Hyeon-Soo Park

Korean Scutellaria baicalensis water extract inhibits cell cycle G1/S transition by suppressing cyclin D1 expression and matrix-metalloproteinase-2 activity in human lung cancer cells

AIM OF THE STUDY Scutellaria baicalensis Georgi is a widely used medicinal herb in several Asian countries including Korea. The various medicinal properties attributed to Scutellaria baicalensis include anti-bacterial, anti-viral, anti-inflammatory and anti-cancer effects. The present study investigated the cytotoxicity of Scutellaria baicalensis water extract (SBWE) on A549 non-small-cell-lung cancer cells and the A549 expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and matrix metalloproteinase-2 (MMP-2), and the effects of SBWE on cell cycle progression, especially the G1/S phase, and on cell motility. MATERIALS AND METHODS SBWE cytotoxicity was assessed by a standard colorimetric assay utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and expression of cyclin D1 and CDK4 protein in SBWE-treated A549 cells was assessed by Western blot analysis. Flow cytometry analysis was performed to determine the effect of SBWE on A549 cell cycle progression. A549 cell MMP-2 activity was examined by zymography. Cell motility and migration was assessed by a scratch wound healing assay. RESULTS SBWE was not cytotoxic. The production of Cyclin D1, CDK4 and MMP-2 activity were significantly decreased in a SBWE dose-dependent manner, with maximum inhibition occurring at SBWE concentrations of 250 μg/ml and 500 μg/ml. SBWE inhibited cell cycle progression in the G1/S phase and significantly inhibited the motility of A549 cells. CONCLUSIONS Cyclin D1 protein may be associated with MMP-2 activity and cell motility. Thus, SBWE promotes a strong protective effect against MMP-2 mediated metastasis and cell proliferation through the down-regulation of cyclin D1. SBWE may be a useful chemotherapeutic agent for lung cancer.

Polyphenolic extract isolated from Korean Lonicera japonica Thunb. induce G2/M cell cycle arrest and apoptosis in HepG2 cells: involvements of PI3K/Akt and MAPKs.

Lonicera japonica Thunb. (L. japonica T.) has been used in Korean traditional medicine for long time because of its anti-cancer and hepatic protective effect. In this study, we investigated polyphenolic extract in L. japonica T. using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) and its anti-cancer effect on hepatocarcinoma cells. Human HepG2 cell line was treated with various concentrations of polyphenolic extract. Apoptosis was detective by cell morphology, cell cycle analysis and immunoblot analysis. Polyphenolic extract inhibited cell proliferation at 48h in a dose-dependent manner. Polyphenolic extract affected HepG2 cell viability by inhibiting cell cycle progression at the G2/M transition and inducing apoptosis. Polyphenolic extract also decreased the expression of CDK1, CDC25C, cyclin B1, pro-caspases-3 and -9 and poly ADP ribose polymerase, and affected the levels of mitochondrial apoptotic-related proteins. The phosphorylation of extracellular signal-related kinase ½ (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38 mitogen-activated protein kinases (MAPKs) were increased in HepG2 cells treated with polyphenolic extract, whereas Akt was dephosphorylated. These results indicate that inhibition of PI3K/Akt and activation of MAPKs are pivotal in G2/M cell cycle arrest and apoptosis of human hepatocarcinoma cells mediated by polyphenolic extract.

Flavonoids Isolated from Korea Citrus aurantium L. Induce G2/M Phase Arrest and Apoptosis in Human Gastric Cancer AGS Cells

Aim of the Study. Citrus species is used in traditional medicine as medicinal herb in several Asian countries including Korea. Flavonioids became known as various properties, such as anti-oxidants, anti-inflammation and anti-cancer, and so forth. The present study, the anti-cancer effect of flavonioids isolated from Citrus aurantium L. in human gastric cancer AGS cells has been investigated. Materials and Methods. The anti-proliferative activity was assayed using MTT assay. Cell cycle analysis was done using flow cytometry and apoptosis detection was done using by hoechst fluorescent staining and Annexin V-propidium iodide double staining. Western blot was used to detect the expression of protein related with cell cycle and apoptosis. Results. Flavonoids isolated from Citrus aurantium L. have the effect of anti proliferation on AGS cells with IC50 value of 99 μg/mL. Flavonoids inhibited cell cycle progression in the G2/M phase and decrease expression level of cyclin B1, cdc 2, cdc 25c. Flavonoids induced apoptosis through activate caspase and inactivate PARP. Conclusions. Flavonoids isolated from Citrus aurantium L. induced G2/M phase arrest through the modulation of cell cycle related proteins and apoptosis through activation caspase. These finding suggest flavonoids isolated from Citrus aurantium L. were useful agent for the chemoprevention of gastric cancer.

Naringin induces autophagy-mediated growth inhibition by downregulating the PI3K/Akt/mTOR cascade via activation of MAPK pathways in AGS cancer cells

Naringin, one of the major bioflavonoid of Citrus, has been demonstrated as potential anticancer agent. However, the underlying anticancer mechanism still needs to be explored further. This study investigated the inhibitory effect of Naringin on human AGS cancer cells. AGS cell proliferation was inhibited by Naringin in a dose- and time-dependent manner. Naringin did not induce apoptotic cell death, determined by no DNA fragmentation and the reduced Bax/Bcl-xL ratio. Growth inhibitory role of Naringin was observed by western blot analysis demonstrating downregulation of PI3K/Akt/mTOR cascade with an upregulated p21CIPI/WAFI. Formation of cytoplasmic vacuoles and autophagosomes were observed in Naringin-treated AGS cells, further confirmed by the activation of autophagic proteins Beclin 1 and LC3B with a significant phosphorylation of mitogen activated protein kinases (MAPKs). Collectively, our observed results determined that anti-proliferative activity of Naringin in AGS cancer cells is due to suppression of PI3K/Akt/mTOR cascade via induction of autophagy with activated MAPKs. Thus, the present finding suggests that Naringin induced autophagy- mediated growth inhibition shows potential as an alternative therapeutic agent for human gastric carcinoma.

The polysaccharides from Ganoderma lucidum: Are they always inhibitors on human hepatocarcinoma cells?

The antitumor activity of intracellular polysaccharides from submerged fermentation of Ganoderma lucidum was investigated focusing on the inhibition on human liver cancer cells. The polysaccharides inhibited human hepatocarcinoma cell HepG2 during earlier phase with lower dosage but obviously became less functional in later phase regardless of the dosage applied. However, apoptosis of the drugged HepG2 cells appeared in later incubation phase with high dosage, and the apoptosis could be enhanced by supplemental dose of the intracellular polysaccharides. Nevertheless, the intracellular polysaccharides inhibited other human hepatocarcinoma cells such as BEL-7402 and Huh-7 but luckily stimulated human normal liver cell L02 only in a positive dose- and time-dependent manner; so did the sulfated extracellular polysaccharides when it inhibited HepG2 and L02 cells. However, the toxicity of sulfated extracellular polysaccharides to L02 cells can be eliminated by the intracellular polysaccharides.

Regulation of Proinflammatory Mediators via NF-κB and p38 MAPK-Dependent Mechanisms in RAW 264.7 Macrophages by Polyphenol Components Isolated from Korea Lonicera japonica THUNB

Lonicera japonica THUNB., which abundantly contains polyphenols, has been used as a traditional medicine for thousands of years in East Asian countries because of the anti-inflammation properties. This study aimed to investigate the anti-inflammatory mechanism of polyphenol components isolated from Korea L. japonica T. by nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) pathway. Polyphenols significantly decreased lipopolysaccharide- (LPS-) induced mRNA and protein expression of inducible nitric oxide synthase and cyclooxygenase-2, as well as mRNA expression of tumor necrosis factor-alpha, interleukin- (IL-) 1β, and IL-6. Moreover, polyphenols inhibited nuclear translocation of NF-κB p65, phosphorylation/degradation of the inhibitor of κB, and phosphorylation of p38 MAPK, whereas the extracellular signal-regulated kinase and Janus N-terminal kinase were not affected. These results indicate that polyphenol components isolated from Korea L. japonica T. should have anti-inflammatory effect on LPS-stimulated RAW 264.7 cells through the decrease of proinflammatory mediators expression by suppressing NF-κB and p38 MAPK activity.

Suppressive Effect on Lipopolysaccharide-Induced Proinflammatory Mediators by Citrus aurantium L. in Macrophage RAW 264.7 Cells via NF-κB Signal Pathway

Citrus fruits have been used as an edible fruit and a traditional medicine since ancient times. In particular, the peels of immature citrus fruits are used widely in traditional herbal medicine in Korea, as they are believed to contain bioactive components exerting anti-inflammatory activity. This study examined whether the crude methanol extract of Citrus aurantium L. (CME) has a suppressive effect on inducible enzymes and proinflammatory cytokines by inhibiting the NF-κB pathway in LPS-stimulated macrophage RAW 264.7 cells. The cells were pretreated with the indicated concentrations of CME (5, 10, 20, and 50 μg/mL) and then treated with LPS (1 μg/mL). The results showed that CME (10, 20, and 50 μg/mL) inhibited the LPS- (1 μg/mL) induced mRNA and protein expression of iNOS in macrophage Raw 264.7 cells. In addition, the expression of COX-2 was inhibited at the mRNA and protein levels by CME in a dose-dependent manner. The mRNA expression of proinflammatory cytokines, such as TNF-α and IL-6, were markedly reduced by CME (10, 20, and 50 μg/mL). Moreover, CME clearly suppressed the nuclear translocation of the NF-κB p65 subunits, which was correlated with its inhibitory effect on I-κB phosphorylation. These results suggest that CME has anti-inflammatory properties by modulating the expression of COX-2, iNOS, and proinflammatory cytokines, such as TNF-α and IL-6, in macrophage RAW 264.7 cells via the NF-κB pathway.

Flavonoids Identified from Korean Scutellaria baicalensis Georgi Inhibit Inflammatory Signaling by Suppressing Activation of NF-κB and MAPK in RAW 264.7 Cells

Scutellaria baicalensis Georgi has been used as traditional medicine for treating inflammatory diseases, hepatitis, tumors, and diarrhea in Asia. Hence, we investigated the anti-inflammatory effect and determined the molecular mechanism of action of flavonoids isolated from Korean S. baicalensis G. in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to examine cytotoxicity of the flavonoids at various concentrations of 10, 40, 70, and 100 µg/mL. No cytotoxicity was observed in RAW 264.7 cells at these concentrations. Furthermore, the flavonoids decreased production of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6, and tumor necrosis factor-alpha and inhibited phosphorylation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-induced RAW 264.7 cells. Moreover, to identify the differentially expressed proteins in RAW 264.7 cells of the control, LPS-treated, and flavonoid-treated groups, two-dimensional gel electrophoresis and mass spectrometry were conducted. The identified proteins were involved in the inflammatory response and included PRKA anchor protein and heat shock protein 70 kD. These findings suggest that the flavonoids isolated from S. baicalensis G. might have anti-inflammatory effects that regulate the expression of inflammatory mediators by inhibiting the NF-κB signaling pathway via the MAPK signaling pathway in RAW 264.7 cells.

Suppressive effect of flavonoids from Korean Citrus aurantium L. on the expression of inflammatory mediators in L6 skeletal muscle cells.

Citrus fruits (Citrus aurantium L.) have long been used as a traditional herbal medicine. The benefits of the flavonoids found in Citrus aurantium L. include anti‐inflammation, anti‐cancer, anti‐viral and anti‐bacterial activities, and enhancement of the immune response. The study investigated the effect of the flavonoids isolated from Citrus aurantium L. native to Korea on the production of pro‐inflammatory mediators by blocking signal transduction mediated by nuclear factor‐kappa B (NF‐κB) and mitogen‐activated protein kinases (MAPKs) in lipopolysaccharide (LPS)‐induced L6 skeletal muscle cells. The flavonoids decreased the production of inducible nitric oxide synthase, cyclooxygenase‐2, interleukin‐6 and tumor necrosis factor‐alpha by suppressing NF‐κB and MAPKs signal pathways in LPS‐induced L6 skeletal muscle cells. These findings suggest that the flavonoids isolated from Korea Citrus aurantium L. might have anti‐inflammatory effects that regulate the expression of inflammatory mediators in L6 skeletal muscle cells. Copyright

Flavonoids identified from Korean Scutellaria baicalensis induce apoptosis by ROS generation and caspase activation on human fibrosarcoma cells.

The effects of flavonoids from Korean Scutellaria baicalensis on fibrosarcoma HT1080 cells and their underlying molecular mechanism were investigated in this study. Flavonoids affected HT1080 cell proliferation by interrupting cell cycle progress, obviously augmenting the proportion of sub-G1 and diminishing that of G1 phase, and undergoing apoptosis at the tested dosage (100-400 μg/mL). In addition, the mediated apoptosis was mainly caused by total reactive oxygen species (ROS) generation and by up-regulating the ratio of Bax/Bcl-xL, triggering caspase cascades (caspase-3, -9 and -8), and inactivating PARP, dose-dependently. The proteomics results showed that AP-4, ARID 5B, HNRNP K, PLOG, Prdx6, and myosin-1, associated with cell growth, differentiation and development, and overexpressed in gastric cancer, colorectal cancer, pancreatic cancer, etc., were statistically down-regulated after the flavonoids treatment. Taken together, our data demonstrated that flavonoids from Korean S. baicalensis induced apoptosis in HT1080 cells, which involved a hierarchy of cellular pathways and multiple signal proteins, and might be a potential anticancer therapeutic agent.