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Previous results indicate that ursolic acid (UA), a pentacyclic triterpene acid, has strong cytotoxic activity and effectively induces growth arrest in a variety of systems. However, the molecular mechanisms underlying anti-tumorigenic or chemopreventive activities of UA are poorly understood. To further determine the mechanism of UA, we investigated the effects of UA on the growth of human prostate epithelial cells. Upon treatment with UA, a concentration-dependent inhibition of cell viability was observed and cells developed many of the hallmark features of apoptosis, including condensation of chromatin and DNA fragmentation. These apoptotic effects of UA were accompanied by proteolytic cleavage of specific target proteins such as PARP, beta-catenin and Rad51 proteins suggesting the possible involvement of caspases. Western blotting and in vitro assay demonstrated that processing/activation of at least four caspases (caspase-1, -3, -8 and -9) accompanies the generation of UA-mediating apoptotic cell death. In addition to activation of caspases, the down-regulation of c-IAPs family proteins, which suppress the apoptotic death signaling by the direct inhibition of activated caspases, was also observed. However, UA did not affect both the level of p53 expression and the alteration of the balance between Bcl-2 and Bax expression. These data suggest that apoptotic signals evoked by UA treatment may converge caspases activation through down-regulation of c-IAPs family and without mitochondrial dysfunction.

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 microM 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 G(0) and G(1) phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21(WAF1), a cell-cycle regulator, was increased by UA, indicating that p21(WAF1) 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 p21(WAF1) expression, which induces the release of cytochrome c and the activation of caspase-3, leading to apoptosis of HepG2 cells., (Copyright 2000 Wiley-Liss, Inc.)

We examined the effects of the purified ginseng components, panaxadiol (PD) and panaxatriol (PT), on the expression of matrix metalloproteinase-9 (MMP-9) in highly metastatic HT1080 human fibrosarcoma cell line. A significant down-regulation of MMP-9 by PD and PT was detected by Northern blot analysis. However, the expression of MMP-2 was not changed by treatment with PD and PT. Quantitative gelatin based zymography confirmed a markedly reduced expression of MMP-9, but not MMP-2 in the treatment of PD and PT. To investigate whether the reduced level of MMP-9 by PD and PT affects the invasive capacity of HT1080 cells, we conducted an in vitro invasion assay with PD and PT treated cells. The results of the in vitro invasion assay revealed that PD and PT reduced tumor cell invasion through a reconstituted basement membrane in the transwell chamber. Because of the similarity of chemical structure between PD, PT and dexamethasone (Dexa), a synthetic glucocorticoid, we investigated whether the down-regulation of MMP-9 by PD and PT were mediated by the nuclear translocation of glucocorticoid receptor (GR). Increased GR in the nucleus of HT1080 human fibrosarcoma cells treated by PD and PT was detected by immunocytochemistry. Western blot and gel retardation assays confirmed the increase of GR in the nucleus after treatment with PD and PT. These results suggest that GR-induced down-regulation of MMP-9 by PD and PT contributes to reduce the invasive capacity of HT1080 cells.

We investigated the effects of triterpene acids (TAs), ursolic acid (UA) and oleanolic acid (OA), on the induction of proliferation and differentiation of normal rat mammary epithelial cells (RMEC) or organoids cultured in Matrigel or primary culture system. To elucidate the effects, we tested their differentiation inducing activities with intercellular communication ability, cell cycle patterns, induction of apoptosis, and morphological differentiation in the three dimensional extracellular culture system. To study the changes of RMEC subpopulation in culture, the cultured cells were isolated, immunostained with peanut lectin (PNA) and anti-Thy-1.1 antibody and then analyzed with flow cytometry. Four different subpopulations, such as PNA and Thy-1.1 negative cells (B-), PNA positive cells (PNA+), Thy-1.1 positive cells (Thy-1.1+), PNA and Thy-1.1 positive cells (B+), were obtained and the size of each subpopulation was changed in culture with time in the presence of TAs. Intercellular communication was observed in culture for 7 days in TAs-treated cells, but not in culture for 4 days with scrape-loading dye transfer technique. G2/M phase cells and the number of apoptotic population were increased in TAs-treated groups in cell cycle analyses. S phase fractions were reduced and the change of G1 phase cells was not observed. The colonies with distinct multicellular structures, such as stellate, ductal, webbed, squamous, lobulo-ductal colonies, were observed in Matrigel culture and the frequencies of each colony were changed in the presence of TAs. These results suggest that UA and OA have differentiation inducing effects on rat mammary epithelial cells in primary or in Matrigel culture.

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.

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.

An attempt was made to isolate the active component of Eriobotrya japonica, which inhibits aflatoxin B1-induced mutagenicity in the Salmonella assay system. The number of revertants per plate was significantly decreased when a MeOH extract of Eriobotrya japonica was added to the assay system using Salmonella typhimurium TA100 or TA98. Furthermore, we examined the effect of each fraction purified from the MeOH extract, and an EtOAc fraction was found to be the most effective. Ursolic acid isolated from the EtOAc fraction markedly and significantly decreased the numbers of Salmonella typhimurium TA100 revertants per plate, thus showing antimutagenic activity.

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 beta (RAR beta) 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 RAR beta 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 gel-shift 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.