Hong-jung Woo

Activation of AMP‐activated Protein Kinase and Phosphorylation of Glycogen Synthase Kinase3 β Mediate Ursolic Acid Induced Apoptosis in HepG2 Liver Cancer Cells

Despite the antitumour effect of ursolic acid observed in several cancers, the underlying mechanism remains unclear. Thus, in the present study, the roles of AMP‐activated protein kinase (AMPK) and glycogen synthase kinase 3 beta (GSK3β) were examined in ursolic acid induced apoptosis in HepG2 hepatocellular carcinoma cells. Ursolic acid significantly exerted cytotoxicity, increased the sub‐G1 population and the number of ethidium homodimer and terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling positive cells in HepG2 cells. Also, ursolic acid enhanced the cleavages of poly‐ADP‐ribose polymerase (PARP) and caspase3, attenuated the expression of astrocyte elevated gene (AEG1) and survivin in HepG2 cells. Interestingly, ursolic acid increased the phosphorylation of AMPK and coenzyme A carboxylase and also enhanced phosphorylation of GSK3β at inactive form serine 9, whereas ursolic acid attenuated the phosphorylation of AKT and mTOR in HepG2 cells. Conversely, AMPK inhibitor compound C or GSK3β inhibitor SB216763 blocked the cleavages of PARP and caspase 3 induced by ursolic acid in HepG2 cells. Furthermore, proteosomal inhibitor MG132 suppressed AMPK activation, GSK3β phosphorylation, cleaved PARP and deceased AEG‐1 induced by ursolic acid in HepG2 cells. Overall, our findings suggest that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3β phosphorylation as a potent chemopreventive agent. Copyright

Effect of Orostachys japonicus on Cell Growth and Apoptosis in Human Hepatic Stellate Cell Line LX2

Orostachys japonicus (O. japonicus), used to treat diseases such as various cancers, gastric ulcers, fever, hepatitis, arthritis, eczema, for hemostasis, and intoxication in folk medicine, has been an important constituent in many herbal formulae. We demonstrated that the water extract of O. japonicus led to growth inhibition of LX2 cells by inducing apoptosis through the caspase activation, related to the MAPK pathway. O. japonicus inhibited proliferation of LX2 cells in a dose- and time-dependent manner, increased the apoptosis fraction at cell cycle progression with an accompanying DNA fragmentation, and resulted in a significant decrease in Bcl-2 and an increase in Bax mRNA levels. Exposure of LX2 cells to O. japonicus induced caspase-3 activation, however when the LX2 cells were also treated with the pan-caspase inhibitor Z-VAD-FMK and the caspase-3 inhibitor Z-DEVE-FMK, apoptosis was blocked. O. japonicus inhibited anti-apoptotic Mcl-1 protein and MEK/ERK phosphorylation in LX2 cells. The results indicate that O. japonicus inhibits the cell growth of LX2 cells by inducing apoptosis through caspase activity. O. japonicus down-regulated Mcl-1 protein levels and inhibited the phosphorylation of MEK/ERK, suggesting that it mediates cell death in LX2 cells through the down-regulation of Mcl-1 protein via a MEK/ERK-independent pathway.

Orostachys japonicus Inhibits Expression of the TLR4, NOD2, iNOS, and COX-2 Genes in LPS-Stimulated Human PMA-Differentiated THP-1 Cells by Inhibiting NF-κB and MAPK Activation.

Orostachys japonicus is traditionally used as an inflammatory agent. In this report, we investigated the effects of O. japonicus extract on the expression of genes encoding pathogen-recognition receptors (TLR2, TLR4, NOD1, and NOD2) and proinflammatory factors (iNOS, COX-2, and cytokines) in LPS-stimulated PMA-differentiated THP-1 cells and the NF-κB and MAPK pathways. O. japonicus induced toxicity at high concentrations but had no effect at concentrations lower than 25 μg/mL. O. japonicus inhibited LPS-induced TLR4 and NOD2 mRNA levels, suppressed LPS-induced iNOS and COX-2 transcription and translocation, and downregulated LPS-induced proinflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) mRNA levels. In addition, O. japonicus inhibited LPS-induced NF-κB activation and IκBα degradation and suppressed LPS-induced JNK, p38 MAPK, and ERK phosphorylation. Overall, our results demonstrate that the anti-inflammatory effects of O. japonicus are mediated by suppression of NF-κB and MAPK signaling, resulting in reduced TLR4, NOD2, iNOS, and COX-2 expression and inhibition of inflammatory cytokine expression.

Chelidonium majus L. extract induces apoptosis through caspase activity via MAPK-independent NF-κB signaling in human epidermoid carcinoma A431 cells

Chelidonium majus L. (C. majus L.) is known to possess certain biological properties such as anti-inflammatory, antimicrobial, antiviral and antitumor activities. We investigated the effects of C. majus L. extract on human epidermoid carcinoma A431 cells through multiple mechanisms, including induction of cell cycle arrest, activation of the caspase-dependent pathway, blocking of nuclear factor-κB (NF-κB) activation and involvement in the mitogen-activated protein kinase (MAPK) pathway. C. majus L. inhibited the proliferation of A431 cells in a dose- and time-dependent manner, increased the percentage of apoptotic cells, significantly decreased the mRNA levels of cyclin D1, Bcl-2, Mcl-1 and survivin and increased p21 and Bax expression. Exposure of A431 cells to C. majus L. extract enhanced the activities of caspase-3 and caspase-9, while co-treatment with C. majus L., the pan-caspase inhibitor Z-VAD-FMK and the caspase-3 inhibitor Z-DEVE-FMK increased the proliferation of A431 cells. C. majus L. extract not only inhibited NF-κB activation, but it also activated p38 MAPK and MEK/ERK signaling. Taken together, these results demonstrate that C. majus L. extract inhibits the proliferation of human epidermoid carcinoma A431 cells by inducing apoptosis through caspase activation and NF-κB inhibition via MAPK-independent pathway.

Artemisia capillaris Thunb. inhibits cell growth and induces apoptosis in human hepatic stellate cell line LX2

SUMMARY Artemisia capillaris (A. capillaries) is known to play roles in many cellular events, such as cellproliferation, differentiation, and apoptosis. We investigated the antifibrogenic efficacy of A.capillaris in the immortalized human hepatic stellate cell line LX2. Cell proliferation wasdetermined by the MTT assay. Cell cycle was analyzed by the flow cytometry. Apoptotic cellswere measured using a cell death detection ELISA. Caspase activity was detected by acolorimetric assay. The mRNA level of Bcl-2 and Bax mRNA were measured by real-time PCR.MEK and ERK protein were detected by Western blot analysis. We provide evidence that A.capillaris induces cell cycle arrest, apoptosis, and potently inhibits the mitogen-activatedprotein kinase pathway. A. capillaris inhibited cell proliferation of LX2 cells in a dose- andtime-dependent manner, increased the apoptosis fraction at cell cycle analysis with anaccompanying DNA fragmentation, and resulted in a significant decrease in Bcl-2 mRNA levelsand an increase in Bax expression. Exposure of LX2 cells to A. capillaris induced caspase-3activation, but co-treatment of A. capillaris with the pan-caspase inhibitor Z-VAD-FMK, andthe caspase-3 inhibitor Z-DEVE-FMK, blocked apoptosis. A. capillaris down-regulated Mcl-1protein levels and inhibited phosphorylation of MEK/ERK, suggesting that it mediates celldeath in LX2 cells through the down-regulation of Mcl-1 protein via a MEK/ERK-independentpathway.Key words: Artemisia capillaris Thunb.; Apoptosis; Caspase activity; Immortalized human hepaticstellate cells LX2; MEK/ERK*Correspondence: Youngchul Kim, Department of InternalMedicine, College of Oriental Medicine, Kyung HeeUniversity, Seoul 130-702, Korea. Tel: +82-2-958-9236; fax:+82-2-958-9120; E-mail: yckim@khmc.or.kr