The Inhibition of DMBA-Induced Carcinogenesis by CHL in Hamster Buccal Pouch

Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been used for the treatment of several abnormal human conditions without apparent toxicity. Recent studies have revealed that CHL is antimutagenic and anticarcinogenic against various carcinogens in vitro and in vivo. In the present study, CHL exhibited dose-related inhibition of His+ reversion in Salmonella typhimurium TA98 induced by 7,12-dimethylbenz[a]anthracene (DMBA). Formation of DNA adducts from DMBA was also attenuated in the presence of CHL. Topical application of CHL prior to the topical application of DMBA resulted in significant reduction in both incidence and multiplicity of tumors in hamster buccal pouch. These results suggest that CHL may act as a potential chemopreventive agent against oral cancer.ope

Genetic Polymorphisms of Carcinogen-Metabolizing Enzymes from Oral Epithelial Cells of Healthy Koreans by Mouthwash Method

Xenobiotic-metabolizing machinery contains two main types of enzymes: the phase I enzymes mediating oxidative metabolism, cytochrome P450 (CYPs) and phase II conjugating enzymes involved in detoxification, glutathione-S-transferase (GSTs). Genes that encode these enzymes are polymorphic, and such polymorphisms are associated with the susceptibility to the hazardous action of chemicals such as carcinogens in some individuals. Moreover, genetic polymorphisms of xenobiotic-metabolizing enzymes have been shown considerable ethnic differences in gene structure and allelic distribution. In this study, we investigated the polymorphic genotypes of CYP1A1 (MspI and exon 7 polymorphisms), CYP2E1 and GSTM1 from oral epithelial cells of 225 healthy Koreans collected by mouthwash method. The genomic DNA was purified by phenol-chloroform extraction and the genotypes of CYP1A1, CYP2E1 and GSTM1 were determined by polymerase chain reaction-based assay. CYP1A1 MspI genotypes m1/m1, m1/m2 and m2/m2 were found in 36.3%, 55.7% and 8.0% of healthy Koreans and exon 7 genotypes were 16.8% for ile/ile, 74.8% for ile/val and 8.4% for val/val. The frequency of CYP1A1 m1/m2 was higher in Koreans than Europeans as compared with the previous studies. CYP1A1 ile/val genotype in Koreans was found to be significantly high in comparison with Japanese as well as Europeans and Caucasian. The c1/c1, c1/c2 and c2/c2 genotypes of CYP2E1 were observed as 53.3%, 43.8% and 2.9%. The presence of c2 allele was significantly more frequent in Koreans (24.3%) than in Swedish (5.0%), European- American (1.0%) and African-American (4.0%). GSTM1 wild and null genotypes were found in 39.0% and 61.0%, respectively and this was not significantly different from other ethnicope

Nitric oxide induces expression of cyclooxygenase-2 in mouse skin through activation of NF-kappa B

Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are frequently overexpressed in tumor tissues or transformed cells. In the present work, we assessed the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of iNOS and COX-2 in mouse skin. Topical application to the dorsal skin of female ICR mice of 10 nmol TPA led to maximal induction of iNOS and COX-2 protein expression at ∼2 and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, the NOS inhibitor aminoguanidine (AG) inhibited the expression of COX-2 protein at the pharmacologically effective dose. Pretreatment with a more specific iNOS inhibitor, NG-nitro-L-arginine-methyl ester, also suppressed TPA-induced COX-2 expression. Immunohistochemical analysis of TPA-treated mouse skin using an anti-nitrotyrosine antibody reveals enhanced levels of nitrotyrosine protein localized in epidermal and dermal layers. Topical application of NO donors, such as sodium nitroprusside (SNP) and S-nitroso-N-acetyl-D,L-penicillamine, induced expression of COX-2 in mouse skin, which was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide. SNP treatment stimulated NF-κB activation in mouse skin, which was associated with the degradation of IκBα. Topical application of inhibitors of NF-κB, such as pyrrolidine dithiocarbamate or N-α-p-tosyl-L-lysine chloromethylketone, inhibited the SNP-induced COX-2 expression. SNP induced a weak but concentration-related increase in COX-2 expression in cultured mouse keratinocytes, which was abolished by treatment with SN50, a specific inhibitor of nuclear translocation of NF-κB. Mouse keratinocytes treated with SNP exhibited an elevated NF-κB-driven COX-2 promoter activity. Topical application of AG (10 µmol) prior to each TPA treatment after initiation reduced the multiplicity of papillomas by 44% at 22 weeks. In conclusion, up-regulation of COX-2 by NO may be mediated by activation of NF-κB in mouse skin, which provides a molecular mechanism by which COX-2 is induced during tumor promotion.ope

Inhibition of Phorbol Ester-induced Mouse Skin Tumor Promotion and COX-2 Expression by Celecoxib: C/EBP as a Potential Molecular Target

PURPOSE: Inflammation acts as a driving force for the development of cancer. Multiple lines of evidence suggest that nonsteroidal anti-inflammatory drugs, especially those that specifically target cyclooxygenase-2 (COX-2), are effective in preventing certain cancers. The present study was aimed at investigating the antitumor promoting potential of celecoxib in chemically induced mouse skin tumorigenesis, as well as elucidating the underlying molecular mechanisms. MATERIALS AND METHODS: To study the antitumor promoting effects of celecoxib, we used the classical two-stage mouse skin tumorigenesis model that involves initiation with a single application of 7,12-dimethylbenz [alpha]anthracene (DMBA) followed by promotion with repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The effects of celecoxib on the expression of COX-2, vascular endothelial growth factor (VEGF), p65 and the different isoforms of CCAAT/enhancer binding protein (C/EBP) were examined by performing Western blot analysis. Electrophoretic mobility gel shift assay was used to examine the effects of celecoxib on the TPA-induced DNA binding activities of various transcription factors. RESULTS: Our study revealed that topical application of celecoxib (10micromol) significantly reduced the multiplicity of papillomas in DMBA-initiated and TPA-promoted mouse skin. Pretreatment with celecoxib also diminished the expression of COX-2 and VEGF in the mouse skin papillomas. Pretreatment with celecoxib attenuated DNA binding of transcription factor (C/EBP) in the TPA-stimulated mouse skin. Moreover, celecoxib suppressed the TPA-induced nuclear expression of C/EBPdelta, but not C/EBPbeta, in mouse skin in vivo. CONCLUSION: Our study demonstrates the inhibitory effects of celecoxib on mouse skin tumor promotion, which was associated with a decreased expression of COX-2 and VEGF, as well as inhibition of C/EBP activation.ope

The Inhibitory Effects of Forsythia Koreana Extracts on the Metastatic Ability of Breast Cancer Cells and Bone Resorption by Osteoclasts

BACKGROUND: Breast cancer is the most common malignant disease in women. The patients with advanced breast cancer develop metastasis to bone. Bone metastasis and skeletal-related events by breast cancer are frequently associated with the invasiveness of breast cancer cells and osteoclasts-mediated bone resorption. Forsythia koreana is used in oriental traditional medicine to treat asthma, atopy, and allergic diseases. The aim of this study was to evaluate the inhibitory effects of F. koreana extracts on the invasion of breast cancer cells and bone resorption by osteoclasts. METHODS: Cell viability was measured by an MTT assay and the migration and invasion of MDA-MB-231 cells were detected by a Boyden chamber assay. The formation of osteoclasts and pit was detected using tartrate-resistant acid phosphatase staining and calcium phosphate-coated plates, respectively. The activities of matrix metalloproteinases (MMPs) and cathepsin K were evaluated by gelatin zymography and a cathepsin K detection kit. RESULTS: The fruit and leaf extracts of F. koreana significantly inhibited the invasion of MDA-MB-231 cells at noncytotoxic concentrations. The fruit extract of F. koreana reduced the transforming growth factor β1-induced migration, invasion and MMPs activities of MDA-MB-231 cells. In addition, the fruit, branch, and leaf extracts of F. koreana also inhibited the receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation and osteoclast-mediated bone-resorbing activity by reducing the activities of MMPs and cathepsin K. CONCLUSIONS: The extracts of F. koreana may possess the potential to inhibit the breast cancer-induced bone destruction through blocking invasion of breast cancer cells, osteoclastogenesis, and the activity of mature osteoclasts.ope

Repression of human telomerase reverse transcriptase using artificial zinc finger transcription factors

Telomerase activation is a key step in the development of human cancers. Expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), represents the limiting factor for telomerase activity. In this study, we have used artificial zinc finger protein (ZFP) transcription factors (TF) to repress the expression of hTERT in human cancer cell lines at the transcriptional level. We have constructed four-fingered ZFPs derived from the human genome which binds 12-bp recognition sequences within the promoter of the hTERT gene and fused them with a KRAB repressor domain to create a potent transcriptional repressor. Luciferase activity was decreased by >80% in all of the transcriptional repressors with luciferase reporter assay. When they were transfected into the telomerase-positive HEK293 cell line, a decrease of mRNA level and telomerase activity together with shortening of telomere length was observed. Actual growth of HEK293 cells was also inhibited by transfection of artificial ZFP-TFs. The repression was maintained for 100 days of culture. The repression of telomerase expression by artificial ZFP-TFs targeting the promoter region of the hTERT presents a new promising strategy for inhibiting the growth of human cancer cellsope

Kalopanaxsaponin A Inhibits the Invasion of MDA-MB-231 Human Metastatic Breast Cancer Cells

Invasion and metastasis of cancer cells are the leading cause of death for cancer patients, and matrix metalloproteinases (MMPs) play an important role in these steps. In this study, we determined the effect of kalopanaxsaponin A (KPS-A), isolated from the stem bark of Kalopanax pictus Nakai, on the invasion of MDA-MB-231 human metastatic breast cancer cells. KPS-A significantly inhibited the viability and PMA-induced invasion of MDA-MB-231 cells in dose-related manner. We also found that PMA-induced invasion was suppressed by KPS-A through decreasing the MMP-9 secretion. In addition, KPS-A remarkably reduced PMA-induced DNA binding and transcriptional activity of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), and blocked PMA-induced phosphorylation of ERK1/2 and Akt, not p38 MAPK. Furthermore, we confirmed that PMA-induced MMP-9 activity and transcriptional activity of NF-κB and AP-1 were regulated by p38 MAPK, Akt, ERK1/2. Taken together, the suppression of MMP-9 activity through ERK1/2/AP-1 and Akt/NF-κB pathway may contribute to the anti-invasion activity of KPS-A in PMA-stimulated MDA-MB-231 cells.ope

Kalopanaxsaponin A inhibits PMA-induced invasion by reducing matrix metalloproteinase-9 via PI3K/Akt- and PKCdelta-mediated signaling in MCF-7 human breast cancer cells

Induction of matrix metalloproteinase (MMP)-9 is particularly important for the invasiveness of breast cancers. We investigated the inhibitory effect of kalopanaxsaponin A (KPS-A) on cell invasion and MMP-9 activation in phorbol 12-myristate 13-acetate (PMA)-treated MCF-7 human breast cancer cells. KPS-A inhibited PMA-induced cell proliferation and invasion. PMA-induced cell invasion was blocked in the presence of a primary antibody of MMP-9, and KPS-A suppressed the increased expression and/or secretion of MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1. Using specific inhibitors, we confirmed that PMA-induced cell invasion and MMP-9 expression is primarily regulated by nuclear factor-kappa B (NF-kappaB) activation via phosphatidylinositol 3-kinase (PI3K)/Akt and activator protein-1 (AP-1) activation via extracellular signal-regulated kinase (ERK)1/2. KPS-A decreased PMA-induced transcriptional activation of NF-kappaB and AP-1 and inhibited PMA-induced phosphorylation of ERK1/2 and Akt. Treatment with the protein kinase C (PKC)delta inhibitor rottlerin caused a marked decrease in PMA-induced MMP-9 secretion and cell invasion, as well as ERK/AP-1 activation, and KPS-A reduced PMA-induced membrane localization of PKCdelta. Furthermore, oral administration of KPS-A led to a substantial decrease in tumor volume and expression of proliferating cell nuclear antigen, MMP-9, TIMP-1 and PKCdelta in mice with MCF-7 breast cancer xenografts in the presence of 17beta-estradiol. These results suggest that KPS-A inhibits PMA-induced invasion by reducing MMP-9 activation, mainly via the PI3K/Akt/NF-kappaB and PKCdelta/ERK/AP-1 pathways in MCF-7 cells and blocks tumor growth and MMP-9-mediated invasiveness in mice with breast carcinoma. Therefore, KPS-A may be a promising anti-invasive agent with the advantage of oral dosingope

Expression of matrix metalloproteinase-1 and -2 mRNA in retrodiscal tissue of the temporomandibular joint

Matrix metalloproteinases (MMPs) play an important role in the normal morphogenesis, maintenance, and repair of matrix and also have important functions in pathologic conditions characterized by excessive degradation of extracellular matrix, such as rheumatoid arthritis, osteoarthritis, periodontitis and in tumor invasion and metastasis. In this study, expression of MMP-1 and -2 mRNA in retrodiscal tissue of the temporomandibular joint (TMJ) was examined and compared with magnetic resonance imaging (MRI) and surgical findings. MMP mRNAs in the retrodiscal tissue samples were detected by reverse transcription - polymerase chain reaction. TMJ internal derangement (ID) was categorized as normal disc position, disc displacement with reduction, early stage of disc displacement without reduction (DDsR) and late stage of DDsR. TMJ osteoarthrosis (OA) was classified with normal, mild and advanced OA. The amount of synovial fluid collection was divided into not detected, small, large and extremely large amount on MR T2-weighted imaging. Perforation and adhesion were examined during open surgery of the TMJ. Six out of 37 samples were excluded because of little amount of extracted total mRNA. MMP-2 mRNA was detected whole joints, and so the MMP-2 mRNA seems to be expressed normally in retrodiscal tissue. However, MMP-1 mRNA was expressed in 8 of 31 joints. Frequencies of MMP-1 mRNA expression according to the TMJ IDs, amount of synovial fluid and surgical findings made no significant difference. MMP-1 mRNA was detected more frequently in OA groups (7/16 joints, 43.8%) than in normal bony structure group (1/15 joints, 6.7%). Expression of MMP-1 mRNA in retrodiscal tissue might be related with OA of the TMJ.ope

Antitumor promotional effects of a novel intestinal bacterial metabolite (IH-901) derived from the protopanaxadiol-type ginsenosides in mouse skin

Epidemiological studies have demonstrated that ginseng intake decreases the risk of cancer. Ginseng saponins (ginsenosides) have been regarded as principal components responsible for the majority of pharmacological activities exerted by ginseng. IH-901 [20-O-beta-d-glucopyranosyl-20(S)-protopanaxadiol], an intestinal bacterial metabolite derived from protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess antitumor effects, including inhibition of invasion, metastasis and angiogenesis and induction of tumor cell apoptosis. Tumor promotion often accompanies an elevated ornithine decarboxylase (ODC) activity, acute inflammation and induction of cyclooxygenase-2 (COX-2) activity. Here we examined the effects of IH-901 on tumor promotion and related molecular events in mouse skin in vivo. Mouse ear edema induced by the prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was repressed by IH-901 pre-treatment in a dose-dependent manner. Topical application of IH-901 onto shaven backs of female ICR mice led to the inhibition of TPA-induced expression of COX-2 and production of prostaglandin E(2). The eukaryotic transcription factor NF-kappaB has been involved in intracellular signaling pathways associated with inflammation and carcinogenesis. IH-901 pre-treatment inhibited TPA-induced epidermal NF-kappaB DNA binding in mouse skin, which appeared to be mediated by blocking phosphorylation and subsequent degradation of IkappaBalpha. In an attempt to elucidate the molecular mechanisms by which IH-901 inactivates NF-kappaB, its effects on activation of upstream signaling kinases were explored. IH-901 also inhibited the activation of ERK1/2 and Akt signaling. When IH-901 was treated topically prior to TPA, expression and activity of ODC were inhibited dose-dependently. In addition, IH-901 given prior to each topical dose of TPA markedly lowered the number of papillomas in mouse skin induced by 7,12-dimethylbenz[a]anthracene. Taken together, these findings suggest that IH-901 exerts anti-inflammatory effects by inhibiting TPA-induced COX-2 expression, which may contribute to its antitumor-promoting effects on mouse skin carcinogenesis.ope