Mangiferin induces immune responses and evaluates the survival rate in WEHI-3 cell generated mouse leukemia in vivo.

Mangiferin is a naturally occurring polyphenol, widely distributed in Thymeraceae families, and presents pharmacological activity, including anti-cancer activities in many human cancer cell lines. Mangiferin has also been reported to affect immune responses; however, no available information concerning the effects of mangiferin on immune reactions in leukemia mice in vivo. In the present study, we investigated the effects of mangiferin on leukemia WEHI-3 cell generated leukemia BLAB/c mice. Overall, the experiments were divided into two parts, one part was immune responses experiment and the other was the survival rate experiment. The immune responses and survival rate study, 40 mice for each part, were randomly separated into five groups (N = 8): Group I was normal animals and groups II-V WEHI-3 cell generated leukemia mice. Group II mice were fed normal diet as a positive control; group III, IV, and V mice received mangiferin at 40, 80, and 120 mg/kg, respectively, by intraperitoneal injection every 2 days for 20 days. Leukocytes cell population, macrophage phagocytosis, and NK cell activities were analyzed by flow cytometry. Isolated splenocytes stimulated with lipopolysaccharide (LPS) and concanavalin A (Con A) were used to determine the proliferation of B and T cells, respectively, and subsequently were analyzed by flow cytometry. Results indicated that mangiferin significantly increased body weight, decreased the liver and spleen weights of leukemia mice. Mangiferin also increased CD3 T-cell and CD19 B cell population but decreased Mac-3 macrophage and CD11b monocyte. Furthermore, mangiferin decreased phagocytosis of macrophages from PBMC and peritoneal cavity at 40, 80, and 120 mg/kg treatment. However, it also increased NK cell activity at 40 and 120 mg/kg treatment. There were no effects on T and B cell proliferation at three examined doses. In survival rate studies, mangiferin significantly elevated survival rate at 40 and 120 mg/kg treatment of leukemia mice in vivo.

Ouabain promotes immune responses in WEHI-3 cells to generate leukemia mice through enhancing phagocytosis and natural killer cell activities in vivo.

Ouabain, a cardiotonic steroid, was used for the treatment of heart failure and atrial fibrillation and induces cancer cell apoptosis in many human cancer cells including human leukemia cells. However, there are no reports to show the effects on immune responses in a leukemia mouse model. In this study, WEHI-3 cell generated leukemia mice were developed and treated by oral ouabain at 0, 0.75, 1.5, and 3 mg/kg for 15 days. Results indicated that ouabain did not affect body appearance, but decreased liver and spleen weights, B- and T-cell proliferation at all three doses treatment and increased CD19 cells at 3.0 mg/kg treatment, decreased CD3, CD11b, and Mac-3 cells levels compared with positive control. Furthermore, ouabain increased the macrophage phagocytosis from peripheral blood mononuclear cell and peritoneal cavity at all three doses treatment and increased NK cell activities. Ouabain restored GOT, GPT and LDH levels in WEHI-3 leukemia mice in vivo.

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.

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.

Antitumor effects with apoptotic death in human promyelocytic leukemia HL-60 cells and suppression of leukemia xenograft tumor growth by irinotecan HCl.

Irinotecan HCl (CPT-11) is an anticancer prodrug, but there is no available information addressing CPT-11-inhibited leukemia cells in in vitro and in vivo studies. Therefore, we investigated the cytotoxic effects of CPT-11 in promyelocytic leukemia HL-60 cells and in vivo and tumor growth in a leukemia xenograft model. Effects of CPT-11 on HL-60 cells were determined using flow cytometry, immunofluorescence staining, comet assay, real-time PCR, and Western blotting. CPT-11 demonstrated a dose- and time-dependent inhibition of cell growth, induction of apoptosis, and cell-cycle arrest at G0/G1 phase in HL-60 cells. CPT-11 promoted the release of AIF from mitochondria and its translocation to the nucleus. Bid, Bax, Apaf-1, caspase-9, AIF, Endo G, caspase-12, ATF-6b, Grp78, CDK2, Chk2, and cyclin D were all significantly upregulated and Bcl-2 was down-regulated by CPT-11 in HL-60 cells. Induction of cell-cycle arrest by CPT-11 was associated with changes in expression of key cell-cycle regulators such as CDK2, Chk2, and cyclin D in HL-60 cells. To test whether CPT-11 could augment antitumor activity in vivo, athymic BALB/c(nu/nu) nude mice were inoculated with HL-60 cells, followed by treatment with either CPT-11. The treatments significantly inhibited tumor growth and reduced tumor weight and volume in the HL-60 xenograft mice. The present study demonstrates the schedule-dependent antileukemia effect of CPT-11 using both in vitro and in vivo models. CPT-11 could potentially be a promising agent for the treatment of promyelocytic leukemia and requires further investigation.

Demethoxycurcumin Promotes Macrophage Cell Population and Phagocytosis in WEHI-3 Cell-generated Leukemia BALB/c Mice In Vivo.

BACKGROUND/AIM: Demethoxycurcumin (DMC), one of the components of curcuminoids, has antitumor activities in many human cancer cells and is known to induce apoptosis in human leukemia cells. However, there are no reports showing the effects of DMC on the immune response in leukemia mice in vivo. Herein, we evaluated the impact of DMC on immune responses in WEHI-3-generated leukemia mice in vivo. MATERIALS AND METHODS: Fifty male BALB/c mice were separated randomly into five groups. Group I is normal mice, and groups II-V mice of generated leukemia by WEHI-3 cells. Group II-V mice were intraperitoneally injected with dimethyl sulfoxide (DMSO, as the positive control), 15, 30, and 60 mg/kg of DMC, respectively, every two days for 14 days. The body weight, blood, peritoneal fluid, liver, and spleen were individually analyzed. RESULTS: DMC did not significantly affect animal appearance and body weight. It decreased liver and spleen weight at a high dose. DMC did not affect the cluster of differentiation 3 (CD3) and CD19 cell populations but induced decrease of CD11b at 30 mg/kg treatment. However, DMC at low dose significantly increased the cluster of macrophage (Mac-3) cell populations, but at high dose it decreased them. DMC increased macrophage phagocytosis from peripheral blood mononuclear cells at 15 mg/kg treatment and peritoneal cavity at 15, 30 and 60 mg/kg of DMC treatments. DMC did not significantly affect the cytotoxic activity of natural killer (NK) cells. Furthermore, DMC decreased B and T cell proliferation at high doses. CONCLUSION: DMC elevated macrophage phagocytosis in leukemia mice in vivo.

Demethoxycurcumin Suppresses Proliferation, Migration, and Invasion of Human Brain Glioblastoma Multiforme GBM 8401 Cells via PI3K/Akt Pathway.

BACKGROUND/AIM: Demethoxycurcumin (DMC), one of the derivatives of curcumin, has been shown to induce apoptotic cell death in many human cancer cell lines. However, there is no available information on whether DMC inhibits metastatic activity in human glioblastoma cancer cells in vitro. MATERIALS AND METHODS: DMC at 1.0-3.0 µM significantly decreased the proliferation of GBM 8401 cells; thus, we used 2.0 µM for further investigation regarding anti-metastatic activity in human glioblastoma GBM 8401 cells. RESULTS: The internalized amount of DMC has reached the highest level in GBM 8401 cells after 3 h treatment. Wound healing assay was used to determine cell mobility and results indicated that DMC suppressed cell movement of GBM 8401 cells. The transwell chamber assays were used for measuring cell migration and invasion and results indicated that DMC suppressed cell migration and invasion in GBM 8401 cells. Gelatin zymography assay was used to examine gelatinolytic activity (MMP-2) in conditioned media of GBM 8401 cells treated by DMC and results demonstrated that DMC significantly reduced MMP-2 activity. Western blotting showed that DMC reduced the levels of p-EGFR(Tyr1068), GRB2, Sos1, p-Raf, MEK, p-ERK1/2, PI3K, p-Akt/PKBα(Thr308), p-PDK1, NF-κB, TIMP-1, MMP-9, MMP-2, GSK3α/ß, ß-catenin, N-cadherin, and vimentin, but it elevated Ras and E-cadherin at 24 h treatment. CONCLUSION: DMC inhibited cancer cell migration and invasion through inhibition of PI3K/Akt and NF-κB signaling pathways in GBM 8401 cells. We suggest that DMC may be used as a novel anti-metastasis agent for the treatment of human glioblastoma cancer in the future.

Curcumin-induced DNA damage and inhibited DNA repair genes expressions in mouse-rat hybrid retina ganglion cells (N18).

Curcumin is reported to be a potent inhibitor of the initiation and promotion of many cancer cells. We investigated to examine whether or not curcumin induce DNA damage in mouse-rat hybrid retina ganglion cell line N18 cells. The Comet assay showed that incubation of N18 cells with 10, 25 and 30 microM of curcumin led to a longer DNA migration smear (Comet tail). The DNA gel electrophoresis showed that 20 microM of curcumin for 24 and 48 h treatment induced DNA damage and fragments in N18 cells. The real time PCR analysis showed that 20 microM of curcumin for 48 h treatment decreased ATM, ATR, BRCA1, 14-3-3sigma, DNA-PK and MGMT mRNA, and ATM and MGMT mRNA expression were inhibited in a time-dependent manner. Our results indicate that curcumin caused DNA damage and inhibited DNA repair genes which may be the factors for curcumin-inhibited cell growth.

Casticin Promotes Immune Responses, Enhances Macrophage and NK Cell Activities, and Increases Survival Rates of Leukemia BALB/c Mice.

Casticin, derived from Fructus Viticis, has anticancer properties in many human cancer cells, however, there is no report to show that casticin promotes immune responses and affects the survival rate of leukemia mice in vivo. The aim of this study is to evaluate the effects of casticin on immune responses and the survival rate of WEHI-3 cells generated in leukemia mice in vivo. Animals were divided into six groups: normal control mice, leukemia control mice, mice treated with ATRA (all-trans retinoic acid), and casticin (0.1, 0.2, and 0.4 mg/kg) treated mice. All animals were treated for 14 days and then measured for body weights, total survival rate, cell markers, the weights of liver and spleen, phagocytosis of spleen cells, NK cell activities and cell proliferation. Results show that casticin did not affect animal appearances, however, it increased body weights and decreased the weights of liver at 0.2 mg/kg and 0.4 mg/kg treatment. Casticin also decreased spleen weight at 0.2 mg/kg and 0.4 mg/kg treatment, increased CD3 at 0.1, 0.2 and 0.4 mg/kg doses and increased CD19 at 0.2 mg/kg treatment but decreased CD11b and Mac-3 at 0.1, 0.2 and 0.4 mg/kg treatment. Casticin (0.1, 0.2 and 0.4 mg/kg) increased macrophage phagocytosis from PBMC (peripheral blood mononuclear cell) and peritoneal cavity. Furthermore, casticin increased NK cells' cytotoxic activity and promoted T cell proliferation at 0.1-0.4 mg/kg treatment with or without concanavalin A (Con A) stimulation, but only increased B cell proliferation at 0.1 mg/kg treatment. Based on these observations, casticin could be used as promoted immune responses in leukemia mice in vivo.

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.

Laminarin Promotes Immune Responses and Reduces Lactate Dehydrogenase But Increases Glutamic Pyruvic Transaminase in Normal Mice In Vivo.

BACKGROUND/AIM: Laminarin, a typical component of fungal cell walls, has been shown to induce immune responses in both adult and larval locusts. We investigated the effects of laminarin on immune response and glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) levels in normal mice. MATERIALS AND METHODS: Thirty-six normal BALB/c mice were randomly divided into four groups and treatments were provided by gavage. Group I mice acted as normal control; mice of groups II-IV received laminarin at different doses (100 µl at 1, 2.5 and 5.0 mg/mouse in double-distilled water, respectively). All animals were treated for 14 days and were weighed, blood was collected for determination of cell markers, liver and spleen samples were weighed. Spleens were used for phagocytosis and determination of natural killer (NK) cell activity and cell proliferation by flow cytometric assay. RESULTS: Laminarin reduced the body weights and weights of liver and spleen. Laminarin increased CD3, CD19 and Mac-3 cell populations at 2.5 and 5 mg/mouse, however, these did not affect CD11b marker levels. Laminarin (1 and 5 mg/mouse) reduced macrophage phagocytosis from peripheral blood mononuclear cells, but did not affect phagocytosis by macrophages from the peritoneal cavity. At an effector:target ratio of 50:1, laminarin reduced NK cell cytotoxic activity at all levels, but at a ratio of 25:1, only at 1 mg treatment. Laminarin did not affect T-cell and B-cell proliferation. Laminarin increased the level of GPT and reduced that of LDH at all doses, indicating laminarin can protect against liver injury. Laminarin is worthy of investigation in future experiments on improving immune responses.

Bufalin Enhances Immune Responses in Leukemic Mice Through Enhancing Phagocytosis of Macrophage In Vivo.

BACKGROUND/AIM: Bufalin, bufadienolide present in Chan Su, has been shown to induce cancer cell apoptosis in many human cancer cells, including human leukemia cells, but its effects on immune responses are unknown. MATERIALS AND METHODS: This study investigated whether bufalin affected immune responses of mice with WEHI-3 cell-generated leukemia in vivo. BALB/c mice were intraperitoneally injected with WEHI-3 cells to develop leukemia and then were treated with oral treatment with bufalin at different doses (0, 0.1, 0.2 and 0.4 mg/kg) for 2 weeks. At the end of treatment, all mice were weighted and blood was collected; liver and spleen tissues were collected for cell marker, phagocytosis, natural killer (NK) cell activity and T- and B-cell proliferation measurements by using flow cytometric assays. RESULTS: When compared with the leukemia control group, bufalin increased the body weight, but reduced liver and spleen weights, and reduced CD3, CD16 and Mac-3 cell markers at 0.4 mg/kg treatment and increased CD11b marker at 0.1 and 0.2 mg/kg treatment. Furthermore, bufalin at 0.4 mg/kg increased phagocytosis by macrophages isolated from peripheral blood mononuclear cells and at 0.1 mg/kg by those from the peritoneal cavity. Bufalin (0.2 and 0.4 mg/kg) increased NK cell cytotoxic activity at effector:target ratio of 50:1. Bufalin increased B-cell proliferation at 0.1 and 0.2 mg/kg treatment but only increased T-cell proliferation at 0.1 mg/kg. Bufalin increased glutamate oxaloacetate transaminase level at all dose treatments, increased glutamic pyruvic transaminase level only at 0.1 mg/kg treatment, but reduced the level of lactate dehydrogenase at all dose levels in mice with WEHI-3 cell-induced leukemia in vivo. CONCLUSION: Bufalin increased immune responses by enhancing phagocytosis in mice with leukemia mice.

ROS mediates baicalin-induced apoptosis in human promyelocytic leukemia HL-60 cells through the expression of the Gadd153 and mitochondrial-dependent pathway.

BACKGROUND: Chemotherapy agents, particularly those that can induce apoptosis, are the major intervening strategy in the treatment of leukemia. In this study, we investigated the effects of baicalin (a compound obtained from Scutellaria baicalensis Georgi and S. rivularis Benth Labiateae) on the viability, induction of apoptosis and associated mechanism in human leukemia HL-60 cells. MATERIALS AND METHODS: The cell viability and apoptosis was examined by flow cytometric analysis. The results showed that baicalin induced cytotoxicity in a dose- and time-dependent manner through the activation of caspase-3, as shown by treatment of HL-60 cells with an inhibitor of caspase-3 (z-VAD-fmk). Baicalin increased the levels of ROS, Ca2+ and decreased mitochondrial membrane potential in HL-60 cells. Western blot demonstrated that baicalin promoted the levels of Gadd153, Bax, cytochrome c and caspase-3 and -12, but decreased the levels of Grp78 and Bcl-2 in HL-60 cells. CONCLUSION: Baicalin was found to induce apoptosis in HL-60 cells through multiple pathways.

(-)-Menthol inhibits WEHI-3 leukemia cells in vitro and in vivo.

(-)-Menthol ([1-alpha]-5-methyl-2-[1-methylethyl]-cyclohexanol), is a widely used flavoring ingredient in mouthwash, foods, toothpaste and cigarettes. The studies reported here revealed that (-)-menthol induced cytotoxicity against murine leukemia WEHI-3 cells in vitro in a dose-dependent manner. The effects of (-)-menthol on WEHI-3 cells in vivo (BALBIc mice) were also examined, and it was observed that the Mac-3 and CD11b markers were decreased, indicating inhibition of differentiation of the precursor of macrophage and granulocyte. The weights of liver and spleen samples from mice treated with (-)-menthol were found to be decreased compared to untreated animals.

The role of Ca2+ in (-)-menthol-induced human promyelocytic leukemia HL-60 cell death.

A human promyelocytic leukemia HL-60 cell line was selected to examine the effect of (-)-Menthol on cell death. Based on the results from morphological changes and the percentage of viable cells in HL-60 cells after treatment with various concentrations of (-)-Menthol, it was shown that (-)-Menthol induced cell death through necrosis, not apoptosis. No cell cycle arrest was found in HL-60 cells examined by flow cytometry analysis. Also, the DNA gel electrophoresis method showed that (-)-Menthol did not induce apoptosis in HL-60 cells. However, it was found that (-)-Menthol induced the production of Ca2+ in these examined cells, dose-dependently. When HL-60 cells were pretreated with the chelator (BAPTA) of Ca2+ for 3 hours before addition of (-)-Menthol to the culture, a decrease of Ca2+ production was observed. Under the same conditions, the percentage of viable HL-60 cells was increased. Apparently Ca2+ production is associated with the induction of (-)-Menthol-induced cell death.

Primary cardiac lymphoma.

Primary cardiac lymphoma (PCL) has rarely been reported in Chinese populations. PCL mostly occurs in the right atrium. The clinical manifestations may be variable and are attributed to its location, the presence of congestive heart failure, pericardial effusion, arrhythmia, and cardiomegaly. The prognosis is usually poor because it is usually found too late and therefore, clinicians should be aware of PCL. Imaging examinations are the best methods for initial diagnosis and include echocardiography, computed tomography (CT) scan, magnetic resonance imaging (MRI), and radioisotope scan. However, the final diagnosis is made by pathology, such as cytologic examination of the effusive fluid and tissue biopsy. Because the tumors are difficult to resect, the main treatment for the disease is chemotherapy, which can be successful. Here, we report a 58-year-old man who had a tumor measuring 8 x 5 cm in the right atrium. By clinical staging, including chest X-ray, echocardiography, CT scan of the abdomen, MRI of the heart, whole body tumor Gallium scan, and gastrointestinal series, no metastatic lesion or involvement was found in other parts of the body. Pathologic findings including cytology of pericardial effusion and heart tumor biopsy revealed the case as a diffuse large B-cell lymphoma. After chemotherapy with COP (cyclophosphamide + vincristine + prednisone) and CHOPBE (COP + doxorubicin + bleomycin + etoposide) regimens, the intracardiac tumor had disappeared, but the patient survived for 12 months in total, despite additional radiotherapy over the pericardial lesions. It was presumed that because the tumor was very large and involved all 3 layers of the heart, it did not respond as well to the therapy as expected.

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.

Casticin induces DNA damage and inhibits DNA repair-associated protein expression in B16F10 mouse melanoma cancer cells.

Casticin, a polymethoxyflavone, has been demonstrated to possess anticancer activities, yet no study has shown in detail that casticin induces DNA damage in lung cancer cells. The purpose of this study was to investigate the possible molecular mechanisms of casticin which induce DNA damage and nuclear condensation in murine melanoma cancer B16F10 cells. In this study, by examining and capturing images using phase contrast microscopy, we found that casticin induced cell morphological changes. Moreover, it decreased the total number of viable cells which was measured by flow cytometry. Casticin-induced DNA damage and nuclear DNA condensation were measured by DAPI staining, respectively. Western blotting indicated that casticin decreased the protein levels of O6­methylguanine-DNA methyltransferase (MGMT), breast cancer 1, early onset (BRCA1), mediator of DNA damage checkpoint 1 (MDC1), DNA-dependent protein kinase (DNA-PK) but increased phospho-p53 tumor suppressor protein (p-p53), phospho-ataxia telangiectasia mutated kinase (p-ATM), phospho-histone H2A.X (Ser139) and poly(ADP-ribose) polymerase (PARP) in the B16F10 cells. Furthermore, we used confocal laser system microscopy to examine the protein expression levels and we found that casticin increased the expression of p-p53 and p-H2A.X in the B16F10 cells. Collectively, casticin induced DNA damage and affected DNA repair proteins in the B16F10 cells in vitro.

Evaluation of Hirsutella sinensis mycelium for antifatigue effect.

The aim of this study was to investigate whether Hirsutella sinensis mycelium (HSM) has any antifatigue effect, using a forced swimming model in rats. Forty rats were randomly divided into five groups, each containing eight animals. The control group received 2 ml/kg body weight of distilled water and a positive control group was administered 1.13 ml/kg Quaker Essence of Chicken. The treated swimming groups were administered HSM powder manufactured by Chang Gung Biotechnology Corporation, Ltd., at doses of 63 mg/kg, 189 mg/kg or 378 mg/kg body weight/day, respectively for a period of six weeks. The above experiment was repeated with another 40 rats but for a period of eight weeks. At the end of the experiments, rats were placed in a swimming apparatus and the total swimming time until exhaustion was recorded. Pre-/post-exercise concentrations of serum urea nitrogen (BUN) and lactic acid were also determined. There were no deaths during the study. Physical and behavioral examinations did not reveal any treatment-related adverse effects after dosing. Changes in lactate levels were dose-dependent for the 8- but not the 6-week treatment. BUN levels were more affected by the 8-week treatment of HSM but not significantly altered in the 6-week treatment groups. The 8-week treatment groups showed a significant increase in swimming time to exhaustion compared to the control groups, which was not dose-dependent. For the 6-week treatment, only the middle and high doses increased swimming time to exhaustion. Conjugated diene contents were significantly higher in rats treated at any HSM dose for 8-weeks than the control groups. Swimming did not alter levels of liver glycogen when compared to the control sub-groups. Results of this study demonstrate that HSM improves physical endurance, which may be beneficial in treating conditions where fatigue is a factor and other antifatigue treatments are contraindicated.

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.