Inhibitory effects of dried longan (Euphoria longana Lam.) seed extract on invasion and matrix metalloproteinases of colon cancer cells.

The critical step in colorectal cancer progression and associated mortality is cancer invasion, which depends on two key gelatinase enzymes, matrix metalloproteinases-2 and -9. Dried longan ( Euphoria longana Lam.) seed is a rich natural source of antioxidant polyphenols.This study evaluated the effect of dried longan seeds on colon cancer cell invasion via gelatinase function and expression. Three dried longan seed fractions were collected by Sephadex LH-20 column chromatography. They showed a potent inhibitor on colorectal cancer cell invasion and gelatinase activity. The antigelatinase activities of fractions 1 and 2 were a direct effect via Zn²âº chelation, whereas fraction 3 modulated indirectly through suppression of zymogen activators. Among the fractions, only fraction 3 reduced the gelatinase expression, which was correlated with the levels of tissue inhibitor of metalloproteinase-1 and may as well involve the p38 mitogen-activated protein kinases and the c-Jun N-terminal kinase signaling pathways. This primary research has manifested and encouraged the anticancer properties of dried longan seed extracts with potential inhibitory effects on cancer cell invasion as well as antigelatinase activity and expression in colon cancer cells.

Antiproliferation and apoptosis on RKO colon cancer by Millingtonia hortensis.

Millingtonia hortensis is a medicinal plant widely used in many Asian countries. An aqueous crude extract of this plant has been shown the apoptosis induction on RKO colon cancer cells. However, its mechanism remains unknown. To learn more about this plant extract, we partially purified the crude extract using Sephadex LH-20 and three aqueous fractions were collected. Each fraction was investigated for cytotoxicity using MTT assay. Fraction 1 showed antiproliferative effect on RKO cells with dose-dependent manner, while fraction 2 and 3 had no effect. Induction of apoptosis was determined using flow cytometry and DNA fragmentation method. Apoptotic cell numbers and the appearance of fragmented DNA increased with dose-dependent manner after treatment with fraction 1 for 48 h. We further investigated the expression of apoptotic protein by western blot analysis. Fraction 1 decreased the expression of anti-apoptotic protein, Bcl-xL and p-Bad, while pro-apoptotic protein Bad, was not changed. Fraction 1 also decreased the expression of p-Akt and slightly increased the level of total Akt. These results indicated that fraction 1 is able to inhibit cell proliferation and induce apoptosis on RKO cells by decreasing the expression of Bcl-xL, p-Bad and p-Akt which are involving in survival of cancer cells.

Inhibitory effects of caraway (Carum carvi L. ) and its component on N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenicity.

To elucidate the mechanism of antimutagenicity of caraway, we examined the effects of caraway seed extract on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis in DNA methyltransferase-deficient Salmonella typhimurium strains, O6-methylguanine DNA adduct formation, and thiol content in S. typhimurium cells. MNNG was highly mutagenic for ogt- strains YG7104 (ogt- ada+) and YG7108 (ogt- ada-), and it showed slightly higher mutagenicity in strain YG7100 (ogt+ ada-) than in strains TA100 and TA1535. Hot water extract of caraway seeds inhibited MNNG-induced mutation only in the ogt+ strains. In the presence of caraway extract, O6-methylguanine DNA adducts in strain YG7100 were decreased in proportion to the decrease of MNNG-induced mutagenesis. Although MNNG is known to degrade in the presence of thiols to produce methyl cation which can react with DNA, caraway had no effect on cellular concentrations of acid-soluble thiols. These results indicate that caraway does not directly inactivate MNNG and that Ogt-O6-methylguanine-DNA methyltransferase may be involved in the antimutagenic activity of caraway.

Induction of apoptosis in RKO colon cancer cell line by an aqueous extract of Millingtonia hortensis.

Millingtonia hortensis is an important medicinal plant in Southeast Asia, used for the treatment of asthma, sinusitis and as a cholagogue and tonic. The aim of this study was to compare the effects of aqueous and ethanol extracts of Millingtonia hortensis on the induction of apoptosis in an RKO human colon cancer cell line. Viability of RKO cells was assessed by MTT reduction assay. The aqueous extract, but not the ethanol extract of M. hortensis inhibited cell growth and proliferation in a dose- and time-dependent manner. Apoptotic cells were determined by flow cytometry and DNA fragmentation assay. Apoptotic cell numbers increased in a dose-dependent manner after treatment with aqueous extract. DNA ladders were clearly observed in RKO cells treated with 200, 300 and 400 ?g/ml of the aqueous extract of M. hortensis for 48 h. These results indicate that the aqueous extract of M. hortensis inhibited cell proliferation in an RKO colon cancer cell line via the apoptosis pathway.

Alteration of protein expression pattern of vascular endothelial growth factor (VEGF) from soluble to cell-associated isoform during tumourigenesis.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells, and its expression has been correlated with increased tumour angiogenesis. Although numerous publications dealing with the measurement of circulating VEGF for diagnostic and therapeutic monitoring have been published, the relationship between the production of tissue VEGF and its concentration in blood is still unclear. The aims of this study were to determine: 1) The expression pattern of VEGF isoforms at the protein level in colorectal and lung adenocarcinoma in comparison to the pattern in corresponding adjacent normal tissues 2) The relationship between the expression pattern of VEGF and total level of circulating VEGF in the blood to clarify whether the results of measuring circulating VEGF can be used to predict VEGF expression in tumour tissues. METHODS: Ninety-four tissue samples were obtained from patients, 76 colorectal tumour tissues and 18 lung tumour tissues. VEGF protein expression pattern and total circulating VEGF were examined using western blot and capture ELISA, respectively. RESULTS: Three major protein bands were predominately detected in tumour samples with an apparent molecular mass under reducing conditions of 18, 23 and 26 kDa. The 18 kDa VEGF protein was expressed equally in both normal and colorectal tumour tissues and predominately expressed in normal tissues of lung, whereas the 23 and 26 kDa protein was only detected at higher levels in tumour tissues. The 18, 23 and 26 kDa proteins are believed to represent the VEGF121, the VEGF165 and the VEGF189, respectively. There was a significant correlation of the expression of VEGF165 with a smaller tumour size maximum diameter < 5 cm (p < 0.05), and there was a significant correlation of VEGF189 with advanced clinical stage of colorectal tumours. The measurement of total circulating VEGF in serum revealed that cancer patients significantly (p < 0.001) possessed a higher level of circulating VEGF (1081 +/- 652 pg/ml in colorectal and 1,251 +/- 568 pg/ml in lung) than a healthy volunteer group (543 +/- 344 pg/ml). No correlation between the level of circulating VEGF and the pathologic features of tumours was observed. CONCLUSION: Our findings indicate that the expression patterns of VEGF isoforms are altered during tumourigenesis as certain isoform overexpression in tumour tissues correlated with tumour progression indicating their important role in tumour development. However, measurement of VEGF in the circulation as a prognostic marker needs to be carefully evaluated as the cell-associated isoform (VEGF189), but not the soluble isoform (VEGF121 and VEGF165) appears to play important role in tumour progression.

Immunomodulatory activities of Centella asiatica and Rhinacanthus nasutus extracts.

Centella asiatica (CA) and Rhinacanthus nasutus (RN )have been used for treatment of various illnesses, but the mechanisms of action remain largely unknown. This study focused on the influence of CA and RN extracts on cell-mediated and humoral immune responses. In human peripheral blood mononuclear cells (PBMCs), CA (water extract) and RN (water and ethanol extracts) significantly increased proliferation and the production of IL-2 and TNF-alpha. In contrast, an ethanol extract of CA inhibited human PBMC mitogenesis and the production of IL-2 and TNF-alpha. BALB/c mice treated with CA extracts (100 mg/kg bw) showed higher responses to both primary and secondary antibodies against BSA when compared with non-treated group. Only the secondary antibody response was increased in RN extract-treated mice. The present study revealed immunomodulating activity of CA and RN extracts with regard to both non-specific cellular and humoral immune responses. The data available to date suggest that they may have chemopreventive or anticancer potential.

Antimutagenicity and DT-diaphorase inducing activity of Gynostemma pentaphyllum Makino extract.

The hot water extract of the herbal tea, Gynostemma pentaphyllum Makino, was not found to be mutagenic in Salmonella mutation assay with or without metabolic activation. However, the extract had both DT-diaphorase inducing activity in the murine hepatoma (Hepa1c1c7) cell line and antimutagenic properties towards chemical-induced mutation in Salmonella typhimurium strains TA98 and TA100. Mutagenicity of aflatoxin B1 (AFB1), 2-amino-6-methyldipyrido [1, 2-a: 3', 2', 3-d] imidazole (Glu-P-1), 2-aminodipyrido [1, 2-a: 3', 2', 3-d] imidazole (GIu-P-2), 2-amino-1, 4-dimethyl-5H-pyrido [4, 3-b] indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido [4, 3-b] indole (Trp-P-2), 2-amino-3-methylimidazo [4, 5-f] quinoline (IQ) and Benzo [a] pyrene (B[a]P) was inhibited by the extract of Gynostemma pentaphyllum Makino in a dose-dependent manner, but no effect was found on the mutagenic activity of 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2). However, the extract enhanced the mutagenicity induced by 2-aminoanthracene (2AA), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG).

Inhibitory effects of asiatic acid and CPT-11 on growth of HT-29 cells.

Asiatic acid is a pentacyclic triterpene contained in medicinal plants. The cytotoxic effect of this compound and its augmentative effect on the anticancer drug irinotecan hydrochloride (CPT-11) were investigated in the human colon adenocarcinoma cell line HT-29. Asiatic acid dose-dependently showed cytotoxicity in HT-29 cells. DNA fragmentation, annexin-positive apoptotic cells, and caspase-3 activation were observed in a dose-dependent manner. A caspase-3 inhibitor suppressed the DNA ladder formation in a concentration-dependent manner. Bcl-2 and Bcl-XL proteins were decreased by asiatic acid treatment. These results indicate that asiatic acid induced apoptosis in HT-29 cells via caspase-3 activation. Cytotoxic effects of combined treatment with CPT-11 and asiatic acid on HT-29 cells were further examined. Simultaneous treatment or sequential exposure first to asiatic acid and then to CPT-11 showed an additive effect. Synergism was observed when cells were first exposed to CPT-11 and then to asiatic acid. These results suggest that asiatic acid can be used as an agent for increasing sensitivity of colon cancer cells to treatment with CPT-11 or as an agent for reducing adverse effects of CPT-11.

Expression of cyclooxygenase-1 and -2 and clinicopathologic features of colorectal cancer in northern Thailand.

Two isoforms of cyclooxygenase, COX-1 and COX-2, have been identified and shown to be involved in tumorigenesis. Although, overexpression of COX-2 in human cancers has been repeatedly reported, no data have hitherto been available for Thai patients. To cast light on the role(s) of COX enzymes in the development and progression of colorectal cancers and to determine the incidence of COX-2 overexpression, the expression levels of COX-1 and COX-2 proteins using Western blot analysis in tumor tissues and adjacent normal tissues obtained from 44 Thai patients with colorectal cancer. Compared with paired normal tissues, COX-2 was overexpressed in 13 of 44 colorectal tumor tissues (29.5%). Overall, COX-2 levels in colorectal tumor specimens were significantly correlated with histological differentiation, in particular in the tumors with poor differentiation (p<0.05). In addition, overexpression of COX-2 was found more frequently in colorectal tumors with lymphatic invasion, regional lymph node metastasis and larger size, although without statistical significance. In contrast to the relatively consistent alteration in COX-2 expression, the level of COX-1 expression was quite varied in tumor tissues. Forty-eight percent of colorectal tumors exhibited a decreased level of COX-1 in comparison to normal tissues and overexpressed in 23%. Thus both isoforms may both play roles in promoting tumorigenesis. However, there was no significant relationship between the alteration of COX-1 protein levels and any pathological features of tumors.

Particulate matter, PM 10 & PM 2.5 levels, and airborne mutagenicity in Chiang Mai, Thailand.

Daily levels of particulate matter (PM) in the ambient air (PM 2.5 and PM 10) were measured in a northern city of Thailand (Chiang Mai) from March 1998 to October 1999. Twenty-four-hour air particulate matter samples were collected each day with Airmetric Minivol portable air samplers. Monthly averages of PM 2.5 from four stations in Chiang Mai varied from 15.39 to 138.31microg/m(3) and 27.29 to 173.40 microg/m(3) for PM 10. The PM 2.5 annual average was 58.48 mg/m(3) and PM 10, 86.38 microg/m(3). Daily PM 2.5 (24h values) during the winter months in Chiang Mai frequently exceeded 200-300 microg/m(3). The maximum concentrations of PM 2.5 (24h average) in Chiang Mai air from December 1998 to April 1999 were 2.8-, 3.5-, 4.2-, 6.5- and 3.2-fold higher than the US Environmental Protection Agency (US EPA), PM 2.5, 24h standard of 65 microg/m(3). From May to October, the mean 24h levels of PM 2.5 and PM 10 were at acceptable levels. The data shows that during the winter season (December to March), levels of PM 2.5 and PM 10 in the Chiang Mai atmosphere are very high, and there may be significant health implications associated with these high concentrations. During the summer season, the fine particles were generally within the acceptable levels. To our knowledge, these are the first measurements of PM 2.5 to be reported for the city of Chiang Mai and they indicate considerable ambient fine particle exposures to the Chiang Mai population. In addition, dichloromethane extracts of airborne particulate matter PM 2.5 or PM 10 collected in the months of winter in the city of Chiang Mai were mutagenic to Salmonella typhimurium strain TA100 without metabolic activation. The mutagenicity appeared to track particle concentrations and increased in the presence of S9 mix.

Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats.

Effects of lemon grass extract (LGE) on hepatocarcinogenesis were examined in male Fischer 344 rats, administered diethylnitrosamine (DEN) at three weekly intraperitoneal doses of 100 mg/kg body weight and partially hepatectomized at the end of week 5. LGE was given at dietary concentrations of 0, 0.2, 0.6 or 1.8% from the end of week 4 for 10 weeks. All rats were sacrificed at the end of week 14. LGE reduced the number of putatively preneoplastic, glutathione S-transferase placental form-positive lesions and the level of oxidative hepatocyte nuclear DNA injury, as assessed in terms of 8-hydroxydeoxyguanosine production. In contrast, LGE did not affect the size of the preneoplastic lesions, hepatocyte proliferative activity, activities of phase II enzymes or hepatocyte extra-nuclear oxidative injury. These results suggest inhibitory effects of LGE on the early phase hepatocarcinogenesis in rats after initiation with DEN.

Antimutagenicity of Murdannia loriformis in the Salmonella mutation assay and its inhibitory effects on azoxymethane-induced DNA methylation and aberrant crypt focus formation in male F344 rats.

An 80% ethanol extract of Murdannia loriformis, a Thai medicinal plant, was examined for antimutagenic activity and cancer chemopreventive activity. In the Salmonella mutation assay, the extract showed antimutagenicity against 2-amino-3-methylimidazo [4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, 2-aminodipyrido[1,2-a:3',2'-d]imidazole, 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol acetate and reduced their mutagenicities to 31.4-67.9% at the dose of 10 mg/plate. However, it did not inhibit the mutagenicities of 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9 H-pyrido[2,3-b]indole, benzo[a]pyrene,N-ethyl-N'-nitro-N-nitrosoguanidine and 1-nitropyrene. The extract itself showed no mutagenicity. The chemopreventive activity of M. loriformis was examined using azoxymethane (AOM)-induced aberrant crypt focus (ACF) formation in the colon of F344 rats. The extract at doses of 0.1-1.0 g/kg wt significantly inhibited ACF formation in the initiation stage (21-51%), although it was more effective at a lower dose. In the post-initiation stage, the extract also tended to inhibit ACF formation (12-27%) and significantly decreased the number of larger ACFs that have more than 3 aberrant crypts per focus. The extract inhibited the formation of O6-methylguanine and N7-methylguanine in the colonic mucosa and muscular layers but not or increased in the liver. These results indicate that M. loriformis extract has antimutagenic activity toward various known mutagens and that it inhibits AOM-induced ACF formation both in the initiation and post-initiation stages in the rat colon.