Kyu-Won Kim

The effect of age on cyclooxygenase-2 gene expression: NF-κB activation and IκBα degradation

Increased oxidative stress resulting in the activation of NF-kappaB is thought to play a crucial role in the expression of the cyclooxygenase-2 (COX-2), which is the key enzyme in proinflammatory prostanoid synthesis. In the current study, we investigated whether the aging process affects the status of the redox-sensitive NF-kappaB in rat kidney, and how this age-related modulation is related to COX-2 gene expression and COX-derived reactive oxygen species (ROS). We found that the aging process strongly enhanced the activation of NF-kappaB and its DNA-binding activity with an increased ROS status. Accompanied with the change in the NF-kappaB activity was a decreased IkappaBalpha as confirmed by the increased nuclear p65 protein. Thus, these data strongly indicated that the aging process increases NF-kappaB activity by downregulating IkappaBalpha. A closer examination further revealed that age-related oxidative status correlated with the increased COX-derived prostanoid biosynthetic process is mediated by the increased NF-kappaB-regulated COX activity. This increase in NF-kappaB activity was accompanied by the increased COX-2 mRNA and protein levels. Based on these data, we concluded that the age-related increase in redox-sensitive NF-kappaB translocation and binding activities are associated with increased ROS, and further that this transactivation was modulated by the age-related decrease of IkappaBalpha.

Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis

Low oxygen and nutrient depletion play critical roles in tumorigenesis, but little is known about how they interact to produce tumor survival and tumor malignancy. In the present study, we investigated the mechanism underlying hypoxia-modulated apoptosis of serum-deprived HepG2 cells. Our results showed that hypoxia blocked the apoptosis, which was accompanied with decreased Bax/Bcl-2 ratio, inhibited cytochrome c release, and reduced caspase-3 activity. More importantly, increased expressions of VEGF and its receptor-2 (KDR) under hypoxic/serum-deprived condition suggest that VEGF may act as a survival factor in a self-promoting manner. Data were further supported by results that recombinant human VEGF (rhVEGF) suppressed the serum deprivation-induced apoptosis, and anti-VEGF neutralizing antibody block anti-apoptotic activity of hypoxia. In addition, inhibitors of receptor tyrosine kinase blocked anti-apoptosis of hypoxia. Our study further showed that rhVEGF or hypoxia induced ERK phosphorylation in serum-deprived cells, and that a specific inhibitor of MAPK/ERK, PD98059 eliminated the anti-apoptotic activity of rhVEGF or hypoxia by increasing Bax/Bcl-2 ratio and caspase-3 activity. Our data led us to conclude that induction of ERK phosphorylation and decrease of Bax/Bcl-2 ratio by rhVEGF implies that hypoxia-induced VEGF prevents apoptosis of serum-deprived cells by activating the MAPK/ERK pathway. Taken together, we propose that hypoxia enhances survival of nutrient-depleted tumor cells by reducing susceptibility to apoptosis, which consequently leads to tumor malignancy.

Anti-angiogenic activity of triterpene acids.

Ursolic acid (UA) and oleanolic acid (OA) were examined for anti-angiogenic activities by using the chick embryo chorioallantoic membrane (CAM) assay. The presence of UA or OA inhibited angiogenesis in a dose-dependent manner; the doses required for half-maximal inhibition (ID50) were 5 micrograms and 40 micrograms per CAM, respectively. UA was a more potent angiogenic inhibitor than OA. We also tested for inhibitory effect on the proliferation of bovine aortic endothelial cell. They effectively inhibited the proliferation of bovine aortic endothelial cell in a concentration-dependent manner. The IC50 values of anti-proliferative effects were determined to be 5 microM for UA and 20 microM for OA. Based on these results, we speculated that the inhibitory effects on bovine aortic endothelial cell proliferation of UA and OA might be important for anti-angiogenesis.

ACTIVATION OF CASPASE- 3 PROTEASE VIA A BCL-2-INSENSITIVE PATHWAY DURING THE PROCESS OF GINSENOSIDE RH2-INDUCED APOPTOSIS

We have demonstrated that ginsenoside Rh2 (G-Rh2), a ginseng saponin with a dammarane skeleton, induces apoptosis of human hepatoma SK-HEP-1 cells as evidenced by analyses of DNA fragmentation, flow cytometry and changes in cell morphology. Ac-YVAD-CMK or Ac-DEVD-CHO effectively prevented G-Rh2-induced DNA fragmentation, indicating the involvement of caspase-like proteases in the process of apoptosis. In addition, G-Rh2 induced the processing of caspase-3 to an active form, p17. In stable Bcl-2 transfectants, G-Rh2 also induced DNA fragmentation, while staurosporine-induced DNA fragmentation was totally blocked. As it did in wild-type cells, G-Rh2 induced the proteolytic activation of caspase-3 protease and subsequent cleavage of PARP in the bcl-2 transfectants. In summary, G-Rh2 contains an apoptotic inducing activity in SK-HEP-1 cells which functions via Bcl-2-insensitive activation of caspase-3, followed by proteolytic cleavage of PARP.

Ursolic acid-induced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells

We have previously reported that ursolic acid, a pentacyclic triterpene acid, inhibited the invasion of HT1080 human fibrosarcoma cells by reducing the expression of matrix metalloproteinase-9. Since the chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid, we investigated whether ursolic acid acts through the glucocorticoid receptor. The expression of matrix metalloproteinase-9 is thought to be regulated similarly with matrix metalloproteinase-1 and matrix metalloproteinase-3 as containing common 2-O-tetradecanoylphorbol-acetate responsible region, where AP-1 proteins can bind. Dexamethasone has been studied to repress the 2-O-tetradecanoylphorbol-acetate-induced expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 through a glucocorticoid receptor-mediated manner. In Northern blot analysis, we found that ursolic acid reduced the expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 induced by 2-O-tetradecanoylphorbol-acetate. Similarly, ursolic acid down-regulated 2-O-tetradecanoylphorbol-acetate-induction of matrix metalloproteinase-9 gene in the same manner of dexamethasone. RU486, a potent glucocorticoid receptor antagonist, was used for identifying that ursolic acid-induced down-regulation of matrix metalloproteinase-9 expression is mediated by its binding to glucocorticoid receptor. The effect of ursolic acid on the matrix metalloproteinase-9 expression was blocked by RU486, suggesting that ursolic acid acts via a glucocorticoid receptor in the regulation of matrix metalloproteinase-9. Western blot analysis and immunocytochemistry showed that ursolic acid increased glucocorticoid receptor fraction in the nucleus, although it decreased the synthesis of glucocorticoid receptor mRNA. In addition, ursolic acid did not decrease the expression of c-jun and DNA-binding activity of AP-1 to its cognate sequences. Taken together, we suggest that ursolic acid may induce the repression of matrix metalloproteinase-9 by stimulating the nuclear translocation of glucocorticoid receptor, and the translocated glucocorticoid receptor probably down-modulating the trans-activating function of AP-1 to 2-O-tetradecanoylphorbol-acetate responsible element of matrix metalloproteinase-9 promoter region.

A novel angiogenic factor derived from Aloe vera gel: beta-sitosterol, a plant sterol.

Aloe vera gel has a beneficial effect on wound healing. Because angiogenesis is an essential process in wound healing, we hypothesized that Aloe vera gel might contain potent angiogenic compounds. Here we demonstrate that Aloe vera gel and its extracts are angiogenic on the chorioallantoic membrane (CAM) of chick embryo. Out of the three compounds purified from the final fraction of Aloe vera gel, β-sitosterol showed a potent angiogenic activity in the CAM assay. In the presence of heparin, β-sitosterol stimulated neovascularization in the mouse Matrigel plug assay and the motility of human umbilical vein endothelial cells in an in vitro wound migration assay. Thus β-sitosterol is a novel plant-derived angiogenic factor which may have potential pharmaceutical applications for the management of chronic wounds.

APOPTOTIC ACTIVITY OF URSOLIC ACID MAY CORRELATE WITH THE INHIBITION OF INITIATION OF DNA REPLICATION

Ursolic acid (UA), a pentacyclic triterpene acid, has been reported to exhibit anti‐tumor activity. In this study, we investigated the pro‐apoptotic effect of UA on HepG2 human hepatoblastoma cells. Treatment with UA decreased the viability of HepG2 cells in a concentration‐ and time‐dependent manner. Furthermore, 30 μM of UA induced DNA fragmentation and subdiploid cells and enhanced the release of cytochrome c and the activation of caspase‐3. These results suggest that UA induces cell death through apoptosis, which may be mediated by cytochrome c‐dependent caspase‐3 activation. In addition, cell‐cycle analysis revealed that UA‐treated cells were arrested predominantly in the G0 and G1 phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21WAF1, a cell‐cycle regulator, was increased by UA, indicating that p21WAF1 might mediate UA‐induced cell‐cycle arrest. However, UA markedly inhibited SV40 DNA replication in the initiation stage in vitro and significantly reduced the DNA cleaving of topoisomerase I and the ssDNA binding activity of replication protein A. These results indicate that the inhibition of DNA replication by UA may result from blockade of the establishment of the replication fork during initiation stage, consequently contributing to UA‐induced cell‐cycle arrest. Taken together, we suggest that UA‐induced cell‐cycle arrest may be mediated by inhibition of DNA replication and the increase of p21WAF1 expression, which induces the release of cytochrome c and the activation of caspase‐3, leading to apoptosis of HepG2 cells. Int. J. Cancer 87:629–636, 2000.

Intracellular Ca2+ release mediates ursolic acid–induced apoptosis in human leukemic HL‐60 cells

The effect of ursolic acid (UA) on tumor cell apoptosis was investigated using HL‐60 human promyelocytic leukemia cells as a model cellular system. Treatment with UA resulted in a concentration‐dependent decreased cell viability assessed by MTT assay. UA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which UA induced cell death was through apoptosis. The intracellular Ca2+ level was increased by treatment with UA. Intracellular Ca2+ inhibitors, such as intracellular Ca2+‐release blockers (dantrolene, TMB‐8 and ruthenium red) and an intracellular Ca2+ chelator (BAPTA/AM), significantly blocked the UA‐induced increased intracellular Ca2+ concentration. These inhibitors also blocked the effects of UA on cell viability and apoptosis. These results suggest that enhanced intracellular Ca2+ signals may be involved in UA‐induced apoptosis in HL‐60 cells. Int. J. Cancer 73:725–728, 1997.

Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models.

Insulin-like growth factor II (IGF-II), highly expressed in a number of human tumours, has been recently known to promote neovascularization in vivo. Yet, the detailed mechanism by which IGF-II induces angiogenesis has not been well defined. In the present study, we explored an angiogenic activity of IGF-II in in vitro angiogenesis model. Human umbilical vein endothelial cells (HUVECs) treated with IGF-II rapidly aligned and formed a capillary-like network on Matrigel. In chemotaxis assay, IGF-II remarkably increased migration of HUVECs. A rapid and transient activation of p38 mitogen-activated protein kinase (p38 MAPK) and p125 focal adhesion kinase (p125FAK) phosphorylation was detected in HUVECs exposed to IGF-II. IGF-II also stimulated invasion of HUVECs through a polycarbonate filter coated with Matrigel. Quantitative gelatin-based zymography identified that matrix metalloproteinase-2 (MMP-2) activity generated from HUVECs was increased by IGF-II. This induction of MMP-2 activity was correlated with Northern blot analysis, showing in HUVECs that IGF-II increased the expression of MMP-2 mRNA, while it did not affect that of TIMP-2, a tissue inhibitor of MMP-2. These results provide the evidence that IGF-II directly induces angiogenesis by stimulating migration and morphological differentiation of endothelial cells, and suggest that IGF-II may play a crucial role in the progression of tumorigenesis by promoting the deleterious neovascularization.

Induction of differentiation in the cultured F9 teratocarcinoma stem cells by triterpene acids

The effects of the triterpene acids, ursolic acid and oleanolic acid, on the differentiation of F9 teratocarcinoma stem cells were studied. These agents caused the morphological change of F9 cells into endoderm cells, as did retinoic acid (RA). Moreover, expression of laminin B1, type IV collagen and retinoic acid receptor β (RARβ) increased in ursolic-and oleanolic-acid treated F9 cells. Since these agents are structurally similar to the glucocorticoid hormone, we studied the effects of dexamethasone, a synthetic glucocorticoid, on F9 cells. Dexamethasone also induced the morphological change and altered the expression of laminin B1, type IV collagen, and RARβ in F9 cells. In addition, transcription of glucocorticoid receptor was detected after treatment with these three agents. According to Southwestern blot analysis, a 94-kDa protein, thought to be a glucocorticoid receptor, was detected in F9 cells treated with these agents. In a gelshift assay, we identified protein factors binding to the glucocorticoid-responsive element (GRE) in the nuclear proteins from F9 cells treated with ursolic or oleanolic acid. The binding activity of the GRE-binding protein disappeared on the addition of unlabeled GRE oligonucleotide. Taken together, these results suggest that UA and OA can induce the differentiation of F9 cells and may regulate the expression of differentiation-specific genes, probably by forming a complex with the glucocorticoid receptor or its analogous nuclear receptor.