Cardamonin induces immune responses and enhances survival rate in WEHI-3 cell-generated mouse leukemia in vivo.

Cardamonin, a monomeric alkaloid, is isolated from Alpinia conchigera Griff and other natural plants. Recently, it has been focused on its anticancer activities, and no information showing its immune effects on leukemia mice was reported. In this study, we investigated the immune effects of cardamonin on WEHI-3 cell-generated leukemia mice. Forty BALB/c mice were randomly divided into four groups: Group I mice were normal animals and groups II-IV were leukemia. Group II mice, as a positive control, were administered with normal diet, and group III and IV mice were treated with 1 and 5 mg/kg of cardamonin, respectively, by intraperitoneal injection every 2 days for 14 days. The population of white blood cells, macrophage phagocytosis, and the proliferations of T and B cells were analyzed by flow cytometry. Another forty mice were also separated randomly into four groups for the determination of survival rate. Results showed that cardamonin did not affect body weight. Cardamonin decreased CD3, CD11b, and Mac-3 cell populations but increased CD19 number. Cardamonin enhanced phagocytic abilities of macrophages from the peripheral blood mononuclear cells of leukemia mice. Furthermore, cardamonin at 1 mg/kg treatment improved the survival rate of leukemia mice in vivo. Therefore, cardamonin could be applied for a leukemia therapeutic reagent at a defined dose.

Cardamonin Induces Cell Cycle Arrest, Apoptosis and Alters Apoptosis Associated Gene Expression in WEHI-3 Mouse Leukemia Cells.

Cardamonin, the chalcone class, is one of the natural components from the spicy herbaceous plant (Alpinia conchigera Griff) and has anticancer activities in many human cancer cell lines. There is, however, no information to show that cardamonin induces cell apoptosis and alters apoptosis associated gene expressions in mouse leukemia cells. Thus, we investigated the effects of cardamonin on the apoptotic cell death and associated gene expression in mouse leukemia WEHI-3 cells in vitro. Results indicated that cardamonin decreased total viable cell number via induced cell morphological changes and apoptotic cell death in WEHI-3 cells that were assay by contrast-phase microscopy and flow cytometry examinations, respectively. The flow cytometry assay indicated that cardamonin increased reactive oxygen species (ROS) and Ca 2+ production, decreased the levels of mitochondrial membrane potential ( ΔΨm) and increased caspase-3, -8 and -9 activities in WEHI-3 cells. Western blotting was performed to analyze expression of relevant pro- and anti-apoptotic proteins and results showed that cardamonin decreased anti-apoptotic protein of Bcl-2 but increased pro-apoptotic protein of Bax in WEHI-3 cells. Furthermore, cardamonin increased cytochrome c, AIF and Endo G release, increased GRP78, caspase-12 that were associated with ER stress and increased Fas, Fas-Ligand and FADD expression. Furthermore, cardamonin increased the gene expressions of DAP (death-associated protein), TMBIM4 transmembrane (BAX inhibitor motif containing 4), ATG5 (autophagy related 5) but decreased the gene expression of DDIT3 (DNA-damage inducible transcript 3), DDIT4 (DNA-damage-inducible transcript 4), BAG6 (BCL2-associated athanogene 6), BCL2L13 [BCL2-like 13 (apoptosis facilitator)] and BRAT1 (BRCA1-associated ATM activator 1) that are associated with apoptosis pathways. Based on those findings, we may suggest cardamonin induced apoptotic cell death through Fas and Fas-Ligand-, caspase- and mitochondria-dependently pathways and also affects the apoptotic gene expression in WEHI-3 cells in vitro.

Ursolic Acid Induces Apoptotic Cell Death Through AIF and Endo G Release Through a Mitochondria-dependent Pathway in NCI-H292 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Ursolic acid (UA), a triterpene compound present in natural plants, has been shown to induce cytotoxic effects on many human cancer cells through induction of cell-cycle arrest and apoptosis. This study investigated the effects of UA on human lung cancer NCI-H292 cells in vitro. MATERIALS AND METHODS: Flow cytometric assay was used to measure the percentage of cell viability, apoptotic cell death by double staining of annexin V and propidium iodide (PI), production of reactive oxygen species (ROS) and Ca2+, and mitochondriaI membrane potential (Ψm). UA-induced chromatin condensation and DNA fragmentation were examined by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis, respectively. Western blotting was used to examine the changes of apoptosis-associated protein expression in NCI-H292 cells. RESULTS: UA reduced cell viability and induced apoptotic cell death. UA increased Ca2+ production, reduced Ψm, but did not affect ROS production in NCI-H292 cells. UA increased apoptosis-inducing factor (AIF) and endonuclease G in NCI-H292 cells. CONCLUSION: Based on these observations, we suggest UA induces apoptotic cell death via AIF and Endo G release through a mitochondria-dependent pathway in NCI-H292 cells.

Laminarin Promotes Immune Responses and Normalizes Glutamic Oxaloacetic Transaminase and Glutamic Pyruvic Transaminase Levels in Leukemic Mice In Vivo.

BACKGROUND/AIM: Laminarin, mainly found in the fronds of Laminaria, has antimicrobial characteristics and induces immune responses. However, there are no available information to show the laminarin effect on glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels in mice with leukemia in vivo. MATERIALS AND METHODS: Fifty normal BALB/c mice were separated randomly into five groups. Group I mice received normal diet as control. Leukemia was generated in groups II-V using WEHI-3 cells: Group II mice received normal diet as positive control; group III, IV and V mice received laminarin at 1, 2.5 and 5 mg/ml with ddH2O, respectively, by oral gavage every 2 days for 14 days (total of seven times). All mice were weighed during the treatment. After treatment, mice were sacrificed, blood was collected for determination of cell markers, liver and spleen samples were weighed, and spleens were used for phagocytosis and natural killer (NK) cell activity and cell proliferation using flow cytometric assay. RESULTS: Laminarin did not affect animal appearances, but increased the body weight at all doses. It reduced the weight of liver at 2.5 and 5 mg/ml and of spleen at 5 mg/ml. Laminarin increased CD3 (2.5 mg/ml) and CD19 (1 and 5 mg/ml) cell populations but reduced CD11b (5 mg/ml) cell populations, however, these did not affect Mac-3 marker level. Laminarin at 1 mg/ml increased phagocytosis by macrophages from peripheral blood mononuclear cell, but did not affect those from the peritoneal cavity. Laminarin increased NK cell cytotoxic activity at all doses and at a target ratio of 25:1 and 50:1. Laminarin did not affect B-cell proliferation, but at 5 mg/ml significantly reduced T-cell proliferation. Laminarin restored glutamate oxaloacetate transaminase (2.5 and 5 mg/ml) and glutamate pyruvate transaminase (2.5 mg/ml) levels. Based on these results, we suggest that laminarin can promote immune responses and protect against liver injury.

Antrodia Cinnamomea Reduces Carbon Tetrachloride-induced Hepatotoxicity In Male Wister Rats.

BACKGROUND/AIM: Antrodia cinnamomea is found with polysaccharides, lipids, vitamins, fibers and ash (minerals) and is well known in Taiwan as a traditional Chinese medicine. Its biological activities have been reported to have anti-inflammatory, anti-fatigue, anti-tumor and immunomodulatory effects, but its protective effects on liver function are still unclear. MATERIALS AND METHODS: We determined if Antrodia cinnamomea was hepatoprotective against carbon tetrachloride (CCl4) toxicity in Wistar rats. Six groups were used in the study: 1) control (no induction by CCl4); 2) negative control (CCl4-induction and no treatment); 3) positive control (silymarin treatment); 4) groups 4-6 were treated with CC14 and different concentrations (350 mg/kg, 1,400 mg/kg, 3,150 mg/kg) of Antrodia cinnamomea. Blood and liver samples of rats were harvested and then detected by biochemical and tissue histochemical analysis. Activity of the antioxidative enzymes glutathione peroxidase, superoxide dismutase and catalase in the liver were also monitored. RESULTS: Only the high-dose treatment was able to decrease serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels and improve liver function. High and medium doses increased total liver protein and reduced hydroxyproline. It was also observed that the high dose treatment reduced lipid peroxidation. Liver sections of CC14 treated animals receiving Antrodia cinnamomea showed less fibrosis compared to the CCl4 control group. CONCLUSION: This finding suggested that Antrodia cinnamomea can either enhance liver recovering from CCl4 damage or attenuate CCl4 toxicity in rats.

Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.

Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN-induced cytotoxic effects and whether or not they went through cell-cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL-60 cells. Cell viability, cell-cycle distribution, sub-G1 (apoptosis), reactive oxygen species (ROS) and Ca2+ production, levels of mitochondrial membrane potential (ΔΨm ), and caspase-3, -8, and -9 activities were assayed by flow cytometry. Apoptosis-associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub-G1 phase development. Furthermore, SFN increased ROS and Ca2+ production and decreased the levels of ΔΨm and activated caspase-3, -8, and -9 activities in HL-60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas-L, caspase-8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl-x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase -8, -3, -4, -6, and -7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL-60 cells via Fas- and mitochondria-dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311-328, 2017.

Sulforaphane promotes immune responses in a WEHI­3­induced leukemia mouse model through enhanced phagocytosis of macrophages and natural killer cell activities in vivo.

Sulforaphane (SFN) is an isothiocyanate, inducing cytotoxic effects in various human cancer cells, including leukemia cells through cell cycle arrest and apoptosis. However, the effect of SFN on the immune responses in a leukemia mouse model remains to be investigated. The present study investigated whether SFN has an effect on the immune responses in a WEHI­3­induced leukemia mouse model in vivo. Normal BALB/c mice were injected with WEHI­3 cells to generate the leukemia mouse model, and were subsequently treated with placebo or SFN (0, 285, 570 and 1,140 mg/kg) for 3 weeks. Following treatment, all mice were weighted and blood samples were collected. In addition, liver and spleen samples were isolated to determine cell markers, phagocytosis and natural killer (NK) cell activities, and cell proliferation was examined using flow cytometry. The results indicated that SFN treatment had no significant effect on the spleen weight, however it decreased liver and body weight. Furthermore, SFN treatment increased the percentage levels of CD3 (T cells) and CD19 (B cell maker), however had no effect on the levels of CD11b (monocytes) or Mac­3 (macrophages), compared with the WEHI­3 control groups. The administration of SFN increased the phagocytosis of macrophages from peripheral blood mononuclear cells and peritoneal cavity, and increased the activity of NK cells from splenocytes. Administration of SFN promoted T and B cell proliferation following stimulation with concanavalin A and lipopolysaccharide, respectively.

Benzyl isothiocyanate (BITC) induces apoptosis of GBM 8401 human brain glioblastoma multiforms cells via activation of caspase-8/Bid and the reactive oxygen species-dependent mitochondrial pathway.

Benzyl isothiocyanate (BITC) is one of member of the isothiocyanate family which has been shown to induce cancer cell apoptosis in many human cancer cells. In the present study, we investigated the effects of BITC on the growth of GBM 8401 human brain glioblastoma multiforms cells. Results indicated that BITC-induced cell morphological changes decreased in the percentage of viable GBM8401 cells and these effects are dose-dependent manners. Results from flow cytometric assay indicated that BITC induced sub-G1 phase and induction of apoptosis of GBM 8401 cells. Furthermore, results also showed that BITC promoted the production of reactive oxygen species (ROS) and Ca2+ release, but decreased the mitochondrial membrane potential (ΔΨm ) and promoted caspase-8, -9, and -3 activates. After cells were pretreated with Z-IETD-FMK, Z-LEHD-FMK, and Z-DEVD-FMK (caspase-8, -9, and -3 inhibitors, respectively) led to decrease in the activities of caspase-8, -9, and -3 and increased the percentage of viable GBM 8401 cells that indicated which BITC induced cell apoptosis through caspase-dependent pathways. Western blotting indicated that BITC induced Fas, Fas-L, FADD, caspase-8, caspase -3, and pro-apoptotic protein (Bax, Bid, and Bak), but inhibited the ant-apoptotic proteins (Bcl-2 and Bcl-x) in GBM 8401 cells. Furthermore, BITC increased the release of cytochrome c, AIF, and Endo G from mitochondria that led to cell apoptosis. Results also showed that BITC increased GADD153, GRP 78, XBP-1, and ATF-6ß, IRE-1α, IRE-1ß, Calpain 1 and 2 in GBM 8401 cells, which is associated with ER stress. Based on these observations, we may suggest that BITC-induced apoptosis might be through Fas receptor, ROS induced ER stress, caspase-3, and mitochondrial signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC-caused growth inhibition and induced apoptotic cell death of GBM 8401 cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1751-1760, 2016.

Evaluation of Hirsutella sinensis mycelium on food safety and anti-hepatoma activity in an animal model.

There is evidence that Hirsutella sinensis may have antitumor activity. The aim of the present study was to determine the anti-hepatoma effects and food safety assessment of Hirsutella sinensis mycelium in vivo and in vitro. Effects on mutagenicity were determined using a bacterial reverse mutation assay employing the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537. There were no dose-dependent increases or decreases in the number of colonies both with and without metabolic S9 activation in Ames tests. Mice were inoculated with SK-Hep 1 cells and those developing tumors were treated with three different concentrations of Hirsutella sinensis mycelium. After six weeks, blood samples were collected and liver pathology was determined. Aspartate aminotransferase levels were significantly different only in the low-dose treatment group (106±27 IU/l, p=0.048), compared to the control group (162±80 IU/l). The tumor weight was significantly different only in the low-dose treatment group. We found that necrosis, hemorrhage and calcifications were presented in both control and experimental groups. Inhibition of tumor growth was observed only at the lowest dose.

Toxicological evaluation of Antrodia cinnamomea in BALB/c mice.

Antrodia cinnamomea is a natural component of some herbal medicines used for treatment of abdominal pain, hypertension and hepatocellular carcinoma in Taiwan and other countries. Subchronic oral toxicity studies of A. cinnamomea extracts in male and female BALB/c mice were performed to evaluate its safety. Three different concentrations of A. cinnamomea (16.67, 833.3 and 1666.67 mg/kg/day) were given orally to groups of mice (10 mice/dose) for 90 consecutive days. All animals survived to the end of the study, and there were no significant differences in body weight among the control and treatment groups. No significant differences were found in hematological and serum biochemical parameters among the control and treatment groups. No abnormalities of internal organs were observed in the treated groups.

Extract of medicinal mushroom Agaricus blazei Murill enhances the non-specific and adaptive immune activities in BALB/c mice.

Agaricus blazei Murill (AbM) is traditionally used against a wide range of conditions such as ulcerative colitis, Crohn's disease, foot-and-mouth disease and chronic hepatitis C infection. In this study, we evaluated the immunomodulatory effects of AbM. For the non-specific immune response experiments, a total of 40 female BALB/c mice were divided into control (group 1) and experimental (groups 2-4) groups of 10 animals each. Groups 2, 3 and 4 were orally-administered high (819 mg/kg), medium (273 mg/kg) and low (136.5 mg/kg) doses of AbM daily for six weeks and then six parameters related to non-specific immune response were detected. For the adaptive immune response experiments, 40 female mice were similarly divided into four groups. After six weeks of treatment, animals were immunized with the OVA immunogen. Two weeks later, splenocytes and sera were collected. Four parameters related to adaptive immune response were evaluated. We found that feeding mice with AbM extract increased the IgG level in serum, promoted phagocytosis of peritoneal macrophages and elevated the activity of Natural killer cells. We also found that the highest dose of AbM increased interleukin-2 (IL-2) levels in splenocytes and that a medium dose increased interferon-γ. The levels of interleukin-4 (IL-4) were reduced or unchanged. T-helper type 1 cytokine levels were increased. AbM increased the humoral immune response and also affected the cellular immune response. These results provide evidence that AbM can modulate innate and adaptive immunity.

Salidroside improves behavioral and histological outcomes and reduces apoptosis via PI3K/Akt signaling after experimental traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) induces a complex sequence of apopototic cascades that contribute to secondary tissue damage. The aim of this study was to investigate the effects of salidroside, a phenolic glycoside with potent anti-apoptotic properties, on behavioral and histological outcomes, brain edema, and apoptosis following experimental TBI and the possible involvement of the phosphoinositide 3-kinase/protein kinase B (PI3K)/Akt signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Mice subjected to controlled cortical impact injury received intraperitoneal salidroside (20, or 50 mg/kg) or vehicle injection 10 min after injury. Behavioral studies, histology analysis and brain water content assessment were performed. Levels of PI3K/Akt signaling-related molecules, apoptosis-related proteins, cytochrome C (CytoC), and Smac/DIABLO were also analyzed. LY294002, a PI3K inhibitor, was administered to examine the mechanism of protection. The protective effect of salidroside was also investigated in primary cultured neurons subjected to stretch injury. Treatment with 20 mg/kg salidroside significantly improved functional recovery and reduced brain tissue damage up to post-injury day 28. Salidroside also significantly reduced neuronal death, apoptosis, and brain edema at day 1. These changes were associated with significant decreases in cleaved caspase-3, CytoC, and Smac/DIABLO at days 1 and 3. Salidroside increased phosphorylation of Akt on Ser473 and the mitochondrial Bcl-2/Bax ratio at day 1, and enhanced phosphorylation of Akt on Thr308 at day 3. This beneficial effect was abolished by pre-injection of LY294002. Moreover, delayed administration of salidroside at 3 or 6 h post-injury reduced neuronal damage at day 1. Salidroside treatment also decreased neuronal vulnerability to stretch-induced injury in vitro. CONCLUSIONS/SIGNIFICANCE: Post-injury salidroside improved long-term behavioral and histological outcomes and reduced brain edema and apoptosis following TBI, at least partially via the PI3K/Akt signaling pathway.

Evaluation of genotoxicity and subclinical toxicity of Agaricus blazei Murrill in the Ames test and in histopathological and biochemical analysis.

This study was conducted in order to assess the safety and tolerability of Agaricus blazei Murrill (ABM) in general toxicological studies by Ames tests in vitro and in 28-day feeding toxicity experiments. There were no dose-dependent increases or decreases in the number of revertant colonies both with and without metabolic activation in Ames tests. Doses of 10, 5 and 0.1 mg/per mouse of ABM daily were administered by oral gavage to mice (n=10) for 28 days. The effects on clinical observations, clinical pathology, and histopathology were evaluated. There were no significant changes in the brain, heart, kidney, liver, spleen, adrenal gland, testes or ovaries visually. With increasing doses, male and female treated mice did not show any gradual elevation of serum concentration in any of the nine items we examined, except for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in females. The AST levels of the treatment by medium or high dose and the ALT levels of the treatment by high dose in females were abnormal in comparison to those of the baseline control group, with significant differences. On studying the histological changes in mice, tissue sections of negative control and experimental groups exhibited no apparent pathological alterations. In summary, the Ames test, pathology determinations, biochemical analysis and routine blood parameters were all normal, except for AST and ALT in females. Results showed that the statistical differences observed in one sex were not observed in the other and were not dose dependent.