WEHI-3 Cells Research Articles

Antineoplastic Activity of Sodium Caseinate in a Cytarabine-Resistant Mouse Acute Myeloid Leukemia Cell Line.

Acute myeloid leukemia (AML) is a hematological neoplasm of rapid and progressive onset, and is the most common form of leukemia in adults. Chemoresistance to conventional treatments such as cytarabine (Ara-C) and daunorubicin is a main cause of relapse, recurrence, metastasis, and high mortality in AML patients. It is known that sodium caseinate (SC), a salt derived from casein, a milk protein, inhibits growth and induces apoptosis in acute myeloid leukemia cells but not in normal hematopoietic cells. However, it is unknown whether SC retains its antileukemic effect in cytarabine-resistant AML cell lines. To evaluate the antineoplastic effect of SC in cytarabine-resistant leukemia models. The SC inhibits the growth and induces apoptosis in parental WEHI-3 AML cells. Here, we generated two cytarabine-resistant sublines, WEHI-CR25 and WEHI-CR50, which exhibit 6- and 16-fold increased resistance to cytarabine, respectively, compared to the parental WEHI-3 cells. Thus, these sublines mimic a chemoresistant model. We demonstrate that WEHI-CR25 and WEHI-CR50 cells retain sensitivity to SC, similar to parental WEHI-3 cells. This sensitivity results in inhibited cell proliferation, induced apoptosis, and increased expression of ENT1 and dCK, molecules involved in the entry and metabolism of Ara-C, while decreasing MDR1 expression. Additionally, we observed that SC prolonged the survival of WEHI-CR50 tumor-bearing mice, despite their resistance to Ara-C. This is the first evidence that SC, a milk protein, may inhibit proliferation and induce apoptosis in cytarabine-resistant cells.

Sodium Caseinate in Combination With Daunorubicin or Cytarabine Improves Survival of Mice With Long-established Leukemia.

Although acute myeloid leukemia (AML) has traditionally been considered an oncological emergency and initiation of therapy is believed to be crucial to minimizing disease-related morbidity and mortality, it has also been suggested that a certain delay in treatment has no negative consequences in terms of response, early mortality, or survival. We aimed to determine the effect of administration of sodium caseinate (SC), a salt of casein, the main milk protein, with cytarabine or with daunorubicin on survival in mice with well-established leukemia. To assay the time of establishment of leukemia in the bone marrow, Balb/c mice were inoculated with 2.5×10 5 WEHI-3 cells/mouse and after 3, 6 and 9 days were euthanized. Bone marrow mononuclear cells (BM-MNCs) of the femur were obtained and cultured for 120 h with or without rmIL-3 and cell proliferation was evaluated by the crystal violet technique. Then, the effect of administrating SC-cytarabine or SC-daunorubicin on survival rates of mice with well-established leukemia was assayed. Another group of Balb/c mice was inoculated with WEHI-3 cell and after 10 days mice were treated with SC-cytarabine or SC-daunorubicin for 40 days. Survival rates were recorded daily and in surviving mice, the prevalence of bone marrow proliferation after treatment was assayed by the crystal violet technique. The assay on the time of establishment of leukemia shows that in 9 days leukemia cells accumulate in the bone marrow in sufficient quantities to sustain an in vitro culture in the absence of growth factors, and we, thus, used this as a criterion of well-established leukemia. When mice with a burden of leukemic cells of more than 9 days were treated with SC-cytarabine or SC-daunorubicin, this resulted in 55% survival for both treatments, and the proliferation assays showed that the bone marrow retained its normal proliferation capacity. SC-cytarabine or SC-daunorubicin treatment prolonged the survival rate of Balb/c mice with a burden of well-established leukemia, and there was no negative impact on bone marrow functionality; however, SC-cytarabine or SC-daunorubicin combination options need to be sought to increase survival beyond 40 days.

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

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. 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. 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. DMC elevated macrophage phagocytosis in leukemia mice in vivo.

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.

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 (ΔΨm ) 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.

MiR-18a Inhibitor Suppresses Leukemia Cell Proliferation by Upregulation of PTEN Expression.

BackgroundLeukemia is common in aging adults and has very high mortality worldwide. The present study was designed to investigate the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP-1 leukemia cells.Material/MethodsThe changes in miR-18a inhibitor-transfected WEHI-3 and THP-1 cell proliferative potential was measured by use of the Cell Counting Kit-8 assay. Apoptotic changes were analyzed by electron microscopy, and evaluation of PI3K, AKT, mTOR, and PTEN expression was assessed by RT-qPCR assay.ResultsTransfection of miR-18a inhibitor significantly (P<0.05) suppressed the proliferative potential of WEHI-3 and THP4 cells. The WEHI-3 cells showed the presence of characteristic apoptotic bodies on transfection with miR-18a inhibitor at 48 h. Flow cytometry showed that miR-18a inhibitor transfection significantly (P<0.05) increased the WEHI-3 cell percentage in G1 phase. The transfection of miR-18a inhibitor significantly (P<0.05) promoted apoptosis in WEHI-3 cells. In WEHI-3 cells, miR-18a inhibitor transfection markedly suppressed the expression of PI3K, AKT, and mTOR mRNA. The expression of PTEN mRNA was significantly (P<0.05) upregulated by miR-18a inhibitor transfection in WEHI-3 cells.ConclusionsThe present study investigated the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP1 leukemia cells. The study demonstrated that miR-18a inhibitor suppressed the proliferative potential of WEHI-3 and THP1 cells and activated apoptotic process through upregulation of PTEN mRNA expression. Therefore, miR-18a inhibitor can be of therapeutic importance for the treatment of leukemia.

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.

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.

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

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

Fisetin Inhibits Cell Proliferation through the Induction of G0/G1 Phase Arrest and Caspase-3-Mediated Apoptosis in Mouse Leukemia Cells.

Fisetin, a naturally occurring flavonoid, is found in common fruits and vegetables and has been shown to induce cytotoxic effects in many human cancer cell lines. No information has shown that fisetin induced cell cycle arrest and apoptosis in mouse leukemia WEHI-3 cells. We found that fisetin decreased total viable cells through G0/G1 phase arrest and induced sub-G1 phase (apoptosis). We have confirmed fisetin induced cell apoptosis by the formation of DNA fragmentation and induction of apoptotic cell death. Results indicated that fisetin induced intracellular Ca increase but decreased the ROS production and the levels of m in WEHI-3 cells. Fisetin increased the activities of caspase-3, -8 and -9. Cells were pre-treated with inhibitors of caspase-3, -8 and -9 and then treated with fisetin and results showed increased viable cell number when compared to fisetin treated only. Fisetin reduced expressions of cdc25a but increased p-p53, Chk1, p21 and p27 that may lead to G0/G1 phase arrest. Fisetin inhibited anti-apoptotic protein Bcl-2 and Bcl-xL and increased pro-apoptotic protein Bax and Bak. Furthermore, fisetin increased the protein expression of cytochrome c and AIF. Fisetin decreased cell number through G0/G1 phase arrest via the inhibition of cdc25c and induction of apoptosis through caspase-dependent and mitochondria-dependent pathways. Therefore, fisetin may be useful as a potential therapeutic agent for leukemia.

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.

Plasmodium yoelii infection inhibits murine leukaemia WEHI-3 cell proliferation in vivo by promoting immune responses

BackgroundLeukaemia is a malignant leukocyte disorder with a high fatality rate, and current treatments for this disease are unsatisfactory. Therefore, new therapeutic strategies for leukaemia must be developed. Malaria parasite infection has been shown to be effective at combating certain neoplasms in animal experiments. This study is to demonstrate the anti-leukaemia activity of malaria parasite Plasmodium yoelii (P. yoelii) infection,.MethodsIn this study, the proportion of CD3, CD19, CD11b and Mac-3 cells was analysed by flow cytometry; the levels of IFN-γ and TNF-α in individual serum samples were measured by enzyme-linked immunosorbent assay, and the phagocytic activity of macrophages and natural killer (NK) cell activity were measured by flow cytometry.ResultsWe found that P. yoelii infection significantly attenuated the growth of WEHI-3 cells in mice. In addition, tumor cell infiltration into the murine liver and spleen was markedly reduced. We also demonstrated that malaria parasite infection elicited anti-leukaemia activity by promoting immune responses, including increasing the surface markers of T cells (CD3) and B cells (CD19); decreasing the surface markers of monocytes (CD11b) and macrophages (Mac-3); inducing the secretion of IFN-γ and TNF-α; and increasing NK cell and macrophage activity.ConclusionsMalaria parasite infection significantly decreases the number of myeloblasts and inhibits neoplasm proliferation in mice. In addition, malaria parasite infection inhibits murine leukaemia by promoting immune responses.

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

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. 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. 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.

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

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. 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. 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. Bufalin increased immune responses by enhancing phagocytosis in mice with leukemia mice.

Beta-mangostin demonstrates apoptogenesis in murine leukaemia (WEHI-3) cells in vitro and in vivo

BackgroundBeta-mangostin (BM) is a xanthone-type of natural compound isolated from Cratoxylum arborescens. This study aimed to examine the apoptosis mechanisms induced by BM in a murine monomyelocytic cell line (WEHI-3) in vitro and in vivo.MethodsA WEHI-3 cell line was used to evaluate the cytotoxicity of BM by MTT. AO/PI and Hoechst 33342 dyes, Annexin V, multiparametric cytotoxicity 3 by high content screening (HCS); cell cycle tests were used to estimate the features of apoptosis and BM effects. Caspase 3 and 9 activities, ROS, western blot for Bcl2, and Bax were detected to study the mechanism of apoptosis. BALB/c mice injected with WEHI-3 cells were used to assess the apoptotic effect of BM in vivo.ResultsBM suppressed the growth of WEHI-3 cells at an IC50value of 14 ± 3 μg/mL in 24 h. The ROS production was increased inside the cells in the treated doses. Both caspases (9 and 3) were activated in treating WEHI-3 cells at 24, 48 and 72 h. Different signs of apoptosis were detected, such as cell membrane blebbing, DNA segmentation and changes in the asymmetry of the cell membrane. Another action by which BM could inhibit WEHI-3 cells is to restrain the cell cycle at the G1/G0 phase. In the in vivo study, BM reduced the destructive effects of leukaemia on the spleen and liver by inducing apoptosis in leukaemic cells.ConclusionBM exerts anti-leukaemic properties in vitro and in vivo.

Mulberry Anthocyanins Induce Leukemia WEHI-3 Cells Apoptosis, Autophagy, Differentiation and prolong leukemic mice survival

Recently, interest in dietary phytochemicals and Chinese herbs for potential cancer chemoprevention has increased substantially owing to their multiple capacities of a combination of several distinct intracellular events (such as cell proliferation, apoptosis, angiogenesis, and metastasis). In our previous studies, we showed that Mulberry anthocyanins (MACs), a natural polyphenol product extracted from Morus, have the antitumor effects on various cancers, such as gastric cancer, melanoma, and hepatoma. Acute myeloid leukemia (AML) patients usually suffer from the side effects of chemotherapy. We investigated effects of MACs on murine Leukemia WEHI-3 cells in vitro. Firstly, 50% of WEHI-3 cells were decreased after treatment with 1.76Â mg/ml and 1.48Â mg/ml for 24 and 48 hr by MTT assay, respectively. The results of flow cytometry assay showed that low does MACs caused the WEHI3 cells apoptosis when they were exposed to high dose MACs. In addition, caspase-9 was activated from pro-caspase to cleaved-caspase in response to treatment MACs with WEHI-3 cells. Immunoflurescence analysis and western blotting assay demonstrated that MACs caused WEHI3 cells death by triggering autophagic flux. By co-treatment with ATRA, MACs also increased CD11b and GM-CSF level, decreased M-CSF expression, which led to WEHI3 cells differentiation. Moreover, co-treated ATRA and MACs cells showed the suppression of MEK, ERK and STAT1 phosphorylation. Altogether, MACs can prevent the growth of WEHI-3 cells for potentially combination chemotherapy therapy to prevent side effects of acute myeloid leukemia.

Anthocyanins from black rice (Oryza sativa) promote immune responses in leukemia through enhancing phagocytosis of macrophages in vivo.

Rice is a staple food in numerous countries around the world. Anthocyanins found in black rice have been reported to reduce the risk of certain diseases, but the effects of crude extract of anthocyanins from Asia University-selected purple glutinous indica rice (AUPGA) on immune responses have not yet been demonstrated. The current study aimed to investigate whether AUPGA treatment could affect immune responses in murine leukemia cells in vivo. Murine acute myelomonocytic leukemia WEHI-3 cells were intraperitoneally injected into normal BALB/c mice to generate leukemia mice. A total of 50 mice were randomly divided into five groups (n=10 in each group) and were fed a diet supplemented with AUPGA at 0, 20, 50 or 100 mg/kg for three weeks. All mice were weighed and the blood, liver and spleen were collected for further experiments. The results indicated that AUPGA did not significantly affect animal body weight, but significantly increased spleen weight (P<0.05) and decreased liver weight (P<0.05) when compared with the control group. AUPGA significantly increased the T cell (CD3) population at treatments of 20 and 100 mg/kg (P<0.05). However, it only significantly increased the B cell (CD19) population at a treatment of 20 mg/kg (P<0.05). Furthermore, AUPGA at 50 and 100 mg/kg significantly increased the monocyte (CD11b) population and the level of macrophages (Mac-3; P<0.05 for both). AUPGA at 50 and 100 mg/kg significantly promoted macrophage phagocytosis in peripheral blood mononuclear cells (P<0.05), and all doses of AUPGA treatment significantly promoted macrophage phagocytotic activity in the peritoneum (P<0.05). AUPGA treatment significantly decreased natural killer cell activity from splenocytes (P<0.05). Finally, AUPGA treatment at 20 mg/kg treatment significantly promoted T cell proliferation (P<0.05), and treatment at 50 and 100 mg/kg significantly decreased B cell proliferation compared with the control group (P<0.05).

Synergistic inhibition of leukemia WEHI-3 cell growth by arsenic trioxide and Hedyotis diffusa Willd extract in vitro and in vivo.

Arsenic trioxide (ATO) is clinically used to treat acute promyelocytic leukemia (APL); however, the therapeutic dose of ATO may prompt critical cardiac side effects. Combination therapy may be used to improve the therapeutic efficiency. To evaluate this possibility, the present study determined the combined effects of Hedyotis diffusa Willd (HDW) extract and ATO in leukemic WEHI-3 cells. The results demonstrated that co-treatment of HDW with ATO resulted in a synergistic augmentation of cytotoxicity in cells at the concentration tested. In order to investigate the potential therapeutic application for leukemia, the combined effects of HDW and ATO were analyzed on the WEHI-3 cell-induced orthotopic leukemia animal model in vivo. The WEHI-3 cells in mice with leukemia were established by injecting murine WEHI-3 cells into BALB/c mice, and treating them with HDW and/or combined with ATO. The results indicated that HDW alone or HDW combined with ATO promoted the total survival rate of mice with leukemia, and these effects are dose-dependent. HDW alone or HDW combined with ATO did not affect the body weight, decreased the spleen weight and did not affect the liver weight. Furthermore, the results demonstrated that HDW alone or HDW combined with ATO resulted in a synergistic augmentation of apoptosis in WEHI-3 cells at the concentration tested. In order to further reveal the detailed mechanism of this synergistic effect on apoptosis, apoptosis-related proteins were also evaluated. The data revealed that HDW alone or HDW combined with ATO induced the expression of death receptor 4 (DR4) and DR5 and the activation of poly adenosine diphosphate ribose polymerase, caspase-3, −8 and −9. Furthermore, HDW alone or HDW combined with ATO decreased the expression levels of B-cell lymphoma 2, B-cell lymphoma-extra large and survivin, and increased the expression levels of Bak and t-Bid. Altogether, the results indicate that the combination of HDW with ATO may be a promising strategy used to increase the clinical efficacy of ATO in the treatment of APL.

Toona sinensis Inhibits Murine Leukemia WEHI-3 Cells and Promotes Immune Response In Vivo.

Toona sinensis (TS) is one of the most popular vegetarian dishes in Taiwan. It has been shown to exhibit antioxidant, antiangiogenic, antiatherosclerotic, and anticancer properties. In this study, we demonstrated the ability of aqueous leaf extracts from TS to promote immune responses in BALB/c mice and to exhibit anti-leukemia activity in murine WEHI-3 cells. BALB/c mice were injected intravenously with WEHI-3 cells and then treated orally with TS (50 mg/kg). In vivo study showed that TS treatment reduced liver and spleen enlargement in WEHI-3 bearing mice compared with the untreated group. Furthermore, TS also decreased white blood cells (WBC), indicating inhibition of differentiation of the precursor of macrophages in WEHI-3 bearing mice. Treatment of WEHI-3 cells with TS (0-75 μg/mL for 24 hours) significantly reduced cell viability. Furthermore, TS treatment–induced late apoptosis was confirmed by Annexin-V/PI staining. Western blot analyses revealed that treatment of WEHI-3 cells with TS statistically increased the protein expression level of cytochrome c in the cytoplasm and activates caspase-3. Notably, TS treatment caused a dramatic reduction in Bcl-2 and increase in Bax protein levels. TS may disturb the Bcl-2 and Bax protein ratio and induce apoptosis. This reports confirms the antitumor activity of this nutritious vegetable potentially against leukemia.

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 (ΔΨm ), 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.