Safrole suppresses murine myelomonocytic leukemia WEHI‐3 cells in vivo,and stimulates macrophage phagocytosis and natural killer cell cytotoxicity in leukemic mice

Many anticancer drugs are obtained from phytochemicals and natural products. However, some phytochemicals have mutagenic effects. Safrole, a component of Piper betle inflorescence, has been reported to be a carcinogen. We have previously reported that safrole induced apoptosis in human oral cancer cells in vitro and inhibited the human oral tumor xenograft growth in vivo. Until now, there is no information addressing if safrole promotes immune responses in vivo. To evaluate whether safrole modulated immune function, BALB/c mice were intraperitoneally injected with murine myelomonocytic WEHI‐3 leukemia cells to establish leukemia and then were treated with or without safrole at 4 and 16 mg/kg. Animals were sacrificed after 2 weeks post‐treatment with safrole for examining the immune cell populations, phagocytosis of macrophages and the natural killer (NK) cells' cytotoxicity. Results indicated that safrole increased the body weight, and decreased the weights of spleen and liver in leukemic mice. Furthermore, safrole promoted the activities of macrophages phagocytosis and NK cells' cytotoxicity in leukemic mice when compared with untreated leukemic mice. After determining the cell marker population, we found that safrole promoted the levels of CD3 (T cells), CD19 (B cells) and Mac‐3 (macrophages), but it did not affect CD11b (monocytes) in leukemic mice. In conclusion, safrole altered the immune modulation and inhibited the leukemia WEHI‐3 cells in vivo. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28:601–608, 2013.     

Phenethyl isothiocyanate promotes immune responses in normal BALB/c mice,inhibits murine leukemia WEHI‐3 cells,and stimulates immunomodulations in vivo

Enhanced cruciferous vegetable consumption is associated with the reduction of cancer incidence as shown in epidemiological studies. Phenethyl isothiocyanate (PEITC), one of the important compounds in cruciferous vegetables, has been shown to induce apoptosis in many types of human cancer cell lines, but there is no available information addressing the effects on normal and leukemia mice in vivo. The purpose of this study is to focus on the in vivo effects of PEITC on immune responses of normal and WEHI‐3 leukemia BALB/c mice in vivo. Influences of PEITC on BALB/c mice after intraperitoneal (i.p.) injection with WEHI‐3 cells and normal mice were investigated. In normal BALB/c mice, PEITC did not affect the body weight when compared to the olive oil treated animals. Moreover, PEITC promoted phagocytosis by macrophages from peripheral blood mononuclear cells (PBMC) and peritoneal cavity, increased the levels of CD11b and Mac‐3, decreased the level of CD19 and promoted natural killer (NK) cell cytotoxic activity, but it did not alter the level of CD3. Also, PEITC enhanced T cell proliferation after concanavalin A (Con A) stimulation. Otherwise, PEITC increased the body weight, but decreased the weight of liver and spleen as compared to the olive oil‐treated WEHI‐3 leukemia mice. PEITC also increased the level of CD19, decreased the levels of CD3 and Mac‐3 rather than influence in the level of CD11b, suggesting that the differentiation of the precursor of macrophages and T cells was inhibited, but the differentiation of the precursor of B cells was promoted in leukemia mice. Furthermore, PEITC enhanced phagocytosis by monocytes and macrophages from PBMC and peritoneal cavity, and also promoted the NK cell cytotoxic activity in comparison with the group of leukemia mice. Based on these observations, the biological properties of PEITC can promote immune responses in normal and WEHI‐3 leukemia mice in vivo. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Alpha‐phellandrene‐induced apoptosis in mice leukemia WEHI‐3 cells in vitro

Although reports have shown that α‐phellandrene (α‐PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α‐PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α‐PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α‐PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI‐3 cells in vitro. Results indicated that α‐PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub‐G1 phase (apoptosis) in WEHI‐3 cells. α‐PA increased the productions of reactive oxygen species (ROS) and Ca²⁺ and decreased the levels of mitochondrial membrane potential (ΔΨ_m) in dose‐ and time‐dependent manners in WEHI‐3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α‐PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI‐3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α‐PA induces cell cycle arrest and apoptosis in WEHI‐3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640–1651, 2016.     

Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI‐3 cells in vitro and promoting immune responses in WEHI‐3 generated leukemia mice in vivo

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti‐inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide‐induced cytotoxicity in murine leukemia WEHI‐3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca²⁺ release and mitochondrial membrane potential (ΔΨ_m), and activations of caspase‐8, ‐9, and ‐3. Triptolide increased protein levels of Fas, Fas‐L, Bax, cytochrome c, caspase‐9, Endo G, Apaf‐1, PARP, caspase‐3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI‐3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC‐3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross‐talk between cross‐interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI‐3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac‐3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550–568, 2017.     

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.     

Arsenic trioxide (As₂O₃) inhibits murine WEHI-3 leukemia in BALB/c mice in vivo

Arsenic trioxide (As₂O₃) is used clinically to treat acute promyelocytic leukemia (APL) and has activity in vitro for induction of apoptosis in several solid tumor cell lines. To investigate the potential therapeutic application of As₂O₃ for leukemia, we analyzed the effects of As₂O₃ on the WEHI‐3 cells‐induced orthotopic leukemia animal model in vivo in this study. We established the WEHI‐3 cells leukemia mice through the injection of murine WEHI‐3 cells into BALB/c mice, and they were then treated with As₂O₃ (0.9 and 4.5 mg kg^?1; p.o.) and/or combined with all‐trans‐retinoic acid (ATRA), (30 mg kg^?1; i.p.). The results indicated that (1) As₂O₃ alone or As₂O₃ combined with ATRA promoted the total survival rate of leukemia mice and these effects are dose‐dependent; (2) As₂O₃ did not affect the body weight but decreased the spleen weight; however, it did not affect liver weight; (3) As₂O₃ alone or As₂O₃ combined with ATRA increased the levels of CD3 and CD19, indicating that the differentiation of T and B cells were promoted; and (4) As₂O₃ alone or As₂O₃ combined with ATRA did not change the levels of Mac‐3 and CD11b markers, indicating that the differentiation of the precursor of macrophage were not inhibited. Based on these observations, As₂O₃ alone or As₂O₃ combined with ATRA have efficacious antileukemia activity in WEHI‐3 cells leukemia in vivo. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Rutin inhibits human leukemia tumor growth in a murine xenograft model in vivo

Numerous studies have shown that rutin has anticancer effects. We have previously reported that rutin induced cell cycle arrest and apoptosis in murine leukemia WEHI‐3 cells in vitro and in vivo. However, there are no data showing that rutin inhibits human leukemia HL‐60 cells in vivo in a murine xenograft animal model. Human leukemia HL‐60 cells were implanted into mice and treated with vehicle (1% DMSO), rutin (120 mg/kg of body weight) or vinblastine (120 μg/kg of body weight). Compounds and agents were injected once every four days intraperitoneally (i.p.) for 36 days. Treatment with 120 mg/kg of rutin or with 120 μg/kg of vinblastine resulted in a reduction of tumor weight and volume when compared with the control groups. Tumor size in xenograft mice treated with 120 mg/kg of rutin was significantly smaller than that in the untreated‐control group. These novel findings indicate that rutin inhibits tumor growth in a xenograft animal model. Rutin may be useful in treating leukemia but certainly much more research is needed. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

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.     

Lactate dehydrogenase downregulation mediates the inhibitory effect of diallyl trisulfide on proliferation,metastasis, and invasion in triple‐negative breast cancer

The Warburg effect plays a critical role in tumorigenesis, suggesting that specific agents targeting Warburg effect key proteins may be a promising strategy for cancer therapy. Previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of breast cancer cells by inducing apoptosis in vitro and in vivo. However, whether the Warburg effect is involved with the apoptosis‐promoting action of DATS is unclear. Here, we show that the action of DATS is associated with downregulation of lactate dehydrogenase A (LDHA), an essential protein of the Warburg effect whose upregulation is closely related to tumorigenesis. Interestingly, inhibition of the Warburg effect by DATS in breast cancer cells did not greatly affect normal cells. Furthermore, DATS inhibited growth of breast cancer cells, particularly in MDA‐MB‐231, a triple‐negative breast cancer (TNBC) cell, and reduced proliferation and migration; invasion was reversed by over‐expression of LDHA. These data suggest that DATS inhibits breast cancer growth and aggressiveness through a novel pathway targeting the key enzyme of the Warburg effect. Our study shows that LDHA downregulation is involved in the apoptotic effect of DATS on TNBC. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1390–1398, 2017.     

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.     

Combinational treatment of 5‐fluorouracil and casticin induces apoptosis in mouse leukemia WEHI‐3 cells in vitro

Leukemia is one of the major diseases causing cancer‐related deaths in the young population, and its cure rate is unsatisfying with side effects on patients. Fluorouracil (5‐FU) is currently used as an anticancer drug for leukemia patients. Casticin, a natural polymethoxyflavone, exerts anticancer activity against many human cancer cell lines in vitro, but no other reports show 5‐FU combined with casticin increased the mouse leukemia cell apoptosis in vitro. Herein, the antileukemia activity of 5‐FU combined with casticin in WEHI‐3 mouse leukemia cells was investigated in vitro. Treatment of two‐drug combination had a higher decrease in cell viability and a higher increase in apoptotic cell death, the level of DNA condensation, and the length of comet tail than that of 5‐FU or casticin treatment alone in WEHI‐3 cells. In addition, the two‐drug combination has a greater production rate of reactive oxygen species but a lower level of Ca²⁺ release and mitochondrial membrane potential (ΔΨ_m) than that of 5‐FU alone. Combined drugs also induced higher caspase‐3 and caspase‐8 activities than that of casticin alone and higher caspase‐9 activity than that of 5‐FU or casticin alone at 48 hours treatment. Furthermore, 5‐FU combined with casticin has a higher expression of Cu/Zn superoxide dismutase (SOD [Cu/Zn]) and lower catalase than that of 5‐FU or casticin treatment alone. The combined treatment has higher levels of Bax, Endo G, and cytochrome C of proapoptotic proteins than that of casticin alone and induced lower levels of B‐cell lymphoma 2 (BCL‐2) and BCL‐X of antiapoptotic proteins than that of 5‐FU or casticin only. Furthermore, the combined treatment had a higher expression of cleaved poly (ADP‐ribose) polymerase (PARP) than that of casticin only. Based on these findings, we may suggest that 5‐FU combined with casticin treatment increased apoptotic cell death in WEHI‐3 mouse leukemia cells that may undergo mitochondria and caspases signaling pathways in vitro.     

Zinc sulfide nanoparticles selectively induce cytotoxic and genotoxic effects on leukemic cells: involvement of reactive oxygen species and tumor necrosis factor alpha

The aim of the present study was to develop zinc sulfide nanoparticles (ZnS NPs) and to study their cytotoxicity against the KG‐1A (human acute myeloid leukemia) cell line. ZnS NPs were synthesized using the pyrolytic method and characterized by X‐ray diffraction, dynamic light scattering, surface zeta potential, scanning electron microscopy and atomic force microscopy. Cell viability study and flow cytometric analysis confirmed the potent cytotoxic effects of ZnS NPs on cancer cells in a dose‐dependent fashion. Successful uptakes of ZnS NPs by leukemic cells were confirmed by phase contrast fluorescence microscopy. pH‐dependent dissolution of ZnS NPs was done using atomic absorption microscopy to understand the cell‐specific internalization of Zn⁺. This internalization of NPs facilitated the generation of excess reactive oxygen species (ROS), followed by tumor necrosis factor alpha (TNF‐α) secretion which caused severe DNA damage as observed in the comet assay and altered the mitochondrial membrane potential (MMP) in leukemic cells. Surprisingly ZnS NPs had no toxic effects on normal lymphocytes at doses up to 50 µg ml^–1. Pre‐treatment with ROS and TNF‐α inhibitor confirmed that these nanoparticles were able to kill leukemic cells by generating an excess amount of ROS and thereby initiated TNF‐α mediated apoptosis pathway. These findings clarify the mechanism with which ZnS NPs induced anticancer activities in vitro. To elicit its utilities and its application to cancer treatment in vivo is under investigation. Copyright © 2014 John Wiley & Sons, Ltd.     

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. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 803–815, 2015.     

cDNA microarray analysis of the gene expression of murine leukemia RAW 264.7 cells after exposure to propofol

Propofol (2,6‐diisopropylphenol) is the most extensively used general anesthetic‐sedative agent and it is employed in clinical patients. It has been shown that propofol exhibits anticancer activities. However, there is no available information to address propofol‐induced cytotoxic effects and affected gene expressions on murine leukemia cells. Therefore, we investigated the effects of propofol on the levels of protein and gene expression, which are associated with apoptotic death in mouse leukemia RAW 264.7 cells in vitro. Results indicated that propofol induced cell morphological changes, cytotoxicity, and induction of apoptosis in RAW 264.7 cells in vitro. Western blot analysis demonstrated that propofol promoted Fas, cytochrome c, caspase‐9 and ?3 active form and Bax levels, but inhibited Bcl‐xl protein level which led to cell apoptosis. Furthermore, cDNA microarray assay indicated that propofol significantly enhanced 5 gene expressions (Gm4884; Gm10883; Lce1c; Lrg1; and LOC100045878) and significantly suppressed 26 gene expressions (Gm10679; Zfp617; LOC621831; LOC621831; Gm5929; Snord116; Gm3994; LOC380994; Gm5592; LOC380994; Gm4638; LOC280487; Gm4638; Tex24; A530064D06Rik; BC094916; EG668725; Gm189; Hist2h3c2; Gm8020; Snord115; Gm3079; Olfr198; Tdh; Snord115; and Olfr1249). Based on these observations, propofol‐altered apoptosis‐related proteins might result from induction of apoptotic gene expression and inhibition of cell growth gene expression, which finally led to apoptosis in a mouse leukemia cell line (RAW 264.7) in vitro. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 471–478, 2013.     

Garlic-derived organosulfur compound exerts antitumor efficacy via activation of MAPK pathway and modulation of cytokines in SGC-7901 tumor-bearing mice

Diallyl trisulfide (DATS), a natural agent derived from garlic, has been tested for its antigastric cancer activities in various preliminary studies. However, more systematic pharmacodymatic (PD) and mechanistic evaluations are clearly needed. The aim of this study was to investigate the antitumor effects of DATS in the treatment of human gastric cancer cell SGC-7901 both in vitro and in vivo using widely recommended study procedures. DATS suppressed cancer cells proliferation and induced cell cycle arrest accompanied by an increase in the expressions of cyclin A2 and cyclin B1 in SGC-7901 cancer cells. DATS also caused an increase in apoptotic cell death, which involved in accumulations of bax, p53, and cytochrome C and reduction of Bcl-2 expressions. Besides, activation of JNK, ERK and p38 phosphorylation in DATS-treated cells suggested that mitogen-activated protein kinase (MAPKs) pathways were involved in DATS-induced apoptosis. Meanwhile, DATS significantly inhibited tumor growth and promoted tumor apoptosis in a xenograft model of gastric cancer cell SGC-7901. DATS inhibited tumor migration and invasion by modulating MMP9 and E-cadherin protein expressions. In addition, DATS treatment evidently increased the cytokine secretions of IL-12, TNF-α and IFN-γ (p < 0.05). Biochemical serum analysis and histopathological examination indicated no obvious side effects in major mouse organs. Therefore, our findings provide a framework for further exploration of DATS as a novel chemotherapeutic for human gastric cancer.     

Regorefenib induces extrinsic/intrinsic apoptosis and inhibits MAPK/NF‐κB‐modulated tumor progression in bladder cancer in vitro and in vivo

The aim of the present study is to investigate anticancer effect and mechanism of regorafenib in bladder cancer in vitro and in vivo. Human bladder cancer TSGH 8301 cells were treated with regorafenib, NF‐κB, AKT, or mitogen‐activated protein kinase (MAPK) inhibitors for different time. The changes of cell viability, NF‐κB activation, apoptotic signaling transduction, and expression of tumor progression‐associated proteins were evaluated with MTT, NF‐κB reporter gene assay, flow cytometry, and Western blotting assay. TSGH 8301 tumor bearing mice were established and treated with vehicle (140 μL of 0.1% DMSO) or regorafenib (10 mg/kg/day by gavage) for 15 days. The changes of tumor volume, body weight, NF‐κB activation, MAPK activation, and tumor progression‐associated proteins (MMP‐9, XIAP, VEGF, and Cyclin‐D1) after regorafenib treatment were evaluated with digital caliper, digital weight, and ex vivo Western blotting assay. Our results demonstrated NF‐κB activation and protein levels of MMP‐9, XIAP, VEGF, and Cyclin‐D1 were significantly reduced by NF‐κB (QNZ), ERK (PD98059), and P38 (SB203580) inhibitors. Regorafenib also significantly induced extrinsic and intrinsic apoptotic signaling transduction in bladder cancer in vitro. In addition, regorafenib significantly inhibited tumor growth, NF‐κB, p38, ERK activation and expression of tumor progression‐associated proteins in bladder cancer in vitro and in vivo. Taken together, these results proved that regorafenib not only induced apoptosis through extrinsic and intrinsic pathways and but suppressed MAPK/ NF‐κB‐modulated tumor progression in bladder cancer.     

In vitro PFOS exposure on immune endpoints in bottlenose dolphins (Tursiops truncatus) and mice

Previous studies in our lab have shown that perfluorooctane sulfonate (PFOS) modulates immune function in mice and correlates with many immune parameters in bottlenose dolphins (Tursiops truncatus). In this study, bottlenose dolphin peripheral blood leukocytes (PBLs) and adult female B6C3F1 mouse splenocytes were exposed to environmentally relevant PFOS concentrations (0–5 µg ml^–1) in vitro; and natural killer (NK) cell activity and lymphocyte proliferation (T and B cell) were assessed using the parallelogram approach for risk assessment. The objectives were: to corroborate results from the correlative studies in bottlenose dolphins with in vitro PFOS exposures; to evaluate the sensitivity of the mouse model as compared with bottlenose dolphins; and to assess risk using the parallelogram approach. In mouse cells, NK cell activity was decreased at in vitro doses of 0.01, 0.5, 0.1, 0.5 and 1 µg PFOS ml^–1 and increased at 5 µg ml^–1. Additionally, B cell proliferation was not altered, but T cell proliferation was decreased at all in vitro PFOS exposures. In dolphin cells, NK cell activity and T cell proliferation were not altered by in vitro PFOS exposure, but B cell proliferation exhibited a positive association in relation to PFOS dose. Overall, the data indicates that: the in vitro exposures of bottlenose dolphin PBLs exhibited results similar to reported correlative fields studies; that mice were generally more sensitive (for these selected endpoints) than were dolphins; and that the parallelogram approach could be used two‐thirds of the time to predict the effects in bottlenose dolphins. Copyright © 2013 John Wiley & Sons, Ltd.     

Impairment of preimplantation and postimplantation embryonic development through intrinsic apoptotic processes by ginsenoside Rg1 in vitro and in vivo

Ginsenoside Rg1, which is the most abundant compound found in Asian ginseng (Panax ginseng), has demonstrated various pharmacological actions, including neuroprotective, immune‐stimulatory, and antidiabetic effects. Pregnant women, especially in the Asian community, consume ginseng as a nutritive supplement. Thus, the effects of ginsenoside‐Rg1 on embryonic development need to be investigated, such as in a mouse model. As previous investigations have found that ginsenoside Rg1 appears to either trigger or prevent apoptosis in different cell lines, the effects of this agent on apoptosis remain to be clarified. In this study, we investigated whether ginsenoside Rg1 exerts a hazardous effect on mouse blastocysts and/or affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25–100 μM ginsenoside Rg1 exhibited significant induction of apoptosis and a corresponding decrease in the inner cell mass (ICM) cell number. Importantly, the implantation rate was lower among ginsenoside Rg1‐treated blastocysts compared to untreated controls. Moreover, embryo transfer assays revealed that blastocysts treated with 100 μM ginsenoside Rg1 exhibited increased resorption of postimplantation embryos and decreased weight among surviving fetuses. In vivo, intravenous injection of mice with ginsenoside Rg1 (2, 4, or 6 mg/kg body weight/day) for 4 days was associated with increased apoptosis of blastocyst‐stage embryos and negatively impacted early embryonic development. Further experiments revealed that these effects may reflect the ability of ginsenoside Rg1 to trigger oxidative stress‐mediated intrinsic apoptotic signaling. Our in vitro results indicate that ginsenoside Rg1 treatment increases intracellular oxidative stress, decreases mitochondrial membrane potential, increases the Bax/Bcl‐2 ratio, and activates caspase‐9 and caspase‐3, but not caspase‐8. Taken together, our study results strongly suggest that ginsenoside Rg1 induces apoptosis and impairs the early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.