Radioprotective effectiveness of a novel delta-tocotrienol prodrug on mouse hematopoietic system against 60Co gamma-ray irradiation through inducing granulocyte-colony stimulating factor production.

Considering the increasing risk of nuclear attacks worldwide, the development of develop potent and safe radioprotective agents for nuclear emergencies is urgently needed. γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have demonstrated a potent radioprotective effect by inducing the production of granulocyte-colony stimulating factor (G-CSF) in vivo. However, their application is limited because of their low bioavailability. The utilization of ester prodrugs can be an effective strategy for modifying the pharmacokinetic properties of drug molecules. In this study, we initially confirmed that DT3 exhibited the most significant potential for inducing G-CSF effects among eight natural vitamin E homologs. Consequently, we designed and synthesized a series of DT3 ester and ether derivatives, leading to improved radioprotective effects. The metabolic study conducted in vitro and in vivo has identified DT3 succinate 5b as a prodrug of DT3 with an approximately seven-fold higher bioavailability compared to DT3 alone. And DT3 ether derivative 8a were relatively stable and approximately 4 times more bioavailable than DT3 prototype. Furthermore, 5b exhibited superior ability to mitigate radiation-induced pancytopenia, enhance the recovery of bone marrow hematopoietic stem and progenitor cells, and promote splenic extramedullary hematopoiesis in sublethal irradiated mice. Similarly, 8a shown potential radiation protection, but its radiation protection is less than DT3. Based on these findings, we identified 5b as a DT3 prodrug, and providing an attractive candidate for further drug development.

Sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production.

To date only four recombinant growth factors, including Filgrastim (rhG-CSF), have been approved by FDA as radiomitigators to ameliorate hematopoietic acute radiation syndrome (H-ARS). These approved agents are not stable under room-temperature, needing to be stored at 2-8 °C, and would not be feasible in a mass casualty scenario where rapid and cost-effective intervention is crucial. Delta-tocotrienol (δ-T3H), the most potent G-CSF-inducing agent among vitamin E isoforms, exhibited efficiency and selectivity on G-CSF production in comparison with TLR and STING agonists in mice. Five-dose δ-T3H was utilized as the optimal therapeutic regimen due to long-term G-CSF production and the best peripheral blood (PB) recovery of irradiated mice. Comparable with rhG-CSF, sequential administration of δ-T3H post-irradiation improved hematologic recovery and accelerated the regeneration of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow (BM) and spleen of 6.5Gy irradiated mice; and consistently enhanced repopulation of BM-HSCs. In 4.0Gy irradiated nonhuman primates, δ-T3H exhibited comparable efficacy as rhG-CSF to promote PB recovery and colony-formation of BM-HPCs. Altogether, we demonstrated that sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production, indicated δ-T3H as a promising radiomitigator for the management of H-ARS, particularly in a mass casualty scenario.

Fluacrypyrim Protects Hematopoietic Stem and Progenitor Cells against Irradiation via Apoptosis Prevention.

Ionizing radiation (IR)-induced hematopoietic injury has become a global concern in the past decade. The underlying cause of this condition is a compromised hematopoietic reserve, and this kind of hematopoietic injury could result in infection or bleeding, in addition to lethal mishaps. Therefore, developing an effective treatment for this condition is imperative. Fluacrypyrim (FAPM) is a recognized effective inhibitor of STAT3, which exhibits anti-inflammation and anti-tumor effects in hematopoietic disorders. In this context, the present study aimed to determine whether FAPM could serve as a curative agent in hematopoietic-acute radiation syndrome (H-ARS) after total body irradiation (TBI). The results revealed that the peritoneally injection of FAPM could effectively promote mice survival after lethal dose irradiation. In addition, promising recovery of peripheral blood, bone marrow (BM) cell counts, hematopoietic stem cell (HSC) cellularity, BM colony-forming ability, and HSC reconstituting ability upon FAPM treatment after sublethal dose irradiation was noted. Furthermore, FAPM could reduce IR-induced apoptosis in hematopoietic stem and progenitor cells (HSPCs) both in vitro and in vivo. Specifically, FAPM could downregulate the expressions of p53-PUMA pathway target genes, such as Puma, Bax, and Noxa. These results suggested that FAPM played a protective role in IR-induced hematopoietic damage and that the possible underlying mechanism was the modulation of apoptotic activities in HSCs.

Single-Dose Administration of Recombinant Human Thrombopoietin Mitigates Total Body Irradiation-Induced Hematopoietic System Injury in Mice and Nonhuman Primates.

PURPOSE: Recombinant human thrombopoietin (rhTPO) has been evaluated as a therapeutic intervention for radiation-induced myelosuppression. However, the immunogenicity induced by a repeated-dosing strategy raises concerns about the therapeutic use of rhTPO. In this study, single-dose administration of rhTPO was evaluated for efficacy in the hematopoietic response and survival effect on mice and nonhuman primates exposed to total body irradiation (TBI). METHODS AND MATERIALS: Survival of lethally (9.0 Gy) irradiated C57BL/6J male mice was observed for 30 days after irradiation. Hematologic evaluations were performed on C57BL/6J male mice given a sublethal dose of radiation (6.5 Gy). Furthermore, in sublethally irradiated mice, we performed bone marrow (BM) histologic evaluation and evaluated BM-derived clonogenic activity. Next, the proportion and number of hematopoietic stem cells (HSCs) were analyzed. Competitive repopulation experiments were conducted to assess the multilineage engraftment of irradiated HSCs after BM transplantation. Flow cytometry was used to evaluate DNA damage, cell apoptosis, and cell cycle stage in HSCs after irradiation. Finally, we evaluated the efficacy of a single dose of rhTPO administered after 7 Gy TBI in male and female rhesus monkeys. RESULTS: A single administration of rhTPO 2 hours after irradiation significantly mitigated TBI-induced death in mice. rhTPO promoted multilineage hematopoietic recovery, increasing peripheral blood cell counts, BM cellularity, and BM colony-forming ability. rhTPO administration led to an accelerated recovery of BM HSC frequency and multilineage engraftment after transplantation. rhTPO treatment reduced radiation-induced DNA damage and apoptosis and promoted HSC proliferation after TBI. Notably, a single administration of rhTPO significantly promoted multilineage hematopoietic recovery and improved survival in nonhuman primates after TBI. CONCLUSIONS: These findings indicate that early intervention with a single administration of rhTPO may represent a promising and effective radiomitigative strategy for victims of radiation disasters.

Dimethyl sulfoxide, a potent oral radioprotective agent, confers radioprotection of hematopoietic stem and progenitor cells independent of apoptosis.

In mass casualty events involving radiation exposure, there is a substantial unmet need for identifying and developing an orally bioavailable agent that can be used to protect the hematopoietic stem cell pool and regenerate hematopoiesis after radiation injury. Dimethyl sulfoxide (DMSO), a free-radical scavenger, has shown therapeutic benefits in many preclinical and clinical studies. This study investigates the radioprotective effects of DMSO on oral administration. Single dose of oral DMSO administrated before irradiation conferred 100% survival of C57BL6/J mice receiving otherwise lethal as well as super-lethal radiation dose, with wide radioprotective time frame (from 15min to 4h). Oral DMSO not only protected radiation-induced acute hematopoietic stem and progenitor cell (HSPC) injury, but also ameliorated long-term BM suppression following irradiation in mice. Mechanistically, DMSO directly protected HSPC survival after irradiation in vitro and in vivo, whereas no radioprotective effect was seen in MLL-AF9-induced leukemia cells. Unexpectedly, DMSO treatment did not inhibit radiation-induced HSPC apoptosis, and the HSPC survival from Trp53-and PUMA-deficient mice after irradiation was also protected by DMSO. In conclusion, our findings demonstrate the radioprotective efficacy of oral DMSO. Given its oral efficacy and little toxicity, DMSO is an attractive candidate for human use in a wide variety of settings, including nuclear accidents and medical radiation.

17α-Ethinyl-androst-5-ene-3ß, 17ß-diol, a Novel Potent Oral Radioprotective Agent, Confers Radioprotection of Hematopoietic Stem and Progenitor Cells in a Granulocyte Colony-Stimulating Factor-Independent Manner.

PURPOSE: The risk of radiation exposure is considered to have increased in recent years. For convenience and simple administration, development of an effective orally administered radioprotective agent is highly desirable. The steroid 5-androstene-3ß, 17ß-diol (5-AED) has been evaluated as both a radioprotector and a radiomitigator in mice and nonhuman primates; however, poor oral bioavailability has limited its development. A variant compound-17α-ethinyl-androst-5-ene-3ß, 17ß-diol (EAD)-exhibits significant oral bioavailability. We investigated the radioprotective effects of EAD via oral administration in mice. METHODS AND MATERIALS: Survival assays were performed in lethally (9.0-10.0 Gy) irradiated mice. Peripheral blood cell counts were monitored in lethally (9.5 Gy) or sublethally (6.5 Gy) irradiated mice. We performed histologic analysis of bone marrow (BM) and frequency and functional analysis of hematopoietic stem and progenitor cells in mice irradiated with 6.5 Gy. To investigate multilineage engraftment of irradiated hematopoietic stem cells after BM transplantation, competitive repopulation assays were conducted. Plasma granulocyte colony-stimulating factor was measured by enzyme-linked immunosorbent assay. RESULTS: Oral administration of EAD on 3 consecutive days before irradiation conferred 100% survival in mice, against otherwise 100% death, at a 9.5-Gy lethal dose of total body irradiation. EAD ameliorated radiation-induced pancytopenia at the same dose. EAD augmented BM cellular recovery and colony-forming ability, promoted hematopoietic stem and progenitor cell recovery, and expanded the pool of functionally superior hematopoietic stem cells in the BM of sublethally irradiated mice. Unlike 5-AED, EAD did not increase granulocyte colony-stimulating factor levels in mice and exhibited no therapeutic effects on hematologic recovery after irradiation; nevertheless, its radioprotective efficacy was superior to that of 5-AED. CONCLUSIONS: Our findings demonstrate the radioprotective efficacy of EAD and reveal that the 17α-ethinyl group is essential for its oral activity. Given its oral efficacy and low toxicity, EAD has potential as an optimal radioprotector for use by first responders, as well as at-risk civilian populations.

Dimethyl Sulfoxide Prevents Radiation-Induced Oral Mucositis Through Facilitating DNA Double-Strand Break Repair in Epithelial Stem Cells.

PURPOSE: Oral mucositis is one of the most prevalent side effects in patients undergoing radiation therapy for head and neck cancers. Current therapeutic agents such as palifermin recombinant human keratinocyte growth factor and amifostine do not efficiently or fully prevent mucositis. Dimethyl sulfoxide (DMSO), a free-radical scavenger, has shown therapeutic benefits in many preclinical and clinical studies. This study aimed to investigate the efficacy of DMSO in a clinically relevant mouse model of acute, radiation-induced oral mucositis. METHODS AND MATERIALS: Oral mucositis was induced by a high single and fractioned irradiation of the head and neck area in C57BL/6J mice, and the effects of DMSO (by intraperitoneal injection) were assessed by macroscopic and histopathological examination. Epithelial stem and progenitor cells were analyzed by immunohistochemical staining of p63 and Ki-67, and DNA double-strand breaks (DSBs) were visualized by immunofluorescence detection of γ-H2AX. Tumor xenograft was obtained using CAL-27 cells. RESULTS: Pretreatment with DMSO protected the oral mucosa from severe acute radiation injury, reduced the extent of radiation-induced weight loss, and had no significant effects on tumor weight in irradiated or nonirradiated xenograft mice. Furthermore, the efficacy of DMSO was superior to that of recombinant human keratinocyte growth factor and amifostine. DMSO treatment prevented the loss of proliferative lingual epithelial stem and progenitor cells upon irradiation. More interestingly, the average levels of γ-H2AX foci were significantly decreased in p63-positive epithelial stem cells at 6 hours, but not at 2 hours, after irradiation, indicating that DMSO facilitated DNA DSB repair rather than suppressing the indirect action of irradiation. CONCLUSIONS: DMSO prevents the loss of proliferative lingual epithelial stem and progenitor cells upon irradiation by facilitating DNA DSB repair, thereby protecting against radiation-induced mucositis without tumor protection. Given its high efficacy and low toxicity, DMSO could be a potential treatment option to prevent radiation-induced oral mucositis.

Vibsanin A sensitizes human acute myeloid leukemia cells to tyrosine kinase inhibitor-induced myeloid differentiation via activation of PKC and upregulation of Lyn.

Differentiation therapies have been proposed to overcome the impaired cell differentiation in acute myeloid leukemia (AML). However, thus far the all-trans retinoic acid-based differentiation therapy has been the only successful modality in treating acute promyelocytic leukemia. Here, we showed that vibsanin A, a novel protein kinase C (PKC) activator, sensitized AML cells to tyrosine kinase inhibitor (TKI)-induced differentiation. Vibsanin A augmented the ability of TKIs to induce growth inhibition and G1 cell cycle arrest of AML cells. Mechanistically, PKC activation was involved in the differentiation-inducing effects of combining vibsanin A with TKIs. Moreover, we found that vibsanin A enhanced TKI-induced Lyn expression and suppression of Lyn interfered with AML cell differentiation, indicating an essential role for Lyn expression in the combination-induced differentiation. Finally, combining vibsanin A and TKIs enhanced the activation of the Raf/MEK/ERK cascade. Together, this is the first study to evaluate the synergy of vibsanin A and TKIs in AML cell differentiation. Our study lays the foundation in assessing new opportunities for the combination of vibsanin A and TKIs as a promising approach for future differentiation therapy.

Succinate ester derivative of δ-tocopherol enhances the protective effects against 60Co γ-ray-induced hematopoietic injury through granulocyte colony-stimulating factor induction in mice.

α-tocopherol succinate (α-TOS), γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have drawn large attention due to their efficacy as radioprotective agents. α-TOS has been shown to act superior to α-tocopherol (α-TOH) in mice by reducing lethality following total body irradiation (TBI). Because α-TOS has been shown to act superior to α-tocopherol (α-TOH) in mice by reducing lethality following total body irradiation (TBI), we hypothesized succinate may be contribute to the radioprotection of α-TOS. To study the contributions of succinate and to identify stronger radioprotective agents, we synthesized α-, γ- and δ-TOS. Then, we evaluated their radioprotective effects and researched further mechanism of δ-TOS on hematological recovery post-irradiation. Our results demonstrated that the chemical group of succinate enhanced the effects of α-, γ- and δ-TOS upon radioprotection and granulocyte colony-stimulating factor (G-CSF) induction, and found δ-TOS a higher radioprotective efficacy at a lower dosage. We further found that treatment with δ-TOS ameliorated radiation-induced pancytopenia, augmenting cellular recovery in bone marrow and the colony forming ability of bone marrow cells in sublethal irradiated mice, thus promoting hematopoietic stem and progenitor cell recovery following irradiation exposure. δ-TOS appears to be an attractive radiation countermeasure without known toxicity, but further exploratory efficacy studies are still required.

Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR.

The protein kinase C agonist prostratin induces differentiation of human myeloid leukemia cells and enhances cellular differentiation by chemotherapeutic agents.

As acute myeloid leukemia (AML) cells are characterized by uncontrolled self-renewal and impaired cellular differentiation, induction of terminal differentiation of leukemia cells by differentiating agents has been proposed as an attractive therapeutic strategy to treat AML. Here, we demonstrated that prostratin, a potent protein kinase C (PKC) activator, inhibited the growth of myeloid leukemia cells by a predominant G1 arrest with variable induction of apoptosis. Conversely, prostratin induced significant differentiation of AML cell lines and primary AML blasts as evidenced by morphology and immunophenotyping. The effects of prostratin were PKC dependent, and activation of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 by PKC was required for prostratin-induced cell differentiation. Consequently, prostratin reprogrammed transcriptional factor expression, and ectopic expression of c-Myc in HL-60 cells significantly eliminated prostratin-mediated cellular differentiation and cell cycle arrest, indicating an essential role for c-Myc suppression in the differentiation-inducing effects of prostratin. Finally, prostratin was able to potentiate cellular differentiation induced by chemotherapeutic agents such as Ara-C. Together, we proposed that prostratin alone or administered with other anticancer agents may be effective in differentiation therapy of AML.

[Study on the effect of vibsane-type diterpenoids of Viburnum odoratissimum on human HepG2 cell growth and its underlying mechanism].

OBJECTIVE: To study the antiproliferation effect on HepG2 cells and its underlying mechanism of the active chemical composition of the Viburnum Odoratissimum. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and trypan blue dye exclusion assay were used to assess the effect of vibsane-type diterpenoids on the proliferation of various tumor cells. Alterations in cell cycle and apoptosis were determined by flowcytometry. The enzymatic activity of caspase-3/7 was measured by Apo-ONE homogeneous Caspase-3/7 Assay kit. RESULTS: Compound 1 #, a vibsane-type diterpenoid, was found to significantly inhibit the growth of HepG2 cells by anticancer proliferation activity screening. It was demonstrated that the modified groups on side chain coupled to C11 site affected the cell growth-inhibition activity of compounds by structure-activity analysis. In addition, HepG2 cell line was most sensitive to compound 1 #, which induced growth arrest of HepG2 cells in a dose- and time-dependent manner. Study on the mechanisms underlying these effects indicated that compound 1 # induced significant G0/G1 phase arrest of HepG2 cells in a time- and concentration-dependent manner. Meanwhile, It was found that higher concentrations of compound (5-10 micromol/L) caused evident increase in the unmber of apoptotic cells and dose-dependent activation of caspase-3/7. CONCLUSION: Vibsane-type diterpenoids could significantly inhibit the growth of HCC HepG2 cells. Induction of cell cycle arrest and apoptosis may play important roles in their anticancer effects.

[Protective effects of WR2721 on early bone marrow hematopoietic function in mice exposed to 6.5 Gy of (60)Co γ-rays].

The aim of this study was to investigate the effect of WR2721(amifostine) against bone marrow hematopoietic damage of mice exposed to 6.5 Gy of (60)Co-γ ray. A total of 60 C57/BL6J mice was divided into 3 groups:normal group (mice were injected with physiological salt solution), irradiation group (mice were injected with physiologic salt solution before irradiation) and WR2721 group (mice were injected with WR2721 before irradiation). The WBC, neutrophil (Neut), Plt and RBC levels in peripheral blood of 3 group mice were counted within 60 days after irradiation; the bone marrow nuclear cells (BMNC) were counted at 2 and 24 hours after irradiation; the hematopoietic stem/progenitor cell (LK/LSK) level and colony formation capability were detected by flow cytometry at 2 and 24 hours after irradiation. The results indicated that the counts of WBC and neut at 4 and 18 days, Plt at 7-18 days and RBC at 10-30 day after irradiation in WR2721 group were higher than those in irradiation group (P < 0.05); the BMNC, LSK and LK levels obviously increased at 24 hours after irradiation (P < 0.05), the CFU-GEMM, CFU-GM, CFU-MK BFU-E and CFU-E all significantly increased at 2 and 24 hours after irradiation (P < 0.01), as compared with irradiation group. It is concluded that WR2721 can effectively alleviate early hematopoietic damage and promote the fast recovery of peripheral blood cells in mice exposed to γ-ray, suggesting that the WR2721 has significant radioprotective effect on hematopoietic system.

Identification of a new Mpl-interacting protein, Atp5d.

Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.

Pennogenin tetraglycoside induces rat myometrial contraction and MLC20 phosphorylation via PLC-IP(3) and RhoA/Rho kinase signaling pathways.

BACKGROUND: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been widely used in China for the treatment of abnormal uterine bleeding. We previously studied the main active constituents of TSSPs and their structure-activity relationships with respect to rat myometrial contractions. Tg (pennogenin tetraglycoside) was identified as one of the active ingredients in TSSPs able to induce rat myometrial contractions. However, the mechanisms underlying the pharmacological actions on uterine activity have not been described clearly. METHODS: Here Tg was screened for effects on contractile activity in isolated uterine strips from estrogen-primed rats and on MLC20 phosphorylation and related signaling pathways in cultured rat myometrial cells as determined by Western blot. Intracellular calcium ([Ca(2+)](i)) was monitored under a confocal microscope using Fluo-4 AM-loaded myometrial cells. RESULTS: Tg dose-dependently stimulated rat myometrial contractions as well as MLC20 phosphorylation in vitro, which could be completely suppressed by an inhibitor of myosin light chain kinase (MLCK). Use of Ca(2+) channel blockers and kinase inhibitors demonstrated that Tg-induced myometrial contractions are mediated by activation of the phospholipase C (PLC)-inositol triphosphate (IP3) signaling pathway, resulting in increased MLC20 phosphorylation. Furthermore, Y27632, a specific inhibitor of Rho kinase (ROK), notably suppressed Tg-stimulated myometrial contractions and decreased MLC20 phosphorylation. CONCLUSIONS: These data provide evidence that rat myometrial contractility induced by Tg results from enhanced MLC20 phosphorylation, while both PLC-IP3 and RhoA/ROK signaling pathways mediate the process. These mechanisms may be responsible for the therapeutic effects of TSSPs on abnormal uterine bleeding.

High dose granulocyte colony-stimulating factor enhances survival and hematopoietic reconstruction in canines irradiated by 2.3 Gy mixed fission neutron and gamma ray.

This study was purposed to evaluate the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on hematopoietic reconstruction and survival in beagles exposed to mixed fission neutron and γ-ray. 13 beagles were unilaterally exposed to single dose of 2.3 Gy 90% neutrons. The experiments were divided into 3 groups: irradiation control group (no any treatment, n = 4), supportive care group (n = 5) and rhG-CSF plus supportive care group (n = 4, abbreviated as rhG-CSF group) in which the beagles were subcutaneously injected with 200 µg/kg of rhG-CSF early at half an hour and 24 hours post-irradiation respectively. The results showed that 2.3 Gy 90% neutron irradiation induced a severe acute radiation sickness of bone marrow type. The administration of rhG-CSF increased the survival rate from 60% in supportive care group to 100%. Twice injection of rhG-CSF in the first 24 hours reduced duration of neutropenia, enhanced neutrophil nadir and promoted neutrophil recovery when compared with control cohort administered clinical support. The number of colony-forming cells (CFU-GM, CFU-E, and BFU-E) in peripheral blood of rhG-CSF treated canines increased 2-to 5-fold relative to those of the supportive care group on day 3. All canines treated with rhG-CSF achieved hematopoietic reconstruction as evidenced by the pathological section of sternum while severe shortage of hemopoietic cells remained in the cohorts given supportive care alone. It is concluded that the combination of supportive care and high-dose rhG-CSF can accelerate hematopoietic recovery and enhance survival of dogs exposed to 2.3 Gy mixed neutron and gamma ray.

RhG-CSF improves radiation-induced myelosuppression and survival in the canine exposed to fission neutron irradiation.

Fission-neutron radiation damage is hard to treat due to its critical injuries to hematopoietic and gastrointestinal systems, and so far few data are available on the therapeutic measures for neutron-radiation syndrome. This study was designed to test the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in dogs which had received 2.3 Gy mixed fission-neutron-γ irradiation with a high ratio of neutrons (~90%). Following irradiation, rhG-CSF treatment induced 100% survival versus 60% in controls. Only two of five rhG-CSF-treated dogs experienced leukopenia (white blood cells [WBC] count < 1.0 × 10(9)/L) and neutropenia (neutrophil [ANC] count < 0.5 × 10(9)/L), whereas all irradiated controls displayed a profound period of leukopenia and neutropenia. Furthermore, administration of rhG-CSF significantly delayed the onset of leukopenia and reduced the duration of leucopenia as compared with controls. In addition, individual dogs in the rhG-CSF-treated group exhibited evident differences in rhG-CSF responsiveness after neutron-irradiation. Finally, histopathological evaluation of the surviving dogs revealed that the incidence and severity of bone marrow, thymus and spleen damage decreased in rhG-CSF-treated dogs as compared with surviving controls. Thus, these results demonstrated that rhG-CSF administration enhanced recovery of myelopoiesis and survival after neutron-irradiation.

Structural requirement of spirostanol glycosides for rat uterine contractility and mode of their synergism.

OBJECTIVES: Total steroidal saponins extracted from the rhizome of Paris polyphylla (TSSP) have been used in China for the treatment of abnormal uterine bleeding. The aim of this study was to analyse the structure-activity relationship of steroidal saponins purified from P. polyphylla Sm. var. yunnanensis on rat myometrial contractions, and investigate the synergism among themselves as well as with known inherent agonists, such as Prostaglandin F(2alpha) (PGF-2alpha). METHODS: In this study, 22 steroidal saponins purified from TSSP were screened for their contractile activity in isolated uterine strips from estrogen-primed rats. KEY FINDINGS: It was shown that spirostanol glycosides exhibited inducible or inhibitory activity in rat uterine contraction based on the difference of their structures, which was not only attributed in part to the number, the length and the position of sugar side chains attached by a glycoside, but also related to the structure of the aglycone. Furthermore, synergistic actions were observed among pennogenin or diosgenin glycosides as well as with the known inherent agonist PGF-2alpha, indicating they may share, at least in part, similar pathways with PGF-2alpha in stimulating myometrial contractions. Finally, the contractile response of rat myometrium to spirostanol glycosides was significantly enhanced with advancing pregnancy. CONCLUSIONS: Together, these data support the possibility that some spirostanol glycosides may represent a new type of contractile agonist for the uterus and their synergism may be responsible for the therapeutic effect of TSSP on abnormal uterine bleeding.

Fluacrypyrim, a novel STAT3 activation inhibitor, induces cell cycle arrest and apoptosis in cancer cells harboring constitutively-active STAT3.

STAT3 protein has an important role in oncogenesis and is a promising anticancer target. Herein, we demonstrate that a novel small molecule fluacrypyrim (FAPM) inhibits the growth of leukemia cells by a predominant G1 arrest with significant decrease of the protein and mRNA levels of cyclin D1. As cyclin D1 is transcriptionally regulated by STAT3, FAPM is then shown to markedly inhibit the STAT3 phosphorylation with marginal effect on the other signal transducers and activators of transcription, and without effect on phosphoinositide-3-kinase and mitogen-activated protein kinase pathways. Further analysis shows that FAPM significantly increases the protein tyrosine phosphatases (PTPs) activity in a dose-dependent manner, and the inhibition of PTP activation by sodium pervanadate reverses FAPM-induced suppression of STAT3 tyrosine phosphorylation, indicating an important role of PTP in the action of FAPM. Finally, FAPM treatment results in selective suppression of STAT3-mediated transcriptional activity and its downstream effectors, and subsequent induction of growth arrest and apoptosis in STAT3-dependent cancer cell lines. This study therefore identifies FAPM as a potent STAT3 activation inhibitor with possible therapeutic potential against malignancies with constitutive STAT3 activation.

Generation and characterization of novel human IRAS monoclonal antibodies.

Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10-120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.