Guiqi Baizhu prescription ameliorates cytarabine-induced intestinal mucositis by targeting JAK2 to inhibit M1 macrophage polarization.

BACKGROUND: Intestinal mucositis (IM) is characterized by damage to the intestinal mucosa resulting from inhibition of epithelial cell division and loss of renewal capacity following anticancer chemotherapy and radiotherapy. Cytarabine (Ara-C), the main chemotherapy drug for the treatment of leukemia and lymphoma, is a frequent cause of IM. Guiqi Baizhu prescription (GQBZP) is a traditional Chinese medicine with anti-cancer and anti-inflammatory effects. PURPOSE: To determine if GQBZP can ameliorate Ara-C induced IM and identify and characterize the pharmacologic and pharmacodynamic mechanisms. STUDY DESIGN AND METHODS: IM was induced in mice with Ara-C and concurrently treated with orally administered GQBZP. Body weight and food intake was monitored, with HE staining to calculate ileal histomorphometric scoring and villus length/crypt depth. Immunoblotting was used to detect intestinal tissue inflammatory factors. M1 macrophages (M1) were labeled with CD86 by flow cytometry and iNOS + F4/80 by immunofluorescence. Virtual screening was used to find potentially active compounds in GQBZP that targeted JAK2. In vitro, RAW264.7 cells were skewed to M1 macrophage polarization by lipopolysaccharide (LPS) and interferon-γ (INF-γ) and treated orally with GQBZP or potential active compounds. M1 was labeled with CD86 by flow cytometry and iNOS by immunofluorescence. ELISA was used to detect inflammatory factor expression. Active compounds against JAK2, p-JAK2, STAT1 and p-STAT1 were identified by western blotting and HCS fluorescence. Molecular dynamics simulations and pharmacokinetic predictions were carried out on representative active compounds. RESULTS: Experimental results with mice in vivo suggest that GQBZP significantly attenuated Ara-C-induced ileal damage and release of pro-inflammatory factors by inhibiting macrophage polarization to M1. Molecular docking was used to identify potentially active compounds in GQBZP that targeted JAK2, a key factor in macrophage polarization to M1. By examining the main components of each herb and applying Lipinski's rules, ten potentially active compounds were identified. In vitro experimental results suggested that all 10 compounds of GQBZP targeted JAK2 and could inhibit M1 polarization in RAW264.7 cells treated with LPS and INF-γ. Among them, acridine and senkyunolide A down-regulated the expression of JAK2 and STAT1. MD simulations revealed that acridine and senkyunolide A were stable in the active site of JAK2 and exhibited good interactions with the surrounding amino acids. CONCLUSIONS: GQBZP can ameliorate Ara-C-induced IM by reducing macrophage polarization to M1, and acridine and senkyunolide A are representative active compounds in GQBZP that target JAK2 to inhibit M1 polarization. Targeting JAK2 to regulate M1 polarization may be a valuable therapeutic strategy for IM.

EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). METHODS: We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). RESULTS: eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. CONCLUSIONS: We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Folate functionalized chitosan nanoparticles as targeted delivery systems for improved anticancer efficiency of cytarabine in MCF-7 human breast cancer cell lines.

Anticancer drug cytarabine, has been widely used for treating haematological malignancies while it has minimal activity against solid tumours, which demands continuous infusion leading to high dose cytarabine toxicity. In this study, folate conjugated chitosan nanoparticles (FCCNP) were used for targeted delivery of cytarabine in breast adenocarcinoma cell lines by making use of the overexpressed folate receptors on the surface of MCF-7. Folate was conjugated to chitosan using carbodiimide. FCCNPs show spherical morphology with a size of<50 nm. Zeta potential of + 45.2 mV and PDI of 0.98 from DLS measurement confirms a stable monodisperse nanoformulation. Cytotoxicity was studied in folate receptor positive, MCF-7 and folate receptor negative, A-549 cell lines. Increased cellular uptake of the drug incorporated nanoparticles was confirmed in MCF-7 cells with fluorophore, squaraine 650 compared to A-549 cells. The relative fold of expression of genes involved in apoptosis such as bax, cyt c and cas 9 were upregulated. The present in vitro study confirms improved cytotoxicity of cytarabine folate conjugated chitosan nanoparticles in MCF-7 cells.

Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

Self-nanoemulsifying system improves oral absorption and enhances anti-acute myeloid leukemia activity of berberine.

BACKGROUND: Recently, we found that berberine (BBR) exerts anti-acute myeloid leukemia activity, particularly toward high-risk and relapsed/refractory acute myeloid leukemia MV4-11 cells in vitro. However, the poor water solubility and low bioavailability observed with oral BBR administration has limited its clinical use. Therefore, we design and develop a novel oil-in-water self-nanoemulsifying system for BBR (BBR SNE) to improve oral bioavailability and enhance BBR efficacy against acute myeloid leukemia by greatly improving its solubility. RESULTS: This system (size 23.50 ± 1.67 nm, zeta potential - 3.35 ± 0.03 mV) was prepared with RH40 (surfactant), 1,2-propanediol (co-surfactant), squalene (oil) and BBR using low-energy emulsification methods. The system loaded BBR successfully according to thermal gravimetric, differential scanning calorimetry, and Fourier transform infrared spectroscopy analyses. The release profile results showed that BBR SNE released BBR more slowly than BBR solution. The relative oral bioavailability of this novel system in rabbits was significantly enhanced by 3.41-fold over that of BBR. Furthermore, Caco-2 cell monolayer transport studies showed that this system could help enhance permeation and prevent efflux of BBR. Importantly, mice with BBR SNE treatment had significantly longer survival time than BBR-treated mice (P < 0.001) in an MV4-11 engrafted leukemia murine model. CONCLUSIONS: These studies confirmed that BBR SNE is a promising therapy for acute myeloid leukemia.

n-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation.

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.

DAPK1-ERK signal mediates oxygen glucose deprivation reperfusion induced apoptosis in mouse N2a cells.

AIMS: Death-associated protein kinase 1 (DAPK1) is a kinase found to promote neuronal apoptosis induced by ischemia. Extracellular signal-regulated kinase (ERK) was identified as a key molecule in DAPK1 signaling. However, the mechanisms of neuronal ischemia reperfusion injury remain unknown. Here, we investigate the influence of DAPK1-ERK signal on neuronal apoptosis following ischemia reperfusion. METHODS: Mouse N2a cells were used in this study and primary cultured neurons along with mice were adopted as supplements. Oxygen glucose deprivation (OGD) or administration of N-methyl-d-aspartate (NMDA) and glycine was performed on cells while middle cerebral artery occlusion (MCAO) model on mice. DAPK1 knocking down was achieved by lentiviral-delivered shRNA. Protein expressions were evaluated by western blots. Protein-protein binding was confirmed by co-immunoprecipitation and immunofluorescent assay. Apoptosis of cells was measured by flow cytometry and lacate dehydrogenase (LDH) leakage assay. RESULTS: Ischemia reperfusion resulted in increased DAPK1 and ERK activation as well as aggravated apoptosis in a time-dependent manner. DAPK1 was proved to bind to ERK during reperfusion following OGD, MCAO and excitotoxicity model. Interception of this binding by knocking down DAPK1 led to nuclear translocation of ERK and reduced apoptosis. CONCLUSION: Our study revealed the DAPK1-ERK signal as a potential mechanism contributing to neuronal apoptosis in response to ischemia reperfusion. Disruption of this signal pathway could be a promising therapeutic target against stroke.

Cafestol, a diterpene molecule found in coffee, induces leukemia cell death.

To evaluate the antitumor properties of Cafestol four leukemia cell lines were used (NB4, K562, HL60 and KG1). Cafestol exhibited the highest cytotoxicity against HL60 and KG1 cells, as evidenced by the accumulation of cells in the sub-G1 fraction, mitochondrial membrane potential reduction, accumulation of cleaved caspase-3 and phosphatidylserine externalization. An increase in CD11b and CD15 differentiation markers with attenuated ROS generation was also observed in Cafestol-treated HL60 cells. These results were similar to those obtained following exposure of the same cell line to cytarabine (Ara-C), an antileukemic drug. Cafestol and Ara-C reduced the clonogenic potential of HL60 cells by 100%, but Cafestol spared murine colony forming unit- granulocyte/macrophage (CFU-GM), which retained their clonogenicity. The co-treatment of Cafestol and Ara-C reduced HL60 cell viability compared with both drugs administered alone. In conclusion, despite the distinct molecular mechanisms involved in the activity of Cafestol and Ara-C, a similar cytotoxicity towards leukemia cells was observed, which suggests a need for prophylactic-therapeutic pre-clinical studies regarding the anticancer properties of Cafestol.

Neural progenitor cell proliferation in the hypothalamus is involved in acquired heat tolerance in long-term heat-acclimated rats.

Constant exposure to moderate heat facilitates progenitor cell proliferation and neuronal differentiation in the hypothalamus of heat-acclimated (HA) rats. In this study, we investigated neural phenotype and responsiveness to heat in HA rats' hypothalamic newborn cells. Additionally, the effect of hypothalamic neurogenesis on heat acclimation in rats was evaluated. Male Wistar rats (5 weeks old) were housed at an ambient temperature (Ta) of 32°C for 6 days (STHA) or 40 days (LTHA), while control (CN) rats were kept at a Ta of 24°C for 6 days (STCN) or 40 days (LTCN). Bromodeoxyuridine (BrdU) was intraperitoneally injected daily for five consecutive days (50 mg/kg/day) after commencing heat exposure. The number of hypothalamic BrdU-immunopositive (BrdU+) cells in STHA and LTHA rats was determined immunohistochemically in brain samples and found to be significantly greater than those in respective CN groups. In LTHA rats, approximately 32.6% of BrdU+ cells in the preoptic area (POA) of the anterior hypothalamus were stained by GAD67, a GABAergic neuron marker, and 15.2% of BrdU+ cells were stained by the glutamate transporter, a glutamatergic neuron marker. In addition, 63.2% of BrdU+ cells in the POA were immunolabeled with c-Fos. Intracerebral administration of the mitosis inhibitor, cytosine arabinoside (AraC), interfered with the proliferation of neural progenitor cells and acquired heat tolerance in LTHA rats, whereas the selected ambient temperature was not changed. These results demonstrate that heat exposure generates heat responsive neurons in the POA, suggesting a pivotal role in autonomic thermoregulation in long-term heat-acclimated rats.

Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation.

Cell proliferation and neuroinflammation in the adult hypothalamus may contribute to the pathogenesis of obesity. We tested whether the intertwining of these two processes plays a role in the metabolic changes caused by 3 weeks of a high-saturated fat diet (HFD) consumption. Compared with chow-fed mice, HFD-fed mice had a rapid increase in body weight and fat mass and specifically showed an increased number of microglia in the arcuate nucleus (ARC) of the hypothalamus. Microglia expansion required the adequate presence of fats and carbohydrates in the diet because feeding mice a very high-fat, very low-carbohydrate diet did not affect cell proliferation. Blocking HFD-induced cell proliferation by central delivery of the antimitotic drug arabinofuranosyl cytidine (AraC) blunted food intake, body weight gain, and adiposity. AraC treatment completely prevented the increase in number of activated microglia in the ARC, the expression of the proinflammatory cytokine tumor necrosis factor-α in microglia, and the recruitment of the nuclear factor-κB pathway while restoring hypothalamic leptin sensitivity. Central blockade of cell proliferation also normalized circulating levels of the cytokines leptin and interleukin 1ß and decreased peritoneal proinflammatory CD86 immunoreactive macrophage number. These findings suggest that inhibition of diet-dependent microglia expansion hinders body weight gain while preventing central and peripheral inflammatory responses due to caloric overload.

N-acetylcysteine supplementation reduces oxidative stress for cytosine arabinoside in rat model.

Cytosine arabinoside (ARA-C) is a pyrimidine analog that may cause keratoconjunctivitis when used in high doses. The underlying mechanism may be the increased amounts of reactive oxygen radicals that may damage the DNA synthesis of corneal and conjunctival epithelial cells. Topical corticosteroids are one of the prophylactic treatments for keratoconjunctivitis induced by ARA-C. Forty Wistar-type albino rats were included in this study the rats were divided into four groups. The first group (Group 1) received only ARA-C, the second group (Group 2) received ARA-C and N-acetylcysteine (NAC), the third group (Group 3) received only NAC and the fourth group (Group 4) was the control group. The total oxidant status (TOS), the total antioxidant capacity and the oxidative stress index (OSI) measurements of the cornea and the conjunctiva were evaluated in these four groups. The mean TOS and OSI value was the highest in Group 1 and the lowest in Group 3. The differences in TOS and OSI values were statistically significant between Group 1 and Group 2. There are decreases in TOS and OSI values in rats which received ARA-C with NAC administration. NAC may have a protective effect on ARA-C-induced keratoconjunctivitis.

NT1721, a novel epidithiodiketopiperazine, exhibits potent in vitro and in vivo efficacy against acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by heterogeneous genetic and epigenetic changes in hematopoietic progenitors that lead to abnormal self-renewal and proliferation. Despite high initial remission rates, prognosis remains poor for most AML patients, especially for those harboring internal tandem duplication (ITD) mutations in the fms-related tyrosine kinase-3 (FLT3). Here, we report that a novel epidithiodiketopiperazine, NT1721, potently decreased the cell viability of FLT3-ITD+ AML cell lines, displaying IC50 values in the low nanomolar range, while leaving normal CD34+ bone marrow cells largely unaffected. The IC50 values for NT1721 were significantly lower than those for clinically used AML drugs (i.e. cytarabine, sorafenib) in all tested AML cell lines regardless of their FLT3 mutation status. Moreover, combinations of NT1721 with sorafenib or cytarabine showed better antileukemic effects than the single agents in vitro. Combining cytarabine with NT1721 also attenuated the cytarabine-induced FLT3 ligand surge that has been linked to resistance to tyrosine kinase inhibitors. Mechanistically, NT1721 depleted DNA methyltransferase 1 (DNMT1) protein levels, leading to the re-expression of silenced tumor suppressor genes and apoptosis induction. NT1721 concomitantly decreased the expression of EZH2 and BMI1, two genes that are associated with the maintenance of leukemic stem/progenitor cells. In a systemic FLT3-ITD+ AML mouse model, treatment with NT1721 reduced tumor burdens by > 95% compared to the control and significantly increased survival times. Taken together, our results suggest that NT1721 may represent a promising novel agent for the treatment of AML.

Initial Testing (Stage 1) of MK-8242-A Novel MDM2 Inhibitor-by the Pediatric Preclinical Testing Program.

BACKGROUND: MK-8242 is an inhibitor of MDM2 that stabilizes the tumor suppressor TP53 and induces growth arrest or apoptosis downstream of TP53 induction. PROCEDURES: MK-8242 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10.0 µM and against the PPTP in vivo xenograft panels using oral gavage on Days 1-5 and Day 15-19 at a dose of 125 mg/kg (solid tumors) or 75 mg/kg (acute lymphoblastic leukemia [ALL] models). RESULTS: The median IC50 for MK-8242 was 0.07 µM for TP53 wild-type cell lines versus >10 µM for TP53 mutant cell lines. MK-8242 induced a twofold or greater delay in time to event in 10 of 17 (59%) of TP53 wild-type solid tumor xenografts, excluding osteosarcoma xenografts that have very low TP53 expression. Objective responses were observed in seven solid tumor xenografts representing multiple histotypes. For the systemic-disease ALL panel, among eight xenografts there were two complete responses (CRs) and six partial responses (PRs). Two additional MLL-rearranged xenografts (MV4;11 and RS4;11) grown subcutaneously showed maintained CR and PR, respectively. The expected pharmacodynamic responses to TP53 activation were observed in TP53 wild-type models treated with MK-8242. Pharmacokinetic analysis showed that MK-8242 drug exposure in SCID mice appears to exceed that was observed in adult phase 1 trials. CONCLUSIONS: MK-8242-induced tumor regressions across multiple solid tumor histotypes and induced CRs or PRs for most ALL xenografts. This activity was observed at MK-8242 drug exposures that appear to exceed those observed in human phase 1 trials.

Comparison of different methods for erythroid differentiation in the K562 cell line.

OBJECTIVE: To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis. RESULTS: Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001). CONCLUSIONS: Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

Interactions Between Nutraceutical Supplements and Standard Acute Myeloid Leukemia Chemotherapeutics.

PURPOSE: Concomitant use of nutraceuticals with chemotherapy is very common. Cancer patients self-medicate to relieve the side effects associated with chemotherapy, improve disease outcome and to regain control of their medical care. However, there is limited empirical evidence on potential drug-nutraceutical interactions and their resulting effect on chemotherapy efficacy. METHOD: To investigate drug-nutraceutical interactions we created and screened a library of commonly used nutraceuticals for their modulatory effects on the activity of cytarabine and daunorubicin, two primary chemotherapeutics used to treat acute myeloid leukemia (AML). Combination screening was performed in 3 AML cell lines (OCI-AML2, KG1a and U937) using the MTS viability assay. Lead compounds were validated using with the Annexin V/ Propidium iodide assay and CalcuSyn drug combination software. RESULTS: We identified zinc as a nutraceutical that enhanced AML chemotherapy efficacy with combination index (CI) values of 0.649, 0.632 and 0.615 at EC 25, 50 and 75, respectively; CI values <0.9, >1.1 or between 0.9-1.1 denote statistical synergy, antagonism or additivity, respectively. In contrast, we show that echinacea hindered AML chemotherapy efficacy by significantly reducing the ability of cytarabine to induce cell death. CONCLUSION: Given the positive and negative effects of nutraceuticals, patients undergoing chemotherapy must consult with their oncologist before consuming over-the-counter supplements. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Effects of cytarabine on activation of human T cells - cytarabine has concentration-dependent effects that are modulated both by valproic acid and all-trans retinoic acid.

BACKGROUND: Cytarabine is used in the treatment of acute myeloid leukemia (AML). Low-dose cytarabine can be combined with valproic acid and all-trans retinoic acid (ATRA) as AML-stabilizing treatment. We have investigated the possible risk of immunotoxicity by this combination. We examined the effects of cytarabine combined with valproic acid and ATRA on in vitro activated human T cells, and we tested cytarabine at concentrations reached during in vivo treatment with high doses, conventional doses and low doses. METHODS: T cells derived from blood donors were activated in vitro in cell culture medium alone or supplemented with ATRA (1 µM), valproic acid (500 or 1000 µM) or cytarabine (0.01-44 µM). Cell characteristics were assessed by flow cytometry. Supernatants were analyzed for cytokines by ELISA or Luminex. Effects on primary human AML cell viability and proliferation of low-dose cytarabine (0.01-0.5 µM) were also assessed. Statistical tests include ANOVA and Cluster analyses. RESULTS: Only cytarabine 44 µM had both antiproliferative and proapoptotic effects. Additionally, this concentration increased the CD4:CD8 T cell ratio, prolonged the expression of the CD69 activation marker, inhibited CD95L and heat shock protein (HSP) 90 release, and decreased the release of several cytokines. In contrast, the lowest concentrations (0.35 and 0.01 µM) did not have or showed minor antiproliferative or cytotoxic effects, did not alter activation marker expression (CD38, CD69) or the release of CD95L and HSP90, but inhibited the release of certain T cell cytokines. Even when these lower cytarabine concentrations were combined with ATRA and/or valproic acid there was still no or minor effects on T cell viability. However, these combinations had strong antiproliferative effects, the expression of both CD38 and CD69 was altered and there was a stronger inhibition of the release of FasL, HSP90 as well as several cytokines. Cytarabine (0.01-0.05 µM) showed a dose-dependent antiproliferative effect on AML cells, and in contrast to the T cells this effect reached statistical significance even at 0.01 µM. CONCLUSIONS: Even low levels of cytarabine, and especially when combined with ATRA and valproic acid, can decrease T cell viability, alter activation-induced membrane-molecule expression and decrease the cytokine release.

Epigallocatechin gallate, ellagic acid, and rosmarinic acid perturb dNTP pools and inhibit de novo DNA synthesis and proliferation of human HL-60 promyelocytic leukemia cells: Synergism with arabinofuranosylcytosine.

Epigallocatechin gallate (EGCG), ellagic acid (EA) and rosmarinic acid (RA) are natural polyphenols exerting cancer chemopreventive effects. Ribonucleotide reductase (RR; EC 1.17.4.1) converts ribonucleoside diphosphates into deoxyribonucleoside diphosphates being essential for DNA replication, which is why the enzyme is considered an excellent target for anticancer therapy. EGCG, EA, and RA dose-dependently inhibited the growth of human HL-60 promyelocytic leukemia cells, exerted strong free radical scavenging potential, and significantly imbalanced nuclear deoxyribonucleoside triphosphate (dNTP) concentrations without distinctly affecting the protein levels of RR subunits (R1, R2, p53R2). Incorporation of (14)C-cytidine into nascent DNA of tumor cells was also significantly lowered, being equivalent to an inhibition of DNA synthesis. Consequently, treatment with EGCG and RA attenuated cells in the G0/G1 phase of the cell cycle, finally resulting in a pronounced induction of apoptosis. Sequential combination of EA and RA with the first-line antileukemic agent arabinofuranosylcytosine (AraC) synergistically potentiated the antiproliferative effect of AraC, whereas EGCG plus AraC yielded additive effects. Taken together, we show for the first time that EGCG, EA, and RA perturbed dNTP levels and inhibited cell proliferation in human HL-60 promyelocytic leukemia cells, with EGCG and RA causing a pronounced induction of apoptosis. Due to these effects and synergism with AraC, these food ingredients deserve further preclinical and in vivo testing as inhibitors of leukemic cell proliferation.

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines.

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.

Hypothalamic neurogenesis is not required for the improved insulin sensitivity following exercise training.

Neurons within the hypothalamic arcuate nucleus (ARC) are important regulators of energy balance. Recent studies suggest that neurogenesis in the ARC is an important regulator of body mass in response to pharmacological stressors. Regular exercise training improves insulin action, and is a primary treatment modality for obesity and type 2 diabetes. We examined whether exercise training causes hypothalamic neurogenesis and whether this contributes to exercise-induced improvements in insulin action. Short-term exercise in adult mice induced a proneurogenic transcriptional program involving growth factors, cell proliferation, and neurogenic regulators in the hypothalamus. Daily exercise training for 7 days increased hypothalamic cell proliferation 3.5-fold above that of sedentary mice, and exercise-induced cell proliferation was maintained in diet-induced obese mice. Colocalization studies indicated negligible neurogenesis in the ARC of sedentary or exercise-trained mice. Blocking cell proliferation via administration of the mitotic blocker arabinosylcytosine (AraC) did not affect food intake or body mass in obese mice. While 4 weeks of exercise training improved whole-body insulin sensitivity compared with sedentary mice, insulin action was not affected by AraC administration. These data suggest that regular exercise training induces significant non-neuronal cell proliferation in the hypothalamus of obese mice, but this proliferation is not required for enhanced insulin action.

Crucial role of heme oxygenase-1 in the sensitivity of acute myeloid leukemia cell line Kasumi-1 to ursolic acid.

Ursolic acid (UA), which has been used extensively as an antileukemic agent in traditional Chinese medicine, is safely edible if originating from food. We found that the apoptotic rate of acute myeloid leukemia (AML) subtype M2 (AML-M2) cell line Kasumi-1 treated by UA was higher than those of other leukemia cell lines, but was not as high as that treated by arabinofuranosyl cytidine (Ara-C), suggesting that UA is an important chemotherapeutic agent to treat AML-M2. Heme oxygenase-1 (HO-1) is a key enzyme exerting potent cytoprotection, cell proliferation, and drug resistance. HO-1 in Kasumi-1 cells was upregulated by being treated with low-dose rather than high-dose UA. Inhibition of HO-1 by zinc protoporphyrin (ZnPP) IX sensitized Kasumi-1 cells to UA, and the apoptotic rate was close to that induced by Ara-C (P<0.01). The sensitizing effect of ZnPP was associated with caspase activation, bcl-2 downregulation, and PARP activation. After silencing HO-1 by siRNA transfection with lentivirus, the cells' proliferation induced by UA was increased as it was by Ara-C. Furthermore, combining ZnPP with UA prolonged the survival of mice bearing the AML subtype M2 tumor with smaller volume of tumor and size of spleen. The results showed that the Kasumi-1 cell line was the most sensitive to UA, but the apoptotic effect was inferior to that treated by Ara-C because of HO-1 upregulation. AML-M2 can feasibly be treated by target-inhibiting HO-1 that enhances the antileukemia effects of UA in vitro and in vivo.