Lead Transiently Promotes Granulocyte-Macrophage Progenitor Differentiation and Subsequently Suppresses Common Myeloid Progenitor Differentiation.

Lead (Pb) is a toxic heavy metal affecting human health; it is known to be harmful to various organs or systems, yet the mechanisms by which Pb influences immune cell development remain to be defined. In this study, we show that Pb exposure (1250 ppm via drinking water) selectively impacted the development of myeloid cells (myelopoiesis). After Pb treatment of adult C57BL/6 mice, the numbers of granulocyte-macrophage progenitors (GMP) were consistently reduced, whereas the numbers of myeloid cells were increased at week (wk) 1 and decreased at wk8 after initiating the Pb exposure. Functional assays indicate that Pb accelerated GMP differentiation in a reactive oxygen species-dependent manner after treatment for 1 week and inhibited common myeloid progenitor differentiation by upregulating interferon regulatory factor 8 (IRF8) expression after treatment for 8 weeks. Consistent with the distinct Pb influences on myeloid cells observed at wk1 and wk8, Pb caused an inflammatory environment in vivo at wk8, but not at wk1. Furthermore, like the observations in mice during the Pb exposure, bloods from humans occupationally exposed to Pb had their numbers of monocytes, neutrophils and GMP negatively associated with the Pb concentration, whereas IRF8 expression in common myeloid progenitor, but not GMP, was positively correlated with the Pb concentration. These data suggest an occupationally relevant level of Pb exposure preferentially influences myelopoiesis involving reactive oxygen species and IRF8, which may contribute to the current understanding of the hematopoietic toxicology of Pb.

In vitro and in vivo effects of JAK2 inhibition in chronic myelomonocytic leukemia.

In chronic myelomonocytic leukemia (CMML), colony-forming units granulocyte/macrophage (CFU-GM), which grow in vitro in the absence of exogenous growth factors, arise from the abnormal clone that is responsible for the overproduction of granulomonocytic cells. Previous in vitro findings including ours suggest that divergent molecular aberrations in CMML seem to converge within the GM-CSF signaling pathway. As JAK2 is a sentinel kinase in this pathway, JAK2 inhibition may be an attractive treatment approach in CMML. We investigated the in vitro effects of the specific JAK2 inhibitor TG101209 on the autonomous CFU-GM formation from peripheral blood mononuclear cells of patients with CMML. TG101209 was found to either block or strongly inhibit spontaneous CFU-GM growth in all 10 patients tested. This inhibitory effect was dose dependent and significantly more pronounced as compared to the inhibitory effect on stimulated CFU-GM growth from normal individuals. In a CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we can demonstrate a spleen response and the disappearance of constitutional symptoms which was associated with a decrease in autonomous CFU-GM formation ex vivo. Pharmacological JAK2 inhibition may be an interesting approach to be systematically studied in patients with CMML.

Extraction and identification of collagen-derived peptides with hematopoietic activity from Colla Corii Asini.

ETHNOPHARMACOLOGICAL RELEVANCE: Colla Corii Asini is a widely used traditional Chinese medicine to treat anemia with a long history due to its stimulating effect in hematopoiesis, but the components contributing to this effect are still unknown. In this study, we aimed to establish a methodology to isolate the bioactive components and provide pharmacological basis for its usage in treating anemia. METHODS: 5-FU and γ-ray radiation induced anemic mice models were generated by treating with 5-FU at 150mg/kg body weight and γ-rays by a 4MV linear accelerator by total body irradiation using female ICR mice respectively. Oral administration of fraction A was performed by gastric lavage at 1g/kg and 2g/kg body weight for 12 days and 25 days and peripheral blood sample was collected from ocular sinus red blood cell (RBC) and white blood cell (WBC) counts every 3 days and 5 days for 5-FU and radiation induced models, respectively. Next, fraction A was separated to A1 and A2 using cation exchange chromatography (IEC) based on ionic strength. Fraction A1 was further separated using reverse phase chromatography (RPC) based on the hydrophobicity first with 0-10% linear gradient, then 20%, 30%, 50% constant gradient of 60% acetonitrile in neutral Na2HPO4 buffer. Peak fractions were pooled, evaporatively dried, and dissolved in ultrapure water. Finally, fraction A11 was analyzed combining tandem mass spectrometry and proteomic tools and two peptides (peptide 11 and 16) were identified. The hematopoietic effects of multiple fractions and the two peptides were measured using colony-forming units-erythroid (CFU-E), an indication of late erythroid progenitor cells and colony-forming units granulocyte-monocyte (CFU-GM), an indication of granulocyte and monocyte progenitor cells respectively on hematopoietic progenitor cells prepared from bone marrow (Till and Mcculloch 1961). RESULTS: Fraction A at 1g/kg and 2g/kg could increase RBC and WBC counts in 5-FU and radiation induced anemic mice models. Fraction A1 at 0.1mg/ml and 0.5mg/ml, exhibited stronger hematopoietic activity than fraction A2, both of which were subfractions from fraction A using IEX, by elevated CFU-E and CFU-GM of mouse bone marrow cells. Furthermore, fraction A11 at 0.1mg/ml showed stronger CFU-E and CFU-GM than fractions A12 to A14 from RPC separation. Finally, peptide 11 and peptide 16 were identified from tandem mass spectrometry and peptide 11 increased CFU-E and CFU-GM in a dose dependent manner. CONCLUSIONS: We combined multiple approaches including chromatography, mass spectrometry, cell-based assays, as well as animal studies to identify and demonstrate that the hematopoietic effect of Colla Corii Asini is at least in part from the peptidic components identified using our methodology. This is the first time to isolate peptidic components from Colla Corii Asini, and to provide molecular basis for its usage in treating anemia, which may particularly have the potential to benefit cancer patients suffering from myelosuppression due to radiotherapy or chemotherapy.

Myelotoxicity of carboplatin is increased in vivo in db/db mice, the animal model of obesity-associated diabetes mellitus.

PURPOSE: Some authors observed increased carboplatin-associated myelotoxicity in obese patients which was exclusively attributed to elevated AUC. To investigate the potential contribution of functional changes of cells primarily responsible for myelopoiesis, granulocyte-macrophage progenitors (CFU-GM) were studied in obesity-associated diabetes mellitus (DMT2). METHODS: The most frequently used animal model of human obesity with DMT2 is db/db mouse. Cellularity, frequency of CFU-GM and total CFU-GM content of femoral bone marrow were measured after 100 mg/kg dose of carboplatin in vivo. To exclude influence of pharmacokinetic changes, direct toxicity of carboplatin on CFU-GM was also determined in vitro and was compared with other anticancer agents, namely doxorubicin, 5-fluorouracil and 4-thiouridylate. RESULTS: After intraperitoneal administration of carboplatin, each measured characteristics of bone marrow function was more significantly suppressed and the induced neutropenia was more serious in db/db mice than in the controls. The increased myelotoxicity seemed to be a direct effect on myeloid progenitor cells since their increased in vitro sensitivity was found in db/db mice. This was not specific for carboplatin, a similar double to fivefold increase in myelotoxicity of each cytotoxic drug with different mechanism of action was observed. Four-thiouridylate, a promising antileukemic molecule with good therapeutic index, was by far the least toxic for CFU-GM of db/db mice. CONCLUSIONS: A serious disorder of CFU-GM progenitors was suggested in obese mice with DMT2, which eventually might lead to more severe myelotoxicity and neutropenia. Weight loss and normalization of glucose homeostasis may be important before chemotherapy of malignant diseases in obesity with DMT2.

Interleukin-10 inhibits autonomous myelopoiesis in patients with myelofibrosis.

The spontaneous formation of colony-forming units granulocyte/macrophage (CFU-GM) in semisolid cultures has been shown to be due to the endogenous release of cytokines and/or to the hypersensitivity of cells against growth factors. We have reported that increased autonomous CFU-GM growth is an in vitro characteristic of myelofibrosis (MF) which may reflect aberrant hematopoiesis in vivo. Because of its cytokine synthesis-inhibiting action, we speculated that interleukin-10 (IL-10) may inhibit pathological overproduction of myeloid cells in MF by suppression of autonomous myelopoiesis. In this study, IL-10 significantly inhibited autonomous CFU-GM formation in vitro from peripheral blood mononuclear cells (PB MNC) in 10 of 11 patients with MF tested. In all patients, there was a mean inhibition of 69% ranging from 35% to 100%. Suppression of autonomous CFU-GM formation by IL-10 was dose dependent and reversible by the addition of anti-IL-10 antibodies. Our results indicate that IL-10 is a potentially useful molecule to affect aberrant myelopoiesis in patients with MF.

Lipopolysaccharide reciprocally alters the stromal cell-regulated positive and negative balance between myelopoiesis and B lymphopoiesis in C57BL/6 mice.

Hematopoiesis in the bone marrow (BM) and spleen is controlled by stromal cells. Inflammation promotes myelopoiesis and simultaneously suppresses B lymphopoiesis. However, the role of the reciprocal regulation of myelopoiesis and B lymphopoiesis by stromal cells during inflammation is not fully understood. We investigated inflammation-induced alteration of hematopoietic regulation in lipopolysaccharide (LPS)-treated mice. C57BL/6 female mice were intravenously injected with a single, 5-µg dose of LPS, which induced a rapid decrease in the number of granulocyte-macrophage progenitors (colony-forming unit granulocyte-macrophage; CFU-GM) and B cell progenitors (CFU-preB) in BM. The CFU-GM count rapidly recovered, whereas the recovery of CFU-preB was delayed. LPS induced a marked increase in the number of CFU-GM but not in the number of CFU-preB in spleen. After LPS treatment, gene expression levels of positive regulators of myelopoiesis such as granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in BM and spleen were markedly upregulated whereas levels of positive regulators for B lymphopoiesis such as stromal cell-derived factor (SDF)-1, stem cell factor (SCF), and IL-7 remained unchanged. Meanwhile, the negative regulator of B lymphopoiesis tumor necrosis factor (TNF)-α was markedly up-regulated. The number of CFU-GM in S-phase in BM increased after LPS treatment, whereas the number of CFU-preB in S-phase decreased. These results suggest that LPS-activated stromal cells induce positive-dominant regulation of myelopoiesis and negative-dominant regulation of B lymphopoiesis, which facilitates emergency myelopoiesis during inflammation by suppressing B lymphopoiesis, thereby contributing to the host defense against infection.

[Effect of GW003 on the granulocyte macrophage colony formation ability of bone marrow cells in vitro].

The aim of this study was to investigate the effect of GW003 on the ability of granulocyte colony forming in vitro of bone marrow cells. The bone marrow samples was collected from normal rhesus, the patients with leukemia in stages of remission and chemotherapy respectively, and the nucleated cells were separated and cultured for 12 days after addition of different concentrations of GW003 or rhG-CSF, or G-CSF mutant. Then the amount of colony-forming unit-granulocyte-macrophage was counted. The results indicated that GW003 could enhance the ability of bone marrow nucleated cells of rhesus to forming CFU-GM in vitro, and its effect was much better than that of rhG-CSF or G-CSF mutant at the same concentration(®). The GW003 showed dose-response relationship to CFU-GM level (r = R(2) = 0.965, P = 0.003, in a certain concentration), the GW003 also could enhance CFU-GM formation of marrow nucleated cells in leukemic patients, especially for patients receiving chemotherapy. The GW003 could relieve the marrow suppression caused by chemotherapy significantly. It is concluded that the GW003 can significantly improve the ability of bone marrow cells to form granulocyte colony in vitro as well as effectively alleviate bone marrow suppression.

High spontaneous granulocyte/macrophage-colony formation in patients with myelofibrosis.

Unstimulated methylcellulose cultures in 25 myelofibrosis (MF) patients were performed to better understand the role of cytokines in the proliferation of MF cells. Compared to controls MF patients show a variable but highly increased spontaneous CFU-GM formation (66 vs 4.8/10(5) PBMNC). There was a marked reduction of autonomous CFU-GM growth by the cytokine-synthesis-inhibiting molecule IL-10 as well as by antibodies against GM-CSF whereas antibodies against IL-3, G-CSF, M-CSF and IL-1ß showed heterogeneous effects. Spontaneous CFU-GM growth >100/10(5) PBMNC predicted shorter survival. Constitutive release of GM-CSF seems to contribute to proliferation of MF cells in vitro and possibly in vivo.

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells.

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

Melatonin improves humoral and cell-mediated immune responses of male golden hamster following stress induced by dexamethasone.

Melatonin is known as an antistress and immunostimulator compound while glucocorticoids have immunosuppressive function. The mechanism of action of both the hormones on immune cells is still a question. We found that melatonin improved the effect of dexamethasone (synthetic glucocorticoid) induced immunosuppression of splenocytes and bone marrow GM-CFU along with increased production of serum IL-2, IgG and the receptor expression for melatonin and glucocorticoid in spleen that might be responsible for the proliferation of immune cells. Thus, seasonal variation in peripheral melatonin might be responsible for the improvement of immune status under different stress conditions experienced by the rodents for better survival.

TTAS a new stilbene derivative that induces apoptosis in Leishmania infantum.

Leishmania parasites are able to undergo apoptosis (programmed cell death), similarly to mammalian cells. Recently it was demonstrated in vitro the anti-leishmanial effect of some natural and synthetic stilbenoids including resveratrol and piceatannol. In this study we evaluated the Leishmanicidal activity of a pool of stilbene derivatives which had previously shown high apoptotic efficacy against neoplastic cells. All the compounds tested were capable to decrease the parasite viability in a dose-dependent manner. Trans-stilbenes proved to be markedly more effective than cis-isomers. This was different from that observed in tumor cells in which cis-stilbenes were more potent cytotoxic agents. Trans-3,4',5-trimethoxy-3'-amino-stilbene (TTAS) was the most active stilbene showing in Leishmania infantum a LD(50) value of 2.6 µg/mL. In contrast TTAS showed a low toxicity when tested on normal hemopoietic cells. This compound induced apoptosis in parasites by disrupting the mitochondrial membrane potential. Moreover it shows the ability to block Leishmania parasites in G(2)-M phase of cell cycle in agreement with the data obtained by affinity chromatography that identify tubulin as the putative target of TTAS. In conclusion, our results indicate that some stilbene derivatives are highly effective as anti-leishmanial agents and TTAS represents a pro-apoptotic agent in Leishmania parasites that merit further in vivo investigation.

Histone deacetylases inhibitor sodium butyrate inhibits JAK2/STAT signaling through upregulation of SOCS1 and SOCS3 mediated by HDAC8 inhibition in myeloproliferative neoplasms.

Constitutive activation of Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT) signaling has an important role in the oncogenesis of myeloproliferative neoplasms (MPNs) and leukemia. Histone deacetylases (HDACs) inhibitors have been reported to possess anticancer activity through different mechanisms. However, whether HDACs inhibitors suppress JAK2/STAT signaling in MPNs is still unknown. In this study, we show that the HDAC inhibitor sodium butyrate (SB) inhibited JAK2/STAT signaling and increased the expression of suppressors of cytokine signaling 1 (SOCS1) and SOCS3, both of which are the potent feedback inhibitors of JAK2/STAT signaling. SB upregulated the expression of SOCS1 and SOCS3 by triggering the promoter-associated histone acetylation of SOCS1 and SOCS3 in K562 and HEL cell lines. Importantly, we found that upon knockdown of each class I HDACs, only knockdown of HDAC8 resulted in the increased expression of SOCS1 and SOCS3. Moreover, overexpression of SOCS1 and SOCS3 significantly inhibited cell growth and suppressed JAK2/STAT signaling in K562 and HEL cells. Furthermore, SB increased the transcript levels of SOCS1 and SOCS3 and inhibited the clonogenic activity of hematopoietic progenitors from patients with MPNs. Taken together, these data establish a new anticancer mechanism that SB inhibits JAK2/STAT signaling through HDAC8-mediated upregulation of SOCS1 and SOCS3. Thus, HDACs inhibitors may have therapeutic potential for the treatment of MPNs.

Improvement of oxidative stress and immunity by melatonin: an age dependent study in golden hamster.

Reactive oxygen species (ROS) have been proposed to play an important role in balancing the pro- and antioxidant homeostasis during aging. Melatonin has been suggested as an effective free radical scavenger that might have a role during the process of aging. We observed, that melatonin administration (25 µg/100 g body weight for 30 days) significantly augments the activity of anti-oxidative enzymes like superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in the plasma, spleen and bone marrow (BM) of young (6 weeks), adult (30 weeks) and old aged (2.5 years) male golden hamster, Mesocricetus auratus. A sharp decline in generation of ROS was observed in peripheral blood mononuclear cells (PBMC) and splenocytes upon melatonin administration in different age group of hamsters. Reduction in the level of thiobarbituric acid-reactive substances (TBARS) and total nitrite and nitrate concentration as metabolites and indicators of nitric oxide (NO) in plasma, spleen and BM were observed along with night time (22:00 h) melatonin concentration in different age group of hamsters after administration of melatonin and compared to the control group (treated with 0.9% saline). General immune parameters like proliferation of splenocytes, PBMC and colony forming ability of GM-CFU were observed following melatonin treatment in different age group, although it was low only in aged hamsters compared to the young and adult. Our data indicates that the age related increase of oxidative load and simultaneously augments the general immunity in aged hamsters.

Delayed myelosuppression with acute exposure to hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and environmental degradation product hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) in rats.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a widely used munitions compound, and hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), its N-nitroso product of anaerobic microbial nitroreduction, are contaminants of military sites. Previous studies have shown MNX to be the most acutely toxic among the nitroreduced degradation products of RDX and to cause mild anemia at high dose. The present study compares hematotoxicity with acute oral exposure to MNX with parent RDX. Both RDX and MNX caused a modest decrease in blood hemoglobin and ~50% loss of granulocytes (NOAELs=47 mg/kg) in female Sprague-Dawley rats observed 14 days post-exposure. We explored the possibility that blood cell loss observed after 14 days was delayed in onset because of toxicity to bone marrow (BM) progenitors. RDX and MNX decreased granulocyte/macrophage-colony forming cells (GM-CFCs) at 14, but not 7, days (NOAELs=24 mg/kg). The earliest observed time at which MNX decreased GM-CFCs was 10 days post-exposure. RDX and MNX likewise decreased BM burst-forming units-erythroid (BFU-Es) at 14, but not 7, days. Granulocyte-erythrocyte-monocyte-megakaryocyte (GEMM)-CFCs were unaffected by RDX and MNX at 7 days suggesting precursor depletion did not account for GM-CFC and BFU-E loss. MNX added to the culture media was without effect on GM-CFC formation indicating no direct inhibition. Flow cytometry showed no differential loss of BM multilineage progenitors (Thy1.1(+)) or erythroid (CD71(+)) precursors with MNX suggesting myeloid and erythroid lineages were comparably affected. Collectively, these data indicate that acute exposure to both RDX and MNX caused delayed suppression of myelo- and erythropoiesis with subsequent decrease of peripheral granulocytes and erythrocytes.

Mechanisms of the anti-inflammatory and antifibrotic activity of a sympatholytic agent during toxic pulmonary fibrosis.

The effect of a course treatment with a sympatholytic reserpine on the inflammatory response and connective tissue proliferation in the lungs of C57Bl/6 mice was studied on the model of toxic pulmonary fibrosis induced by intratracheal administration of bleomycin. This sympatholytic reduced infiltration of the alveolar interstitium and alveolar ducts with inflammatory cells (lymphocytes, macrophages, neutrophils, and plasma cells) and prevented connective tissue proliferation in the lungs. The anti-inflammatory effect of reserpine was associated with a decrease in activity of bone marrow granulocyte-erythroid-macrophage-megakaryocyte and granulocyte precursors (proliferation and mobilization). The antifibrotic effect of reserpine was due to a decrease in the number of committed precursors for mesenchymopoiesis.

In vitro effects of sporobacterin probiotic on the function of donor granulocyte-macrophage cells.

The effects of sporobacterin probiotic (Bakoren Company) on oxidative activity of donor granulocyte-macrophage cells (GMC) were studied in vitro by luminol-dependent chemiluminescent method, and the effects of the probiotic on the production of pro- and anti-inflammatory cytokines were evaluated by ELISA. The probiotic dose-dependently stimulated spontaneous production of free radicals by GMC; combined treatment with immunomodulators likopid, polyoxydonium, and IFN-α2a produced a more potent effect. Sporobacterin stimulated the production of pro- and anti-inflammatory cytokines in cell cultures. These data confirmed the immunomodulatory effect of sporobacterin, an important component in the phagocytic system cells.

Toxicity of cytotoxic agents to granulocyte-macrophage progenitors is increased in obese Zucker and non-obese but insulin resistant Goto-Kakizaki rats.

Increased risk of anticancer chemotherapy in seriously obese patients is known. Obesity may be among factors that predict treatment-related toxicity during chemotherapy. We investigated whether functional changes in granulopoiesis may also contribute to increased myelotoxicity in addition to the known alterations of pharmacokinetic parameters in obesity. Hemopoiesis - as measured by cellularity, frequency of granulocyte-macrophage progenitors (CFU-GM) and total CFU-GM content of the femoral bone marrow - did not differ in obese, insulin resistant Zucker rats compared with Wistar rats. Nevertheless increased sensitivity of their CFU-GM progenitor cells to cytotoxic drugs was found by culturing them in vitro in the presence of carboplatin, doxorubicin and 5-fluorouracil. All drugs were more toxic on CFU-GM progenitor cells of insulin resistant Zucker rats than on CFU-GM cells of the control strain. This might be based on metabolic disorders, at least in part, because we could demonstrate a similar increase in toxicity of the studied anticancer drugs to the CFU-GM progenitors originated from the non-obese but insulin resistant Goto-Kakizaki rats in the same dose ranges. After in vivo administration of rosiglitazone, an insulin sensitizer, the anticancer drug sensitivity of CFU-GM progenitors of Goto-Kakizaki rats was decreased concurrently with improvement of insulin resistance. Although the increased treatment-related myelotoxicity and mortality are well-known among obese patients with malignant diseases, only the altered half lives, volumes of distribution and clearances of cytotoxic drugs are thought to be the underlying reasons. According to our knowledge the results presented here, are the first observations about an impaired granulopoiesis in obese animals.

Paclitaxel and CYC3, an aurora kinase A inhibitor, synergise in pancreatic cancer cells but not bone marrow precursor cells.

BACKGROUND: Amplification of aurora kinase A (AK-A) overrides the mitotic spindle assembly checkpoint, inducing resistance to taxanes. RNA interference targeting AK-A in human pancreatic cancer cell lines enhanced taxane chemosensitivity. In this study, a novel AK-A inhibitor, CYC3, was investigated in pancreatic cancer cell lines, in combination with paclitaxel. METHODS: Western blot, flow cytometry and immunostaining were used to investigate the specificity of CYC3. Sulforhodamine B staining, time-lapse microscopy and colony-formation assays were employed to evaluate the cytotoxic effect of CYC3 and paclitaxel. Human colony-forming unit of granulocyte and macrophage (CFU-GM) cells were used to compare the effect in tumour and normal tissue. RESULTS: CYC3 was shown to be a specific AK-A inhibitor. Three nanomolar paclitaxel (growth inhibition 50% (GI(50)) 3 nM in PANC-1, 5.1 nM in MIA PaCa-2) in combination with 1 µM CYC3 (GI(50) 1.1 µM in MIA PaCa2 and 2 µM in PANC-1) was synergistic in inhibiting pancreatic cell growth and causing mitotic arrest, achieving similar effects to 10-fold higher concentrations of paclitaxel (30 nM). In CFU-GM cells, the effect of the combination was simply additive, displaying significantly less myelotoxicity compared with high concentrations of paclitaxel (30 nM; 60-70% vs 100% inhibition). CONCLUSION: The combination of lower doses of paclitaxel and CYC3 merits further investigation with the potential for an improved therapeutic index in vivo.

Spanish black radish (Raphanus sativus L. Var. niger) diet enhances clearance of DMBA and diminishes toxic effects on bone marrow progenitor cells.

Vegetables of the Cruciferae family contain high levels of glucosinolates, metabolites of which are believed to enhance detoxification. Spanish black radishes (SBR) contain 4× more glucosinolates than other crucifers. This study examined whether feeding mice a diet containing 20% SBR for 2 wk could enhance metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) and inhibit DMBA-mediated bone marrow toxicity. Expression of Phase I and II detoxification enzymes was significantly greater for mice fed SBR than control diet. Six hours after DMBA administration, the blood levels of DMBA in mice fed the SBR diet were significantly lower than mice fed a control diet. DMBA reduced bone marrow cells in mice fed control diet to a significantly greater extent than mice fed the SBR diet. Colony forming assays demonstrated that mice on the SBR diet had 1) less reduction in lymphoid CFU-preB progenitor cells, 2) greater recovery of CFU-preB progenitor cells at 168 h, and 3) less reduction of CFU-GM progenitor cells at 6 h. Therefore, mice fed a 20% SBR diet for 2 wk had greater expression of detoxification enzymes, faster metabolism of DMBA, and a reduction in DMBA-induced bone marrow toxicity. Overall, these results support the hypothesis that glucosinolates in SBR are protective against acute toxicity.

Co-exposure of Fusarium mycotoxins: in vitro myelotoxicity assessment on human hematopoietic progenitors.

Mycotoxins such as beauvericin (BEA), deoxynivalenol (DON), enniatin B (ENB), fumonisin B1 (FB1), T-2 toxin and zearalenone (ZEA) can co-occur in food commodities. This aim of this study was to assess the myelotoxicity of these mycotoxins in couple using in vitro human granulo-monocytic (Colony Forming Unit-Granulocyte and Macrophage, CFU-GM) hematopoietic progenitors. Clonogenic assays have been performed in the presence of the following couples of fusariotoxins: DON + BEA, DON + FB1, DON + T-2, DON + ZEA, T-2 + ZEA and BEA + ENB. Co-exposure of human CFU-GM to DON + BEA resulted in synergic myelotoxic effects. The combination of DON + T-2 presented additive or synergic myelotoxic effects. The couples DON + ZEA, T-2 + ZEA and BEA + ENB had additive myelotoxic effects, while the combination of DON + FB1 showed antagonist myelotoxic effects. These in vitro results suggested that the simultaneous presence of mycotoxins in food commodities and diet may be more myelotoxic than the presence of one mycotoxin alone. Diminution of hematopoietic progenitors could give rise to a decrease number of mature blood cells, inducing agranulocytosis and/or thrombocytopenia and in severe cases aplastic anemia.