Hwa-Jin Chung

Down-regulation of c-Src/EGFR-mediated signaling activation is involved in the honokiol-induced cell cycle arrest and apoptosis in MDA-MB-231 human breast cancer cells

Honokiol is a naturally occurring neolignan abundant in Magnoliae Cortex and has showed anti-proliferative and pro-apoptotic effects in a wide range of human cancer cells. However, the molecular mechanisms on the anti-proliferative activity in cancer cells have been poorly elucidated. In this study, we evaluated the growth inhibitory activity of honokiol in cultured estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells. Honokiol exerted anti-proliferative activity with the cell cycle arrest at the G0/G1 phase and sequential induction of apoptotic cell death in a concentration-dependent manner. The honokiol-induced cell cycle arrest was well correlated with the suppressive expression of CDK4, cyclin D1, CDK2, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb) at Ser780. Apoptosis caused by honokiol was also concomitant with the cleavage of caspases (caspase-3, -8, and -9) and Bid along with the suppressive expression of Bcl-2, but it was independent on the expression of Bax and p53. In addition, honokiol-treated cells exhibited the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. In the analysis of signal transduction pathway, honokiol down-regulated the expression and phosphorylation of c-Src, epidermal growth factor receptor (EGFR), and Akt, and consequently led to the inactivation of mTOR and its downstream signal molecules including 4E-binding protein (4E-BP) and p70 S6 kinase. These findings suggest that honokiol-mediated inhibitory activity of cancer cell growth might be related with the cell cycle arrest and induction of apoptosis via modulating signal transduction pathways.

Wnt/β-Catenin Signaling Mediates the Antitumor Activity of Magnolol in Colorectal Cancer Cells

Abnormal activation of the canonical Wnt/β-catenin pathway and up-regulation of the β-catenin/T-cell factor (TCF) response to transcriptional signaling play a critical role early in colorectal carcinogenesis. Therefore, Wnt/β-catenin signaling is considered an attractive target for cancer chemotherapeutic or chemopreventive agents. Small molecules derived from the natural products were used in our cell-based reporter gene assay to identify potential inhibitors of Wnt/β-catenin signaling. Magnolol, a neolignan from the cortex of Magnolia obovata, was identified as a promising candidate because it effectively inhibited β-catenin/TCF reporter gene (TOPflash) activity. Magnolol also suppressed Wnt3a-induced β-catenin translocation and subsequent target gene expression in human embryonic kidney 293 cells. To further investigate the precise mechanisms of action in the regulation of Wnt/β-catenin signaling by magnolol, we performed Western blot analysis, real-time reverse transcriptase-polymerase chain reactions, and an electrophoretic mobility shift assay in human colon cancer cells with aberrantly activated Wnt/β-catenin signaling. Magnolol inhibited the nuclear translocation of β-catenin and significantly suppressed the binding of β-catenin/TCF complexes onto their specific DNA-binding sites in the nucleus. These events led to the down-regulation of β-catenin/TCF-targeted downstream genes such as c-myc, matrix metalloproteinase-7, and urokinase-type plasminogen activator in SW480 and HCT116 human colon cancer cells. In addition, magnolol inhibited the invasion and motility of tumor cells and exhibited antitumor activity in a xenograft nude mouse model bearing HCT116 cells. These findings suggest that the growth inhibition of magnolol against human colon cancer cells can be partly attributed to the regulation of the Wnt/β-catenin signaling pathway.

Growth inhibition of human lung cancer cells via down-regulation of epidermal growth factor receptor signaling by yuanhuadine, a daphnane diterpene from Daphne genkwa.

The growth inhibition and antitumor activities of yuanhuadine (1), a daphnane diterpenoid from the flowers of Daphne genkwa, were investigated in human lung cancer cells. Compound 1 exhibited a relatively selective growth inhibition against human lung cancer cells compared to other solid human cancer cell lines. The potent antiproliferative activity by 1 was associated with cell-cycle arrest and modulation of cell-signaling pathways. Cell-cycle arrest in the G0/G1 and G2/M phase was induced by 1 in A549 human non-small-cell lung cancer cells, and these events were correlated with the expression of checkpoint proteins including the up-regulation of p21 and down-regulation of cyclins, cyclin-dependent kinases 2 (CDK2) and 4 (CDK4), and c-Myc. Compound 1 also suppressed the expression of the Akt/mammalian target of rapamycin (mTOR) and its downstream effector molecules including p70 S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1). Ligand-induced epidermal growth factor receptor (EGFR) and c-Met signaling were also inhibited by 1. The oral administration of 1 (0.5 mg/kg body weight, daily) for 14 days significantly inhibited tumor growth in athymic xenograft nude mouse model bearing human lung A549 cells, without any overt toxicity. Synergistic antiproliferative effects of compound 1 were also found in combination with the EGFR inhibitor gefitinib. Cell-cycle arrest and suppression of Akt/mTOR and EGFR signaling pathways might be plausible mechanisms of actions for the antiproliferative and antitumor activity of 1 in human non-small-cell lung cancer cells.

Suppression of Src/ERK and GSK-3/β-catenin signaling by pinosylvin inhibits the growth of human colorectal cancer cells

Pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, has exhibited a potential cancer chemopreventive activity. However, the growth inhibitory activity against cancer cells and the underlying molecular mechanisms remain to be elucidated. Therefore, the anti-proliferative activity of pinosylvin was investigated in human colorectal HCT 116 cancer cells. Pinosylvin inhibited the proliferation of HCT 116 cells by arresting transition of cell cycle from G1 to S phase along with the downregulation of cyclin D1, cyclin E, cyclin A, cyclin dependent kinase 2 (CDK2), CDK4, c-Myc, and retinoblastoma protein (pRb), and the upregulation of p21(WAF1/CIP1) and p53. Pinosylvin was also found to attenuate the activation of proteins involved in focal adhesion kinase (FAK)/c-Src/extracellular signal-regulated kinase (ERK) signaling, and phosphoinositide 3-kinase (PI3K)/Akt/ glycogen synthase kinase 3β (GSK-3β) signaling pathway. Subsequently, pinosylvin suppressed the nuclear translocation of β-catenin, one of downstream molecules of PI3K/Akt/GSK-3β signaling, and these events led to the sequential downregulation of β-catenin-mediated transcription of target genes including BMP4, ID2, survivin, cyclin D1, MMP7, and c-Myc. These findings demonstrate that the anti-proliferative activity of pinosylvin might be associated with the cell cycle arrest and downregulation of cell proliferation regulating signaling pathways in human colorectal cancer cells.

Inhibition of cell growth and potentiation of tumor necrosis factor-α (TNF-α)-induced apoptosis by a phenanthroindolizidine alkaloid antofine in human colon cancer cells.

Based on the potential of natural products as a source for the development of cancer chemotherapeutic agents, this study was performed to investigate the anti-proliferative and antitumor effects of antofine, a phenanthroindolizidine alkaloid derived from Cynanchum paniculatum. Antofine showed potent anti-proliferative effects in several human cancer cells with IC(50) values in the nanomolar range. Treatment with antofine for 24h did not result in the induction of apoptotic cell death but moderately induced cell cycle arrest at G0/G1 phase and inhibited the expression of cyclin D1, cyclin E, and CDK4. In addition, antofine inhibited the transcriptional activity of β-catenin/Tcf in human colon HCT 116 cells, and the expression level of β-catenin and cyclin D1 was also down-regulated by antofine in human colon SW480 cells. Moreover, antofine potentiated tumor necrosis factor-α (TNF-α)-induced apoptosis, which was demonstrated by the increase of Annexin V-positive cell population and of the cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-8. Antofine also effectively suppressed tumor growth in the HCT 116 implanted xenograft nude mouse model. Taken together, these findings suggest that antofine might be a potential candidate for the development of cancer chemotherapeutic agents derived from natural products.

Alterations in white matter microstructures and cognitive dysfunctions in benign childhood epilepsy with centrotemporal spikes

Although benign childhood epilepsy with centrotemporal spikes (BECTS) is known to have good prognosis, patients often manifest neuropsychological impairments. This study aimed to investigate cognitive dysfunctions and their relationship with white matter microstructural changes in BECTS patients.

Antimetastatic activity of pinosylvin, a natural stilbenoid, is associated with the suppression of matrix metalloproteinases

Metastasis is a major cause of death in cancer patients. Our previous studies showed that pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, exhibited a potential cancer chemopreventive activity and also inhibited the growth of various human cancer cell lines via the regulation of cell cycle progression. In this study, we further evaluated the potential antimetastatic activity of pinosylvin in in vitro and in vivo models. Pinosylvin suppressed the expression of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type 1-MMP in cultured human fibrosarcoma HT1080 cells. We also found that pinosylvin inhibited the migration of HT1080 cells in colony dispersion and wound healing assay systems. In in vivo spontaneous pulmonary metastasis model employing intravenously injected CT26 mouse colon cancer cells in Balb/c mice, pinosylvin (10 mg/kg body weight, intraperitoneal administration) significantly inhibited the formation of tumor nodules and tumor weight in lung tissues. The analysis of tumor in lung tissues indicated that the antimetastatic effect of pinosylvin coincided with the down-regulation of MMP-9 and cyclooxygenase-2 expression, and phosphorylation of ERK1/2 and Akt. These data suggest that pinosylvin might be an effective inhibitor of tumor cell metastasis via modulation of MMPs.

Anti-proliferative activity and suppression of P-glycoprotein by (-)-antofine, a natural phenanthroindolizidine alkaloid, in paclitaxel-resistant human lung cancer cells.

Multidrug resistance (MDR) is a major obstacle in effective chemotherapy for cancer patients. The expression of P-glycoprotein (P-gp) in cancer cells is highly correlated with resistance to chemotherapeutic drugs. (-)-Antofine, a phenanthroindolizidine alkaloid derived from Cynanchum paniculatum, inhibits the growth of various human cancer cells. In this study, we further explored the potential of (-)-antofine to overcome the resistance induced by anti-cancer drugs. To this end, we established the paclitaxel-resistant human lung cancer cell line A549-PA by gradually exposing A549 cells to increasing concentrations of paclitaxel. As a result, the A549-PA cells acquired resistance against paclitaxel treatment and had an IC50 that was more than 200 times that of the parental A549 cells. (-)-Antofine, however, effectively suppressed the growth of both the parental and drug-resistant cells. Additional studies revealed that the anti-proliferative activity of (-)-antofine in A549-PA cells is accompanied by a down-regulation of P-gp mRNA and protein expression. The effect of reversing the multidrug resistance of A549-PA cells via (-)-antofine treatment was demonstrated an increase in intracellular rhodamine-123 accumulation, measured using FACS analysis. These findings suggest an additional chemotherapeutic value of (-)-antofine, that is, regulation of cancer cell drug resistance, in addition to its antitumor effect.

Induction of Cell Cycle Arrest and Apoptosis by Physcion, an Anthraquinone Isolated From Rhubarb (Rhizomes of Rheum tanguticum ), in MDA-MB-231 Human Breast Cancer Cells

Background: Physcion is an anthraquinone from rhubarb (rhizomes of Rheum tanguticum) and has been reported to have anti-inflammatory, hepatoprotective, antifungal, and anti-cancer activities. However, the growth inhibitory activity against human cancer cells and the underlying molecular mechanisms have been poorly determined. This study was designed to investigate the anti-proliferative activity of physcion by induction of cell cycle arrest and apoptosis in human MDA-MB-231 triple negative breast cancer cell line. Methods: MDA-MB-231 cells were treated with physcion, and the anti-proliferative activity was evaluated by the sulforhodamine B assay. The mechanisms of action for the growth inhibitory activity of physcion were evaluated by flow cytometry for cell cycle distribution, and by Western blot for the assessment of potential target proteins. Results: Physcion showed a significant anti-proliferative activity against MDA-MB-231 human breast cancer cells. Flow cytometric analysis indicated that physcion markedly induced the accumulation of cells in the G0/G1 phase and the increase of cell population in the sub-G1 phase. The G0/G1 cell cycle arrest by physcion was associated with the down-regulation of Cyclin D1, Cyclin A, CDK4, CDK2, c-Myc and phosphorylated Rb protein expressions. The increase of sub-G1 peak by physcion was closely correlated with the induction of apoptosis, which was confirmed by the induction of cleaved poly-(adenosine diphosphate ribose) polymerase, activation of Caspases, and suppression of Bid and Bcl-2 expression. Conclusions: The induction of G0/G1 cell cycle arrest and apoptosis might be one of the plausible mechanisms of actions for the anti-proliferative activity of physcion in human breast cancer cells.

Pinosylvin suppresses LPS-stimulated inducible nitric oxide synthase expression via the MyD88-independent, but TRIF-dependent downregulation of IRF-3 signaling pathway in mouse macrophage cells.

Since inhibitors of inducible nitric oxide synthase (iNOS) have been considered as potential anti-inflammatory and cancer chemopreventive agents, we have evaluated the inhibitory effects on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells with natural and synthetic compounds. Pinosylvin (3,5-dihydroxy-trans-stilbene), a stilbenoid mainly found in heartwood of Pinus sylvestris, exhibited the inhibition of iNOS protein and mRNA expression. The plausible mechanisms of pinosylvin on the suppression of iNOS gene expression were found to be associated with the downregulation of interferon regulatory factor 3 (IRF-3) and interferon-β (IFN-β) expression, which are related to Toll/IL-1 receptor domain-containing adapter inducing interferon-β (TRIF)-mediated signaling. Decreased IFN-β expression suppressed a phosphorylation of JAK kinase, and subsequently, the phosphorylation of signal transducer and activator of transcription-1, one of the iNOS transcriptional activators, was inhibited by pinosylvin. In addition, the suppression of poly(I:C)-induced iNOS expression, and the attenuation of iNOS expression under the IRF-3 gene knock-down condition also confirmed that pinosylvin affects TRIF pathway. These findings demonstrate that the suppression of LPS-induced iNOS expression by pinosylvin is associated with the regulation of MyD88-independent, but TRIF-mediated signaling pathway.