Acetylation Enhances the Anticancer Activity and Oral Bioavailability of 5-Demethyltangeretin.

A kind of hydroxylated polymethoxyflavone (PMFs) existing in the citrus genus, 5-Demethyltangeretin (5-DTAN), has been reported to possess several bioactivities in vitro and in vivo. The aim of this study was to investigate whether acetylation could enhance the anticancer activity and oral bioavailability of 5-DTAN. PC-3 human prostate cancer cells were treated with tangeretin (TAN), 5-DTAN, and 5-acetylated TAN (5-ATAN), and the results showed that the cytotoxic effect 5-ATAN (IC50 value of 5.1 µM) on the cell viability of PC-3 cells was stronger than that of TAN (IC50 value of 17.2 µM) and 5-DTAN (IC50 value of 11.8 µM). Compared to 5-DTAN, 5-ATAN treatment caused a more pronounced DNA ladder, increased the sub-G1 phase population, and induced G2/M phase arrest in the cell cycle of PC-3 cells. We also found that 5-ATAN triggered the activation of caspase-3 and the progression of the intrinsic mitochondrial pathway in PC-3 cells, suggesting the induction of apoptosis. In a cell wound healing test, 5-ATAN dose-dependently reduced the cell migration, and the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was decreased after 48 h of 5-ATAN treatment. Moreover, oral administration of 5-ATAN showed a significantly stronger inhibitory effect on tumor size and tumor weight in tumor-bearing nude mice than those of vehicle or the 5-DTAN group (p < 0.05). Furthermore, pharmacokinetic results showed that single-dose oral administration of 5-ATAN exhibited a higher maximum concentration (Cmax) and area under the curve (AUC) of 5-DTAN in plasma than that of 5-DTAN. More extensive distribution of 5-DTAN to most tissues of mice was also observed in mice treated with 5-ATAN for 7 days. In conclusion, acetylation strongly enhances the anticancer activity and oral bioavailability of 5-DTAN and could be a promising strategy to promote the potential bioactivities of natural products.

Freshwater Clam Extract Attenuates Indomethacin-Induced Gastric Damage In Vitro and In Vivo.

Contemporary pharmacological studies have reported that freshwater clam (Corbicula fluminea) can provide a broad spectrum of bioactivities, including antioxidant, anticancer, antihypertensive, hepatoprotective, and hypocholesterolemic effects. The aim of this study was to evaluate the gastroprotective effects of water extract of freshwater clam (WEC) on indomethacin (IND)-induced gastric mucosal cell damage in vitro and gastric ulcer in vivo. The cell viability of rat gastric mucosa RGM-1 cells was markedly decreased by 0.8 mM of IND treatment, and pre-treated with various concentration of WEC significantly restored IND-induced cell damage in a dose-dependent manner. WEC also significantly attenuated the elevated reactive oxygen species (ROS) levels, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, and nuclear factor-κB (NF-κB) p65 nuclear translocation induced by IND. In the in vivo study, IND caused severe gastric ulcer in Wistar rats, while WEC pretreatment effectively reduced the ulcer area and edema in the submucosa. We found that WEC significantly restored glutathione (GSH) content in gastric mucosa in a dose-dependent manner (p < 0.05). The reduction of prostaglandin E2 (PGE2) caused by IND was also improved with higher doses of WEC administration. Moreover, the overexpression of COX-2, iNOS, and tumor necrosis factor-α (TNF-α) proteins in gastric mucosa was downregulated by administration of WEC. Consequently, WEC can be used as a potential nutritional supplement to improve NSAIDs-caused gastric mucosal lesions.

Biological actions and molecular effects of resveratrol, pterostilbene, and 3'-hydroxypterostilbene.

Stilbenes are a class of polyphenolic compounds, naturally found in a wide variety of dietary sources such as grapes, berries, peanuts, red wine, and some medicinal plants. There are several well-known stilbenes including trans-resveratrol, pterostilbene, and 3'-hydroxypterostilbene. The core chemical structure of stilbene compounds is 1,2-diphenylethylene. Recently, stilbenes have attracted extensive attention and interest due to their wide range of health-beneficial effects such as anti-inflammation, -carcinogenic, -diabetes, and -dyslipidemia activities. Moreover, accumulating in vitro and in vivo studies have reported that stilbene compounds act as inducers of multiple cell-death pathways such as apoptosis, cell cycle arrest, and autophagy for chemopreventive and chemotherapeutic agents in several types of cancer cells. The aim of this review is to highlight recent molecular findings and biological actions of trans-resveratrol, pterostilbene, and 3'-hydroxypterostilbene.

Lactobacillus pentosus GMNL-77 inhibits skin lesions in imiquimod-induced psoriasis-like mice.

Psoriasis, which is regarded as a T-cell-mediated chronic inflammatory skin disease, is characterized by hyperproliferation and poor differentiation of epidermal keratinocytes. In this study, we aimed to determine the in vivo effect of a potentially probiotic strain, Lactobacillus pentosus GMNL-77, in imiquimod-induced epidermal hyperplasia and psoriasis-like skin inflammation in BALB/c mice. Oral administration of L. pentosus GMNL-77 significantly decreased erythematous scaling lesions. Real-time polymerase chain reaction showed that treatment with L. pentosus GMNL-77 significantly decreased the mRNA levels of proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin (IL)-6, and the IL-23/IL-17A axis-associated cytokines (IL-23, IL-17A/F, and IL-22) in the skin of imiquimod-treated mice. In addition, we found that L. pentosus GMNL-77 decreased the spleen weights of the imiquimod-treated group and reduced the numbers of IL-17- and IL-22-producing CD4+ T cells in the spleen. In conclusion, the present study provides insight into the potential use of L. pentosus GMNL-77 in the future treatment of psoriasis.

Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo.

BACKGROUND: Lung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC). METHODS: In this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS. RESULTS: Our results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP. CONCLUSIONS: We demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS.

Sinularin induces DNA damage, G2/M phase arrest, and apoptosis in human hepatocellular carcinoma cells.

BACKGROUND: Sinularin isolated from the cultured soft coral Sinularia flexibilis has been reported to exert potent cytotoxic effects against particular types of cancer. This study was carried out to investigate the cytotoxic effects in sinularin-treated human hepatocellular carcinoma cells, HepG2, and to subsequently explore the underlying molecular mechanisms. METHODS: TheMTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl- tetrazolium bromide) method was used to evaluate the cytotoxicity of sinularin on HepG2 and Hep3B cell lines. Furthermore, the cell cycle distribution assay, apoptosis assay, and western blot analysis in vitro were used to explore the possible mechanisms of action. RESULTS: From the results of our study, cell viability was obviously inhibited by sinularin in a dose-dependent manner. In addition, our results suggested that sinularin triggered DNA damage and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser(1981)), p-Chk2 (Tyr(68)), p-cdc2 (Tyr(15)), and p53 coupled with increased expression of downstream proteins p21 and down-regulation of p-cdc25 (Ser(216)). Moreover, the results of the apoptosis assay and western blot analysis indicated that the cytotoxic activity could be related to mitochondrial apoptosis, characterized by decrease of Bcl-2 expression, disruption of mitochondrial membrane potential, and sequential activation of caspases and Poly (ADP-ribose) polymerase (PARP). CONCLUSIONS: This study reveals for the first time the anti-HCC activities of sinularin, the active compound isolated from the cultured soft coral Sinularia flexibilis. We believe that our results warrant further evaluation of sinularin as a new anti-HCC chemotherapeutic agent.

Tangeretin derivative, 5-acetyloxy-6,7,8,4'-tetramethoxyflavone induces G2/M arrest, apoptosis and autophagy in human non-small cell lung cancer cells in vitro and in vivo.

Tangeretin, a major phytochemicals in tangerine peels--an important Chinese herb, has been found to have anti-carcinogenic properties. To improve bioavailability and increase potency of tangeretin, its derivative, 5-acetyloxy-6,7,8,4'-tetramethoxyflavone (5-AcTMF), has been synthesized and shown potent inhibition of proliferation activity against human breast and leukemia cancer cell lines. In this study, we have further investigated the anticancer effects of 5-AcTMF on CL1-5 non-small cell lung cancer cells (NSCLC) both in vitro and in vivo and demonstrated that 5-AcTMF effectively inhibited cancer cell proliferation, induced G2/M-phase arrest associated with cdc2 and CDC25c and increased in the apoptotic cells associated with caspase activation, down regulation of Bcl-2, XIAP and Survivn, inducing release of cytochrome c into the cytosol and disruption of mitochondrial membrane potential. We also found that 5-AcTMF treatment of CL1-5 activated autophagy, indicated by triggered autophagosome formation and increased LC3-II levels and formation of LC3 puncta. Moreover, we also found that 5-AcTMF lowered phophoatidylinositol 3-kinase/AKT/mTOR signaling pathway. Over-expression of AKT by AKT cDNA transfection decreased 5-AcTMF mediated apoptosis and autophagy, supporting the induction of apoptosis and autophagy by inhibition of AKT pathway. In an animal study, 5-AcTMF effectively delayed tumor growth in a nude mouse model of CL1-5 xenografts without observed adverse effect. Immunohistochemistry Analysis indicated that 5-AcTMF induced CL1-5 cell apoptosis and autophagy in vivo. Taken together, these data demonstrate that 5-AcTMF is a novel small molecule agent that can inhibit NSCLC cell proliferation, and induce G(2)/M phase arrest and via the mitochondrial apoptotic pathway and autophagy.

5-demethylnobiletin promotes the formation of polymerized tubulin, leads to G2/M phase arrest and induces autophagy via JNK activation in human lung cancer cells.

5-Demethylnobiletin is a hydroxylated polymethoxyflavone found in citrus plants that shows antiproliferative activities in several cancer cell lines. In this study, we investigated the effects and underlying molecular mechanisms of 5-demethylnobiletin on inhibition of cell growth, apoptosis, cell cycle and autophagy in A549 and CL1-5 lung cancer cells. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay suggested that 5-demethylnobiletin inhibited cell growth in a dose- and time-dependent manner. Flow cytometry results suggested that 5-demethylnobiletin inhibited proliferation in lung cancer cells by inducing G2/M cell cycle phase arrest but predominantly not through apoptosis. Western blot results illustrated that the blockade of the cell cycle was associated with reduced levels of cdc25 and cdc2. Notably, our results indicated that 5-demethylnobiletin induced significant abnormal microtubule dynamics in A549 and CL1-5 cells, a novel finding. Studies conducted with isolated tubulin and docking models suggest that 5-demethylnobiletin promoted the polymerization of microtubules and bound to the taxol site. Additionally, 5-demethylnobiletin might also induce autophagy via activation of the JNK signaling pathway in A549 and CL1-5 cells. Pretreatment of the cells with the autophagy inhibitor 3-methyladenine significantly potentiated 5-demethylnobiletin-induced apoptosis, suggesting that 5-demethylnobiletin-induced autophagy mitigated cell apoptosis. Further investigation revealed that 5-demethylnobiletin inhibition of CL1-5 lung cancer cell growth was reproducible in a nude mouse model. Taken together, these studies suggest that 5-demethylnobiletin has anti-lung cancer efficacy both in vitro and in vivo possibly through induction of G2/M arrest, autophagy and apoptosis.

Rice bran feruloylated oligosaccharides activate dendritic cells via Toll-like receptor 2 and 4 signaling.

This work presents the effects of feruloylated oligosaccharides (FOs) of rice bran on murine bone marrow-derived dendritic cells (BMDCs) and the potential pathway through which the effects are mediated. We found that FOs induced phenotypic maturation of DCs, as shown by the increased expression of CD40, CD80/CD86 and MHC-I/II molecules. FOs efficiently induced maturation of DCs generated from C3H/HeN or C57BL/6 mice with normal toll-like receptor 4 (TLR-4) or TLR-2 but not DCs from mice with mutated TLR4 or TLR2. The mechanism of action of FOs may be mediated by increased phosphorylation of ERK, p38 and JNK mitogen-activated protein kinase (MAPKs) and increased NF-kB activity, which are important signaling molecules downstream of TLR-4 and TLR-2. These data suggest that FOs induce DCs maturation through TLR-4 and/or TLR-2 and that FOs might have potential efficacy against tumor or virus infection or represent a candidate-adjuvant approach for application in immunotherapy and vaccination.

Baicalein Triggers Mitochondria-Mediated Apoptosis and Enhances the Antileukemic Effect of Vincristine in Childhood Acute Lymphoblastic Leukemia CCRF-CEM Cells.

Acute lymphoblastic leukemia (ALL) accounts for approximately 75% of childhood leukemia, and chemotherapy remains the mainstay therapy. Baicalein is an active flavonoid used in traditional Chinese medicine and has recently been found to have anticancer, anti-inflammatory, and antiallergic properties. This study aims to investigate the molecular apoptotic mechanisms of baicalein in CCRF-CEM leukemic cells and to evaluate the combined therapeutic efficacy of baicalein with several commonly used chemotherapeutic drugs in CCRF-CEM cells. Our results demonstrate that baicalein induces mitochondria-dependent cleavage of caspases-9 and -3 and PARP with concomitant decreases in IAP family proteins, survivin, and XIAP. Furthermore, our results present for the first time that baicalein triggers a convergence of the intrinsic and extrinsic apoptotic pathways via the death receptor-caspase 8-tBid signaling cascade in CCRF-CEM cells. In addition, we also present for the first time that the combination of baicalein and vincristine results in a synergistic therapeutic efficacy. Overall, this combination strategy is recommended for future clinical trials in the treatment of pediatric leukemia owing to baicalein's beneficial effects in alleviating the vomiting, nausea, and skin rashes caused by chemotherapy.

Immunomodulatory effects of feruloylated oligosaccharides from rice bran.

Feruloylated oligosaccharides (FOs), the ferulic acid ester of oligosaccharides, can be released either by the enzymatic or mild acid hydrolysis of arabinoxylans present in cereal bran, and are usually considered as natural antioxidants. However, no related research is available to explain their immunomodulatory effects. This report elucidated their immunomodulatory effects through the variations of pro-inflammatory mediators in vitro. FOs were obtained from the mild acid hydrolysis of rice bran. We found that FOs (0.1-100 µg/ml) induced tumour necrosis factor alpha (TNF-α), IL-1ß, IL-6, nitric oxide (NO) and PGE(2) production in unstimulated macrophages, RAW264.7 cells. Furthermore, pre- and post-treated FOs (0.1-100 µg/ml) dose-dependently suppressed TNF-α, IL-1ß, IL-6 and NO production, and induced IL-10 production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells without exerting cytotoxicity. As a result anti-inflammatory and therapeutic activities were revealed. It is noteworthy that prostaglandin E(2) (PGE(2)) production was significantly suppressed at an FO level of 100 µg/ml. The in vitro assessment of inflammatory mediators should be useful in further characterising the effects of FOs on immunomodulation. Moreover, it will create the economical value of rice bran, which has long been considered as conventional agricultural wastes.

Dietary administration of δ- and γ-tocopherol inhibits tumorigenesis in the animal model of estrogen receptor-positive, but not HER-2 breast cancer.

Tocopherol, a member of the vitamin E family, consists of four forms designated as α, ß, γ, and δ. Several large cancer prevention studies with α-tocopherol have reported no beneficial results, but recent laboratory studies have suggested that δ- and γ-tocopherol may be more effective. In two different animal models of breast cancer, the chemopreventive activities of individual tocopherols were assessed using diets containing 0.3% of tocopherol (α-, δ-, or γ-) or 0.3% of a γ-tocopherol rich mixture (γ-TmT). Although administration of tocopherols did not prevent human epidermal growth factor receptor 2 (HER2/neu)-driven tumorigenesis, δ- and γ-tocopherols inhibited hormone-dependent mammary tumorigenesis in N-methyl-N-nitrosourea (NMU)-treated female Sprague-Dawley rats. NMU-treated rats showed an average tumor burden of 10.6 ± 0.8 g in the control group at 11 weeks, whereas dietary administration of δ- and γ-tocopherols significantly decreased tumor burden to 7.2 ± 0.8 g (P < 0.01) and 7.1 ± 0.7 g (P < 0.01), respectively. Tumor multiplicity was also reduced in δ- and γ-tocopherol treatment groups by 42% (P < 0.001) and 32% (P < 0.01), respectively. In contrast, α-tocopherol did not decrease tumor burden or multiplicity. In mammary tumors, the protein levels of proapoptotic markers (BAX, cleaved caspase-9, cleaved caspase-3, cleaved PARP) were increased, whereas antiapoptotic markers (Bcl-2, XIAP) were inhibited by δ-tocopherol, γ-tocopherol, and γ-TmT. Furthermore, markers of cell proliferation (PCNA, PKCα), survival (PPAR-γ, PTEN, phospho-Akt), and cell cycle (p53, p21) were affected by δ- and γ-tocopherols. Both δ- and γ-tocopherols, but not α-tocopherol, seem to be promising agents for the prevention of hormone-dependent breast cancer.

δ- and γ-tocopherols, but not α-tocopherol, inhibit colon carcinogenesis in azoxymethane-treated F344 rats.

The cancer preventive activity of vitamin E has been extensively discussed, but the activities of specific forms of tocopherols have not received sufficient attention. Herein, we compared the activities of δ-tocopherol (δ-T), γ-T, and α-T in a colon carcinogenesis model. Male F344 rats, seven weeks old, were given two weekly subcutaneous injections of azoxymethane (AOM) each at a dose of 15 mg/kg body weight. Starting 1 week before the AOM injection, the animals were maintained on a modified AIN76A diet, or the same diet containing 0.2% of δ-T, γ-T, α-T, or a γ-T-rich mixture of tocopherols (γ-TmT), until the termination of the experiment at 8 weeks after the second AOM injection. δ-T treatment showed the strongest inhibitory effect, decreasing the numbers of aberrant crypt foci by 62%. γ-T and γ-TmT were also effective, but α-T was not. Immunohistochemical analysis showed that δ-T and γ-T treatments reduced the levels of 4-hydroxynonenal and nitrotyrosine and the expression of cyclin D1 in the colon, preserved the expression of PPAR-γ, and decreased the serum levels of prostaglandin E2 and 8-isoprostane. Supplementation with 0.2% δ-T, γ-T, or α-T increased the respective levels of tocopherols and their side-chain degradation metabolites in the serum and colon tissues. Rather high concentrations of δ-T and γ-T and their metabolites were found in colon tissues. Our study provides the first evidence for the much higher cancer preventive activity of δ-T and γ-T than α-T in a chemically induced colon carcinogenesis model. It further suggests that δ-T is more effective than γ-T.

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice.

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.

Pro-oxidative activities and dose-response relationship of (-)-epigallocatechin-3-gallate in the inhibition of lung cancer cell growth: a comparative study in vivo and in vitro.

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. Nevertheless, the dose-response relationship of the inhibitory activity in vivo has not been systematically characterized. The present studies were conducted to address these issues, as well as the involvement of reactive oxygen species (ROS), in the inhibitory action of EGCG in vivo and in vitro. We characterized the inhibitory actions of EGCG against human lung cancer H1299 cells in culture and in xenograft tumors. The growth of tumors was dose dependently inhibited by EGCG at doses of 0.1, 0.3 and 0.5% in the diet. Tumor cell apoptosis and oxidative DNA damage, assessed by the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and phosphorylated histone 2A variant X (gamma-H2AX), were dose dependently increased by EGCG treatment. However, the levels of 8-OHdG and gamma-H2AX were not changed by the EGCG treatment in host organs. In culture, the growth of viable H1299 cells was dose dependently reduced by EGCG; the estimated concentration that causes 50% inhibition (IC(50)) (20 microM) was much higher than the IC(50) (0.15 microM) observed in vivo. The action of EGCG was mostly abolished by the presence of superoxide dismutase (SOD) and catalase, which decompose the ROS formed in the culture medium. Treatment with EGCG also caused the generation of intracellular ROS and mitochondrial ROS. Although EGCG is generally considered to be an antioxidant, the present study demonstrates the pro-oxidative activities of EGCG in vivo and in vitro in the described experimental system.

A gamma-tocopherol-rich mixture of tocopherols inhibits chemically induced lung tumorigenesis in A/J mice and xenograft tumor growth.

The present study investigated the effects of a preparation of a gamma-tocopherol-rich mixture of tocopherols (gamma-TmT) on chemically induced lung tumorigenesis in female A/J mice and the growth of H1299 human lung cancer cell xenograft tumors. In the A/J mouse model, the lung tumors were induced by either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; intraperitoneal injections with 100 and 75 mg/kg on Week 1 and 2, respectively) or NNK plus benzo[a]pyrene (B[a]P) (8 weekly gavages of 2 mumole each from Week 1 to 8). The NNK plus B[a]P treatment induced 21 tumors per lung on Week 19; dietary 0.3% gamma-TmT treatment during the entire experimental period significantly lowered tumor multiplicity, tumor volume and tumor burden (by 30, 50 and 55%, respectively; P < 0.05). For three groups of mice treated with NNK alone, the gamma-TmT diet was given during the initiation stage (Week 0 to 3), post-initiation stage (Week 3 to 19) or the entire experimental period, and the tumor multiplicity was reduced by 17.8, 19.7 or 29.3%, respectively (P < 0.05). gamma-TmT treatment during the tumor initiation stage or throughout the entire period of the experiment also significantly reduced tumor burden (by 36 or 43%, respectively). In the xenograft tumor model of human lung cancer H1299 cells in NCr-nu/nu mice, 0.3% dietary gamma-TmT treatment significantly reduced tumor volume and tumor weight by 56 and 47%, respectively (P < 0.05). In both the carcinogenesis and tumor growth models, the inhibitory action of gamma-TmT was associated with enhanced apoptosis and lowered levels of 8-hydroxydeoxyguanine, gamma-H2AX and nitrotyrosine in the tumors of the gamma-TmT-treated mice. In cell culture, the growth of H1299 cells was inhibited by tocopherols with their effectiveness following the order of delta-T > gamma-TmT > gamma-T, whereas alpha-T was not effective. These results demonstrate the inhibitory effect of gamma-TmT against lung tumorigenesis and the growth of xenograft tumors of human lung cancer cells. The inhibitory activity may be due mainly to the actions of delta-T and gamma-T.

Cytotoxic activities of 9,11-dehydroergosterol peroxide and ergosterol peroxide from the fermentation mycelia of ganoderma lucidum cultivated in the medium containing leguminous plants on Hep 3B cells.

The objective of this study was to investigate the cytotoxicity of the ethanolic extract of mycelia from Ganoderma lucidum (EMG) cultivated in a medium containing leguminous plants Glycine max (L.) Merr. and Astragalus membranaceus on human hepatocellular carcinoma cells (Hep 3B) and to isolate the active components from EMG. The results indicated that EMG induced cytotoxicity in a dose- and time-dependent manner, and the cells treated with EMG for 24, 48, and 72 h had IC(50) values of 156.8, 89.9, and 70.1 microg/mL, respectively. Furthermore, EMG was fractionated into seven fractions (F1-F7). We found that F5 and F6 had higher growth inhibitory effects on Hep 3B cells than the other fractions, and F6 possessed enough amounts (about 2.1 g) to carry out a more detailed study. F6 caused a sub-G1 peak rise and DNA fragmentation in Hep 3B cells and was further separated by high-performance liquid chromatography to obtain two active compounds, 9,11-dehydroergosterol peroxide [9(11)-DHEP] (compound 1) and ergosterol peroxide (EP) (compound 2). The IC(50) values of 9(11)-DHEP and EP based on the cell viability of Hep 3B were 16.7 and 19.4 microg/mL, respectively.