Jongdai Kim

Hexane/ethanol extract of Glycyrrhiza uralensis and its active compound isoangustone A induce G1 cycle arrest in DU145 human prostate and 4T1 murine mammary cancer cells.

Although licorice is known to exert anticarcinogenic effects, it contains large quantities of glycyrrhizin (GL), which causes severe hypertension. We have previously demonstrated that the hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) contains no detectable GL and suppresses doxorubicin-induced apoptosis in H9c2 rat cardiac myoblasts. The principal objective of this study was to determine whether and by what mechanism HEGU and its active component, isoangustone A, inhibit cell-cycle progression in DU145 human prostate and 4T1 mouse breast cancer cells. HEGU and isoangustone A dose-dependently decreased DNA synthesis and induced G1 phase arrest in both DU145 and 4T1 cells. HEGU and isoangustone A reduced the levels of CDK2 and CDK4 as well as cyclin A and cyclin D1 proteins, and also induced a decrease in CDK2 activity. The addition of HEGU to drinking water significantly suppressed the orthotopic growth of 4T1 allografts and the expression of the proliferating nuclear cell antigen, CDK2 and CDK4 proteins in the tumor tissues. These results demonstrate the potential of HEGU containing isoangustone A as an antitumor agent.

3,3'-diindolylmethane suppresses 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and tumor promotion in mouse skin via the downregulation of inflammatory mediators.

3,3′‐Diindolylmethane (DIM) is a major acid‐condensation product of indole‐3‐carbinol and is present in cruciferous vegetables. In this study, we evaluated the effects of DIM on antiinflammatory and antitumor promotion activity in mouse skin and explored the relevant mechanisms. When 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was applied topically to the mouse ear to induce inflammation, DIM pretreatment effectively inhibited TPA‐induced ear edema formation. To evaluate the mechanisms underlying DIMs antiinflammatory effects, DIM was topically treated to the shaved backs of mice 30 min before TPA treatment. DIM inhibited the TPA‐induced increases in the expression of cyclooxygenase (COX)‐2, inducible nitric oxide synthase (iNOS), chemokine (C‐X‐C motif) ligand (CXCL) 5, and interleukin (IL)‐6 in mouse skin. DIM also inhibited nuclear factor‐kappa B (NF‐κB)s DNA binding activity, the nuclear translocation of p65, and the degradation of inhibitor of κB (IκB) α in TPA‐stimulated mouse skin. Furthermore, DIM reduced TPA‐induced increases in the activity of extracellular signal regulated protein kinase (ERK)‐1/2 and IκB kinase (IKK). When mouse skin papillomas were initiated via the topical application of 7,12‐dimethylbenz[α]anthracene (DMBA) and promoted with repeated topical applications of TPA, repeated topical applications of DIM prior to each TPA treatment significantly suppressed the incidence and multiplicity of the papillomas. DIM also reduced the expression of COX‐2 and iNOS, ERK phosphorylation, and the nuclear translocation of p65 in papillomas. Collectively, these results show that DIM exerts antiinflammatory and chemopreventive effects in mouse skin via the downregulation of COX‐2, iNOS, CXCL5, and IL‐6 expression, which may be mediated by reductions in NF‐κB activation.

Hexane-ethanol extract of Glycyrrhiza uralensis containing licoricidin inhibits the metastatic capacity of DU145 human prostate cancer cells.

Licorice extracts are known to exhibit anti-carcinogenic activities. However, chronic licorice consumption can lead to serious side effects due to the presence of considerable quantities of glycyrrhizin, which causes severe hypokalaemia and hypertension. In the present study, we evaluated the effects of a hexane-ethanol extract of Glycyrrhiza uralensis (HEGU), which lacks glycyrrhizin, on the metastatic characteristics of DU145 prostate cancer cells. HEGU inhibited basal and epidermal growth factor-induced cell migration, invasion and adhesion in a dose-dependent fashion. HEGU significantly suppressed the secretion and activation of the matrix metalloproteinase (MMP)-2 and MMP-9. The secretion of tissue inhibitor of metalloproteinase (TIMP)-1 was reduced, but that of TIMP-2 was increased in HEGU-treated cells. HEGU reduced the protein levels of integrin-α2, the intercellular adhesion molecule, and the vascular cell adhesion molecule. An active fraction of HEGU was separated via column chromatography, and the structure of the active component, licoricidin, was identified via 1H NMR and 13C NMR. The treatment of DU145 cells with licoricidin induced a reduction in cell migration and the secretion of MMP-9, TIMP-1, urokinase-type plasminogen activator and vascular endothelial growth factor, as well as in the expression of adhesion molecules. These results indicate that HEGU, which contains licoricidin, is a potent anti-metastatic agent, which can markedly inhibit the metastatic and invasive capacity of malignant prostate cancer cells. The observed reductions in the activation of proteases and the levels of adhesion molecules may constitute a component of the mechanisms by which HEGU inhibits the migration and adhesion of prostate cancer cells.

Isoangustone A present in hexane/ethanol extract of Glycyrrhiza uralensis induces apoptosis in DU145 human prostate cancer cells via the activation of DR4 and intrinsic apoptosis pathway

Glycyrrhiza uralensis (licorice) is one of the most frequently prescribed ingredients in Oriental medicine, and licorice extract has been shown to exert anti-carcinogenic effects. However, its use as a cancer chemopreventive agent is rather limited, due to the fact that its principal component, glycyrrhizin, is known to induce hypertension. This study determined the effects of a hexane/ethanol extract of G. uralensis (HEGU), which contains undetectable amounts of glycyrrhizin, on the apoptosis of androgen-insensitive DU145 cells. HEGU induced apoptosis and increased the levels of cleaved caspase-9, caspase-7, caspase-3 and poly (ADP-ribose) polymerase (PARP). HEGU also induced mitochondrial membrane depolarization and cytochrome c release to the cytosol. HEGU increased the levels of Fas, death receptor 4 (DR4), cleaved caspase-8, Mcl-1S, and truncated Bid proteins. A caspase-8 inhibitor suppressed HEGU-induced apoptosis. An active fraction of HEGU was separated via column chromatography and the structure of the active compound isoangustone A was identified via 1H-NMR and 13C-NMR. Isoangustone A increased apoptotic cells, the cleavage of PARP and caspases, and the levels of DR4 and Mcl-1S. Transfection with DR4 small interfering RNA attenuated HEGU- and isoangustone A-induced apoptosis. These results demonstrate that the activation of DR4 contributes to HEGU- and isoangustone A-induced apoptosis of DU145 cells.

The Hexane Extract of Saussurea lappa and Its Active Principle, Dehydrocostus Lactone, Inhibit Prostate Cancer Cell Migration

Saussurea lappa has been used in Chinese traditional medicine for the treatment of abdominal pain, tenesmus, nausea, and cancer; previous studies have shown that S. lappa also induces G(2) growth arrest and apoptosis in gastric cancer cells. In this study, we investigated the effects of hexane extracts of S. lappa (HESLs) on the migration of DU145 and TRAMP-C2 prostate cancer cells. DU145 and TRAMP-C2 cells were cultured in the presence of 0-4 μg/mL HESL with or without 10 ng/mL epidermal growth factor (EGF). HESL inhibited the basal and EGF-induced migration of prostate cancer cells in a dose-dependent manner, whereas HESL did not influence the viability of these cancer cells under the conditions used in this study. Active fractions of HESL were separated via column chromatography, and the structure of the active principle was determined using (1)H and (13)C nuclear magnetic resonance spectroscopy. The active compound, dehydrocostus lactone (DHCL), in fraction 7 dose-dependently inhibited the basal and EGF-induced migration of prostate cancer cells. HESL and DHCL reduced matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 secretion but increased TIMP-2 levels in both the absence and presence of EGF. Our results demonstrate that the inhibition of MMP-9 secretion and the stimulation of TIMP-2 secretion contribute to reduced migration of DU145 cells treated with HESL and DHCL. These results indicate that HESL containing its active principle, DHCL, has potential as an antimetastatic agent for the treatment of prostate cancer.

Anti-inflammatory Effect of Ethanol Extract from Eupatorium japonicum

Acer barbinerve Maxim belongs to the Aceraceae tree family and is often consumed as an Oriental medicine. In this study, we investigated whether or not ethanol extract from the bark of A. barbinerve Max. (EBA) inhibits lipopolysaccharide (LPS)-induced inflammatory responses in Raw264.7 macrophages. EBA was fractionated using n-hexane, CH2Cl2, ethyl acetate (EtOAc), and water. Raw264.7 cells were treated with 20 μg/mL of EBA and the EBA fractions. EBA inhibited LPS-induced nitric oxide (NO) production. Among the three fractions, EtOAc fraction of EBA (EFEBA) was the most effective in inhibiting LPS-induced NO production without significant cytotoxicity in Raw264.7 cells. EFEBA futher reduced LPS-induced expression of inducible NO synthase (iNOS) proteins and its corresponding mRNA. Additionally, EFEBA decreased the mRNA levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α in LPS-treated Raw264.7 cells. Lastly, EFEBA inhibited LPS-induced degradation of the inhibitor of kappaBalpha (IκBα) as well as phosphorylation of p65 nuclear factor-κB (NF-κB). These results indicate that EFEBA exhibits strong anti-inflammatory effects and can be developed as a potential anti-inflammatory agent.Eupatorium japonicum belongs to a family of Asteraceae plants and flowers of E. japonicum have been consumed as a tea. In this study, we investigated whether E. japonicum extract inhibits lipopolysaccharide (LPS)-induced inflammatory responses in Raw264.7 macrophages. The cells were treated with various concentrations (0, 1, 2.5, 5, or 10 mg/L) of 70% ethanol extract from E. japonicum flowers (EJE) in Raw264.7 cells. LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production were inhibited by EJE up to 67% and 49% of these productions, respectively without any reduction of viable cell numbers. EJE reduced LPS-induced expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 proteins and their corresponding mRNA levels. Additionally, EJE decreased the levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α mRNA. EJE was further fractionated with water, butanol, ethylacetate (EA), hexane, or methylene chloride (MC). Among the resulting five fractions, EA and MC, respectively from EJE significantly inhibited LPS-induced NO production (each inhibition rate was 85.3% of 10 mg/L EA fraction and 97.2% of 10 mg/L MC fraction) without significant cytotoxicity in Raw264.7 cells. These results indicate that EJE exhibits powerful effects of anti-inflammation and can be developed as a potential anti-inflammatory agent.

trans-10,cis-12 conjugated linoleic acid inhibits insulin-like growth factor-I receptor signaling in TSU-Pr1 human bladder cancer cells.

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid; the major isomers are trans-10,cis-12 CLA (t10c12) and cis-9,trans-11 CLA (c9t11). CLA has been demonstrated to exert strong anticarcinogenic effects in a variety of experimental cancer models. We previously observed that CLA (a mixture of isomers) and t10c12 decreased the growth of TSU-Pr1 cells, whereas linoleic acid and c9t11 exerted no effects. In the current study, the mechanisms underlying the t10c12-mediated regulation of the growth of these bladder cancer cells were evaluated. TSU-Pr1 cells were incubated in serum-free medium with various concentrations of t10c12 or c9t11 in the presence or absence of insulin-like growth factor (IGF)-I. The incorporation of [(3)H]thymidine into DNA was decreased, and the number of annexin V-stained cells was increased after t10c12 treatment, whereas c9t11 had no effect on apoptosis or [(3)H]thymidine incorporation. Treatment with exogenous IGF-I alone increased the numbers of viable cells but did not counteract the t10c12-induced growth inhibition of TSU-Pr1 cells. t10c12 effected a dose-dependent reduction in IGF-I receptor (IGF-IR) transcripts and protein levels, whereas c9t11 exerted no effects. Additionally, t10c12 inhibited the IGF-I-induced phosphorylation of IGF-IR, the recruitment of the p85 regulatory subunit of phosphoinositide 3-kinase to IGF-IR, and the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. These results indicate that the inhibition of IGF-IR signaling and the activation of Akt and ERK-1/2 contributed to decreased cell proliferation and increased apoptosis in TSU-Pr1 cells treated with t10c12.

Phenethyl Isothiocyanate Inhibits 12-O-Tetradecanoylphorbol-13-Acetate-Induced Inflammatory Responses in Mouse Skin

Phenethyl isothiocyanate (PITC) is the hydrolysis product of the glucosinolate gluconasturtiin in cruciferous vegetables. This study was conducted to determine whether PITC inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the mouse ear. Topical application of 5 nmol of TPA to mouse ears markedly increased the ear weight, expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein, and phosphorylation of the inhibitor of κB (IκB) α, AKT, and extracellular signal-regulated protein kinase (ERK) 1/2 and reduced IκBα protein levels. Pretreatment with PITC (150-450 nmol) significantly suppressed these TPA-induced inflammatory responses. We also determined whether low concentrations of PITC (0.5-5 μmol/L) inhibited lipopolysaccharide (LPS)-stimulated inflammatory responses in Raw264.7 cells. PITC dose-dependently reduced the LPS-induced secretion of nitric oxide, prostaglandin E(2), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, as well as COX-2 and iNOS protein expression. PITC also attenuated LPS-induced increases in iNOS, COX-2, IL- 6, IL-1β, and TNF-α mRNA levels, as well as the promoter-dependent transcriptional activation of the genes for iNOS and COX-2. PITC inhibited LPS-induced IκBα phosphorylation and degradation and subsequently reduced LPS-induced p65 nuclear translocation and the transcriptional activity of nuclear factor-κB (NF-κB), which was accompanied by a reduction in ERK1/2 and AKT phosphorylation. The results of this study demonstrated that PITC effectively inhibits inflammatory responses in vivo and in vitro, which may be mediated via the inhibition of AKT and ERK1/2 activation, leading to subsequent inhibition of the transcriptional activity of NF-κB.