Sang Kook Lee

Cytotoxic and Antineoplastic Activity of Timosaponin A-III for Human Colon Cancer Cells

The potential antitumor activity of timosaponin A-III (1), a steroidal saponin from the rhizomes of Anemarrhena asphodeloides, was investigated in human colorectal cancer HCT-15 cells both in cell culture and in an in vivo murine xenograft model. Compound 1 inhibited the proliferation of cancer cells with cell-cycle arrest and induction of apoptosis. Cell-cycle arrest in the G0/G1 and G2/M phase by 1 was correlated with the down-regulation of cyclin A, cyclin B1, cyclin-dependent kinase 2 (CDK2), CDK4, proliferating cell nuclear antigen, and c-Myc. The increase of the sub-G1 peak by 1 was also closely related to the induction of apoptosis, which was evidenced by the induction of DNA fragmentation, activation of caspases, induction of cleaved poly-(ADP ribose) polymerase, and suppression of Bcl-xL and Bcl-2 expression. In an in vivo xenograft model, treatment with 1 (2 or 5 mg/kg body weight, three times/week, ip administration) for four weeks significantly suppressed tumor growth in athymic nude mice bearing HCT-15 cells, without any overt toxicity. Cell-cycle arrest and induction of apoptosis might be plausible mechanisms of actions for the observed antineoplastic activity of 1.

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.

The root barks of Morus alba and the flavonoid constituents inhibit airway inflammation

ETHNOPHARMACOLOGICAL RELEVANCEnThe root barks of Morus alba have been used in traditional medicine as an anti-inflammatory drug, especially for treating lung inflammatory disorders.nnnAIM OF STUDYnTo find new alternative agents against airway inflammation and to establish the scientific rationale of the herbal medicine in clinical use, the root barks of Morus alba and its flavonoid constituents were examined for the first time for their pharmacological activity against lung inflammation.nnnMATERIALS AND METHODSnFor in vivo evaluation, an animal model of lipopolysaccharide-induced airway inflammation in mice was used. An inhibitory action against the production of proinflammatory molecules in lung epithelial cells and lung macrophages was examined.nnnRESULTSnAgainst lipopolysaccharide-induced airway inflammation, the ethanol extract of the root barks of Morus alba clearly inhibited bronchitis-like symptoms, as determined by TNF-α production, inflammatory cells infiltration and histological observation at 200-400mg/kg/day by oral administration. In addition, Morus alba and their major flavonoid constituents including kuwanone E, kuwanone G and norartocarpanone significantly inhibited IL-6 production in lung epithelial cells (A549) and NO production in lung macrophages (MH-S).nnnCONCLUSIONSnTaken together, it is concluded that Morus alba and the major prenylated flavonoid constituents have a potential for new agents to control lung inflammation including bronchitis.

Magnolol inhibits angiogenesis by regulating ROS-mediated apoptosis and the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells

Magnolol, a neolignan from the traditional medicinal plant Magnolia obovata, has been shown to possess neuroprotective, anti-inflammatory, anticancer and anti-angiogenic activities. However, the precise mechanism of the anti-angiogenic activity of magnolol remains to be elucidated. In the present study, the anti-angiogenic effect of magnolol was evaluated in mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial-like cells. The endothelial-like cells were obtained by differentiation from mES/EB cells. Magnolol (20 µM) significantly suppressed the transcriptional and translational expression of platelet endothelial cell adhesion molecule (PECAM), an endothelial biomarker, in mES/EB-derived endothelial-like cells. To further understand the molecular mechanism of the suppression of PECAM expression, signaling pathways were analyzed in the mES/EB-derived endothelial-like cells. Magnolol induced the generation of reactive oxygen species (ROS) by mitochondria, a process that was associated with the induction of apoptosis as determined by positive Annexin V staining and the activation of cleaved caspase-3. The involvement of ROS generation by magnolol was confirmed by treatment with an antioxidant, N-acetyl-cysteine (NAC). NAC inhibited the magnolol-mediated induction of ROS generation and suppression of PECAM expression. In addition, magnolol suppressed the activation of MAPKs (ERK, JNK and p38) and the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells. Taken together, these findings demonstrate for the first time that the anti-angiogenic activity of magnolol may be associated with ROS-mediated apoptosis and the suppression of the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells.

6″-Debromohamacanthin A, a Bis (Indole) Alkaloid, Inhibits Angiogenesis by Targeting the VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways

Hamacanthins, bis (indole) alkaloids, are found in a few marine sponges, including Spongosorites sp. Hamacanthins have been shown to possess cytotoxic, antibacterial and antifungal activities. However, the precise mechanism for the biological activities of hamacanthins has not yet been elucidated. In the present study, the anti-angiogenic effects of 6″-debromohamacanthin A (DBHA), an active component of isolated hamacanthins, were evaluated in cultured human umbilical vascular endothelial cells (HUVEC) and endothelial-like cells differentiated from mouse embryonic stem (mES) cells. DBHA significantly inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation, migration and tube formation in the HUVEC. DBHA also suppressed the capillary-like structure formation and the expression of platelet endothelial cell adhesion molecule (PECAM), an endothelial biomarker, in mES cell-derived endothelial-like cells. To further understand the precise molecular mechanism of action, VEGF-mediated signaling pathways were analyzed in HUVEC cells and mES cell-derived endothelial-like cells. DBHA suppressed the VEGF-induced expression of MAPKs (p38, ERK and SAPK/JNK) and the PI3K/AKT/mTOR signaling pathway. In addition, DBHA inhibited microvessel sprouting in mES/EB-derived embryoid bodies. In an ex vivo model, DBHA also suppressed the microvessel sprouting of mouse aortic rings. The findings suggest for the first time that DBHA inhibits angiogenesis by targeting the vascular endothelial growth factor receptor 2 (VEGFR2)-mediated PI3K/AKT/mTOR signaling pathway in endothelial cells.

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.

Extract of Magnoliae Flos inhibits ovariectomy-induced osteoporosis by blocking osteoclastogenesis and reducing osteoclast-mediated bone resorption.

Bone homeostasis is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. Osteoporosis occurs when osteoclast activity surpasses osteoblast activity. Pro-inflammatory cytokines stimulate osteoclast differentiation and activity by increasing production of macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). In this study, we investigated whether Magnoliae Flos (MF), one of the most commonly used Chinese medicinal herbs for managing rhinitis, sinusitis and headache, could effectively inhibit osteoporosis. In ovariectomized (OVX) mice compared to sham mice, the body weight increased and serum levels of alkaline phosphatase (ALP), tartrate resistant acid phosphatase 5b, calcium, and osteocalcin were significantly elevated. However, orally administrated MF extract substantially inhibited the increased body weight and serum levels of bone turnover markers, without any evidence of tissue toxicity. MF extract treatment significantly reversed the morphometric parameters of ovariectomy-induced bone loss, including trabecular bone volume, thickness, number, separation, and bone density, to almost the same levels of the sham mice. Furthermore, MF extract reduced the RANKL-mediated osteoclast differentiation and bone resorption by inhibiting the activities of matrix metalloproteinases (MMPs) and cathepsin K in mouse bone marrow macrophages. MF extract appeared to increase ALP activity in murine osteoblastic cells. Taken together, MF extract may be a beneficial supplement for the blockade of osteoporosis progression, particularly for the management of postmenopausal osteoporosis.

Inhibition of airway inflammation by the roots of Angelica decursiva and its constituent, columbianadin.

ETHNOPHARMACOLOGICAL RELEVANCEnThe roots of Angelica decursiva Fr. Et Sav (Umbelliferae) have been frequently used in traditional medicine as anti-inflammatory, antitussive, analgesic agents and expectorant, especially for treating cough, asthma, bronchitis and upper respiratory tract infections. To establish the scientific rationale for the clinical use of Angelica decursiva and to identify new agents for treating inflammatory lung disorders, pharmacological evaluation of the roots of Angelica decursiva and the isolated constituents was performed.nnnMETHODSnIn vitro study was carried out using two lung cells, lung epithelial cells (A549) and alveolar macrophages (MH-S). The inflammatory markers such as IL-6 and nitric oxide (NO) for each cell line were examined. For in vivo study, a mouse model of lipopolysaccharide (LPS)-induced acute lung injury was used and the effects on lung inflammation were established by measuring the cell numbers in bronchoalveolar lavage fluid (BALF) and by histological observation.nnnRESULTSnWater and 70% ethanol extracts of the roots of Angelica decursiva showed considerable inhibitory activity against LPS-induced lung inflammation in mice following oral administration at a dose of 400 mg/kg. Five coumarin derivatives including columbianadin, umbelliferone, umbelliferone 6-carboxylic acid, nodakenin and nodakenetin were isolated. Among the isolated compounds, columbianadin was found to possess strong inhibitory activity against the inflammatory response of IL-1β-treated A549 cells and LPS-treated MH-S cells. Columbianadin was found to inhibit NO production by down-regulation of inducible NO synthase. Moreover, columbianadin was also proved to possess significant inhibitory activity against LPS-induced lung inflammation following oral administration at a dose of 20-60 mg/kg.nnnCONCLUSIONSnThe roots of Angelica decursiva were proved to be effective in the treatment of lung inflammation. Columbianadin can be a potential new agent for treating inflammatory lung disorders.

β-Caryophyllene alleviates D-galactosamine and lipopolysaccharide-induced hepatic injury through suppression of the TLR4 and RAGE signaling pathways.

Agastache rugosa (A. rugosa, Labiatae), a perennial herb spread throughout Korean fields, is widely consumed as a wild edible vegetable and is used in folk medicine. This study examined the hepatoprotective mechanisms of β-caryophyllene (BCP), a major bicyclic sesquiterpene of A. rugosa, against D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic failure. Mice were given an intraperitoneal injection of BCP (50, 100 and 200 mg/kg) 1 h before GalN (800 mg/kg)/LPS (40 μg/kg) injection and were killed 1 h or 6 h after GalN/LPS injection. GalN/LPS markedly increased mortality and serum aminotransferase activity, both of which were attenuated by BCP. BCP also attenuated increases in serum tumor necrosis factor-α, interleukin 6, and high-mobility group protein B1 levels by GalN/LPS. GalN/LPS significantly increased toll-like receptor (TLR) 4 and receptor for advanced glycation end products (RAGE) protein expression, extracellular signal-related kinase, p38 and c-Jun N-terminal kinase phosphorylation, nuclear factor κB (NF-κB), early growth response protein-1, and macrophage inflammatory protein-2 protein expression. These increases were attenuated by BCP. Furthermore, BCP suppressed increased TLR4 and RAGE protein expression and proinflammatory cytokines production in LPS-treated isolated Kupffer cells. Our findings suggest that BCP protects against GalN/LPS-induced liver injury through down-regulation of the TLR4 and RAGE signaling.

Synthesis, in vitro and in vivo evaluation of 3-arylisoquinolinamines as potent antitumor agents

In the search for potent water-soluble 3-arylisoquinolines, several 3-arylisoquinolinamines were designed and synthesized. Various substituted 3-arylisoquinolinamines exhibited strong cytotoxic activity against eight different human cancer cell lines. In particular, C-6 or C-7 dimethylamino-substituted 3-arylisoquinolinamines displayed stronger potency than the lead compound 7a. Interestingly, compounds 7b and 7c showed more effective activity against paclitaxel-resistant HCT-15 human colorectal cancer cell lines when compared to the original cytotoxic cancer drug, paclitaxel. We analyzed the cell cycle dynamics by flow cytometry and found that treatment of human HCT-15 cells with 3-arylisoquinolinamine 7b blocked or delayed the progression of cells from G0/G1 phase into S phase, and induced cell death. Treatment with compound 7b also significantly inhibited the growth of tumors and enhanced tumor regression in a paclitaxel-resistant HCT-15 xenograft model.