Effects of diallyl trisulfide on induction of apoptotic death in murine leukemia WEHI‐3 cells in vitro and alterations of the immune responses in normal and leukemic mice in vivo

Diallyl trisulfide (DATS), a chemopreventive dietary constituent and extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI‐3 cells in vitro and used WEHI‐3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI‐3 cells through the G0/G1 phase arrest and induction of caspase‐3 activation. In in vivo study DATS decreased the weight of spleen of leukemia mice but did not affect the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI‐3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac‐3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI‐3 cells. DATS also promotes immune responses in leukemia and normal mice in vivo. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1343–1353, 2015.     

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.     

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.     

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.     

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.     

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.     

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 (ΔΨ_m) 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‐6β, IRE‐1α, IRE‐1β, 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.     

Bisdemethoxycurcumin‐induced S phase arrest through the inhibition of cyclin A and E and induction of apoptosis via endoplasmic reticulum stress and mitochondria‐dependent pathways in human lung cancer NCI H460 cells

Curcuminoids are the major natural phenolic compounds found in the rhizome of many Curcuma species. Curcuminoids consist of a mixture of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Although numerous studies have shown that curcumin induced cell apoptosis in many human cancer cells, however, mechanisms of BDMC‐inhibited cell growth and ‐induced apoptosis in human lung cancer cells still remain unclear. Herein, we investigated the effect of BDMC on the cell death via the cell cycle arrest and induction of apoptosis in NCI H460 human lung cancer cells. Flow cytometry assay was used to measure viable cells, cell cycle distribution, the productions of reactive oxygen species (ROS) and Ca²⁺, mitochondrial membrane potential (ΔΨ_m) and caspase‐3, ‐8 and ‐9 activity. DNA damage and condension were assayed by Comet assay and DAPI staining, respectively. Western blotting was used to measure the changes of cell cycle and apoptosis associated protein expressions. Results indicated that BDMC significantly induced cell death through induced S phase arrest and induced apoptosis. Moreover, DMC induced DNA damage and condension, increased ROS and Ca²⁺ productions and decreased the levels of ΔΨ_m and promoted activities caspase‐3, ‐8, and ‐9. Western blotting results showed that BDMC inhibited Cdc25A, cyclin A and E for causing S phase arrest, furthermore, promoted the expression of AIF, Endo G and PARP and the levels of Fas ligand (Fas L) and Fas were also up‐regulated. Results also indicated that BDMC increased ER stress associated protein expression such as GRP78, GADD153, IRE1α, IRE1β, ATF‐6α, ATF‐6β, and caspase‐4. Taken together, we suggest that BDMC induced cell apoptosis through multiple signal pathways such as extrinsic, intrinsic and ES tress pathway. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1899–1908, 2016.     

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 (ΔΨ_m), 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 ΔΨ_m 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.     

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.     

Quercetin simultaneously induces G0/G1‐phase arrest and caspase‐mediated crosstalk between apoptosis and autophagy in human leukemia HL‐60 cells

Quercetin is a plant‐derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL‐60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL‐60 cell line in vitro and in vivo. Quercetin‐induced G ₀/G₁‐phase arrest occurred when expressions of cyclin‐dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)?1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)‐II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3‐methyladenine, significantly enhanced quercetin‐mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL‐60 cells with the pan‐caspase inhibitor, Z‐VAD‐fmk, dramatically reversed quercetin‐mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin‐mediated HL‐60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL‐60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML.     

Fisetin‐induced apoptosis of human oral cancer SCC‐4 cells through reactive oxygen species production,endoplasmic reticulum stress,caspase‐, and mitochondria‐dependent signaling pathways

Oral cancer is one of the cancer‐related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin‐induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin‐induced cell death and associated signal pathways on human oral cancer SCC‐4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca²⁺, mitochondria membrane potential (ΔΨ_m), and caspase‐8, ‐9, and ‐3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca²⁺ production, and decreased the level of ΔΨ_m and increased caspase‐3, ‐8, and ‐9 activities in SCC‐4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin‐induced cell apoptosis in SCC‐4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl‐2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC‐4 cells. We also used ATF‐6α, ATF‐6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria‐, and caspase‐dependent pathways.     

Alpha‐phellandrene‐induced DNA damage and affect DNA repair protein expression in WEHI‐3 murine leukemia cells in vitro

Although there are few reports regarding α‐phellandrene (α‐PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α‐PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α‐PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α‐PA on total cell viability and the results indicated that α‐PA induced cell death. Comet assay and 4,6‐diamidino‐2‐phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α‐PA induced DNA damage and condensation in a concentration‐dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α‐PA induced DNA damage in WEHI‐3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α‐PA increased p‐p53, p‐H2A.X, 14‐3‐3‐σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA‐PK, and BRCA‐1. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1322–1330, 2015.     

Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL‐60 cancer cell xenograft growth in vivo

Genistein, a major isoflavone compound in soybeans, has been shown to have biological activities including anti‐cancer activates. In the present, we investigated the anti‐leukemia activity of genistein on HL‐60 cells in vitro. The percentage of viable cell, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS), and Ca²⁺ production and the level of ΔΨ_m were measured by flow cytometric assay. Cell apoptosis and endoplasmic reticulum (ER) stress associated protein expressions were examined by Western blotting assay. Calpain 1, GRP78, and GADD153 expression were measured by confocal laser microscopy. Results indicated that genistein‐induced cell morphological changes, decreased the total viable cells, induced G₂/M phase arrest and DNA damage and fragmentation (cell apoptosis) in HL‐60 cells. Genistein promoted ROS and Ca²⁺ productions and decreased the level of ΔΨ_m in HL‐60 cells. Western blotting assay demonstrated that genistein increased ER stress‐associated protein expression such as IRE‐1α, Calpain 1, GRP78, GADD153, caspase‐7, caspase‐4, and ATF‐6α at 20‐50 μM treatment and increased apoptosis associated protein expression such as pro‐apoptotic protein Bax, PARP‐cleavage, caspase‐9, and ‐3, but decreased anti‐apoptotic protein such as Bcl‐2 and Bid in HL‐60 cells. Calpain 1, GRP78, and GADD153 were increased in HL‐60 cells after exposure to 40 μM of genistein. In animal xenografted model, mice were intraperitoneally injected with genistein (0, 0.2, and 0.4 mg/kg) for 28 days and the body weight and tumor volume were recorded. Results showed that genistein did not affect the body weights but significantly reduced the tumor weight in 0.4 mg/kg genistein‐treated group. Genistein also increased the expressions of ATF‐6α, GRP78, Bax, Bad, and Bak in tumor. In conclusion, genistein decreased cell number through G₂/M phase arrest and the induction of cell apoptosis through ER stress‐ and mitochondria‐dependent pathways in HL‐60 cells and suppressed tumor properties in vivo.     

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species‐modulated pathways in U‐2 OS human osteosarcoma cells

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U‐2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell‐cycle distribution, apoptotic cells in sub‐G1 phase, reactive oxygen species (ROS), Ca²⁺ levels, and mitochondrial membrane potential (ΔΨ_m). Comet assay and 4′‐6‐diamidino‐2‐phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis‐associated protein levels in U‐2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G₀/G₁ arrest, and apoptosis in U‐2 OS cells. CECF‐stimulated activities of caspase‐8, caspase‐9, and caspase‐3, ROS, and Ca²⁺ production, decreased ΔΨ_m levels of in U‐2 OS cells. CECF increased protein levels of caspase‐3, caspase‐9, Bax, cytochrome c, GRP78, AIF, ATF‐6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell‐cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U‐2 OS cells via ROS‐modulated caspase‐dependent and ‐independent pathways. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 1020–1031, 2014.     

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.