously exerts both anti-obesity and immunostimulatory effects.

Immunostimulatory activity of the aqueous extract from the leaves of Sambucus racemosa subsp. pendula through TLR4­dependent JNK activation in RAW264.7 cells.

Sambucus racemosa subsp. pendula (SRP) is an endemic plant of Korea, exclusively found on Ulleungdo Island. SRP is widely used as both a traditional medicine and food source. However, there is a lack of research on the pharmacological activities of SRP. Therefore, the present study aimed to explore the potential use of SRP leaves (SRPL) as a natural immunostimulant by analyzing its macrophage activation properties and the underlying mechanisms of action. Among the various extraction conditions, SRPL (AE20-SRPL) extracted with 100% distilled water at 20˚C induced the highest nitric oxide (NO) production in RAW264.7 cells. Thus, the further studies were performed using AE20-SRPL. AE20-SRPL increased the production of immunostimulatory factors such as NO, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2, IL-1ß and TNF-α and phagocytosis in a dose-dependent manner in RAW264.7 cells without exhibiting cytotoxicity. Among Toll-like receptor (TLR)2 and TLR4, inhibition of TLR4 significantly reduced AE20-SRPL-mediated increases in the production of immunostimulatory factors and phagocytosis in RAW264.7 cells. Furthermore, in RAW264.7 cells, inhibition of JNK, one of the components of MAPK signaling along with ERK1/2 and p38, attenuated the AE20-SRPL-mediated increases in the production of immunostimulatory factors and phagocytosis. Additionally, AE20-SRPL induced the phosphorylation of JNK and inhibition of TLR4 reduced AE20-SRPL-mediated JNK phosphorylation. These results suggested that AE20-SRPL may enhance the production of immunostimulatory factors and phagocytosis through TLR4-dependent activation of JNK in macrophages. Although the present study is limited to in vitro research using a cell model, AE20-SRPL demonstrated potential as a natural material capable of inducing macrophage activation for immune enhancement.