Rhamnus crenata leaf extracts exhibit anti-inflammatory activity via modulating the Nrf2/HO-1 and NF-κB/MAPK signaling pathways

Objective: To elucidate the potential anti-inflammatory mechanisms of Rhamnus crenata leaf extracts using RAW264.7 cells. Methods: We used 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay to measure cell viability. Nitric oxide (NO) production was measured using Griess reagent. Western blotting and RT-PCR assays were carried out for analyzing the protein and gene expressions of pro-inflammatory mediators, respectively. Moreover, PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and BAY11-7082 (NF-κB inhibitor) were used to evaluate the anti-inflammatory mechanism of Rhamnus crenata leaf extract. Results: Rhamnus crenata leaf extracts significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, COX-2, IL-1β, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Rhamnus crenata leaf extracts also suppressed LPS-induced degradation of IκB-α and nuclear accumulation of p65, which resulted in the inhibition of NF-κB activation in RAW264.7 cells. Additionally, the extracts attenuated the phosphorylation of p38, ERK1/2, and JNK in LPS-stimulated RAW264.7 cells. Moreover, HO-1 expression induced by Rhamnus crenata leaf extracts was significantly downregulated by SB230580, PD98059, SP600125 and BAY11-7082. Conclusions: Rhamnus crenata leaf extract may upregulate HO-1 expression through inhibition of p38, ERK1/2, and NF-κB activation, which may contribute to the anti-inflammatory activity of the extracts. Rhamnus crenata leaf extracts may have great potential for the development of anti-inflammatory drugs to treat acute and chronic inflammatory diseases.

Kahweol from Coffee Induces Apoptosis by Upregulating Activating Transcription Factor 3 in Human Colorectal Cancer Cells

Kahweol as a coffee-specific diterpene has been reported to induce apoptosis in human cancer cells. Although some molecular targets for kahweol-mediated apoptosis have been elucidated, the further mechanism for apoptotic effect of kahweol is not 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 kahweol stimulates ATF3 expression and apoptosis in human colorectal cancer cells. Kahweol increased apoptosis in human colorectal cancer cells. It also increased ATF3 expression through the transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by kahweol was CREB located between −147 to −85 of ATF3 promoter. ATF3 overexpression increased kahweol-mediated cleaved PARP, while ATF3 knockdown attenuated the cleavage of PARP by kahweol. Inhibition of ERK1/2 and GSK3β blocked kahweol-mediated ATF3 expression. The results suggest that kahweol induces apoptosis through ATF3-mediated pathway in human colorectal cancer cells.

Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells

Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-κB-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of β-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA. The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin. In addition, silymarin increased phosphorylation of β-catenin and a point mutation of S33Y attenuated silymarin-mediated β-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of β-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells.

Naringenin-Mediated ATF3 Expression Contributes to Apoptosis in Human Colon Cancer

Naringenin (NAR) as one of the flavonoids observed in grapefruit has been reported to exhibit an anti-cancer activity. Activating transcription factor 3 (ATF3) is associated with apoptosis in human colon cancer cells. This study was performed to investigate the molecular mechanism by which NAR stimulates ATF3 expression and apoptosis in human colon cancer cells. NAR reduced the cell viability and induced an apoptosis in human colon cancer cells. ATF3 overexpression increased NAR-mediated cleaved PARP, while ATF3 knockdown attenuated the cleavage of PARP by NAR. NAR increased ATF3 expression in both protein and mRNA level, and increased the luciferase activity of ATF3 promoter in a dose-dependent manner. The responsible region for ATF3 transcriptional activation by NAR is located between -317 and -148 of ATF3 promoter. p38 inhibition blocked NAR-mediated ATF3 expression, its promoter activation and apoptosis. The results suggest that NAR induces apoptosis through p38-dependent ATF3 activation in human colon cancer cells.

Anti-Proliferative Effect of Naringenin through p38-Dependent Downregulation of Cyclin D1 in Human Colorectal Cancer Cells

Naringenin (NAR) as one of the flavonoids observed in grapefruit has been reported to exhibit an anti-cancer activity. However, more detailed mechanism by which NAR exerts anti-cancer properties still remains unanswered. Thus, in this study, we have shown that NAR down-regulates the level of cyclin D1 in human colorectal cancer cell lines, HCT116 and SW480. NAR inhibited the cell proliferation in HCT116 and SW480 cells and decreased the level of cyclin D1 protein. Inhibition of proteasomal degradation by MG132 blocked NAR-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with NAR. In addition, NAR increased the phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine blocked cyclin D1 downregulation by NAR. p38 inactivation attenuated cyclin D1 downregulation by NAR. From these results, we suggest that NAR-mediated cyclin D1 downregulation may result from proteasomal degradation through p38 activation. The current study provides new mechanistic link between NAR, cyclin D1 downregulation and cell growth in human colorectal cancer cells.