Deping Han

Icaritin Synergistically Enhances the Radiosensitivity of 4T1 Breast Cancer Cells

Icaritin (ICT) is a hydrolytic form of icariin isolated from plants of the genus Epimedium. This study was to investigate the radiosensitization effect of icaritin and its possible underlying mechanism using murine 4T1 breast cancer cells. The combination of Icaritin at 3 µM or 6 µM with 6 or 8 Gy of ionizing radiation (IR) in the clonogenic assay yielded an ER (enhancement ratio) of 1.18 or 1.28, CI (combination index) of 0.38 or 0.19 and DRI (dose reducing index) of 2.51 or 5.07, respectively. These strongly suggest that Icaritin exerted a synergistic killing (?) effect with radiation on the tumor cells. This effect might relate with bioactivities of ICT: 1) exert an anti-proliferative effect in a dose- and time-dependent manner, which is different from IR killing effect but likely work together with the IR effect; 2) suppress the IR-induced activation of two survival paths, ERK1/2 and AKT; 3) induce the G2/M blockage, enhancing IR killing effect; and 4) synergize with IR to enhance cell apoptosis. In addition, ICT suppressed angiogenesis in chick embryo chorioallantoic membrane (CAM) assay. Taken together, ICT is a new radiosensitizer and can enhance anti-cancer effect of IR or other therapies.

The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity

BackgroundChanges in glutamatergic neurotransmission via decreased glutamate transporter (GLT) activity or expression contributes to multiple neurological disorders. Chemokines and their receptors are involved in neurological diseases but the role of chemokines in the expression of glutamate transporters is unclear.MethodsPrimary astrocytes were prepared from neonatal (<24 hours old) SJL/J mouse brains and incubated with 5 μg/ml lipopolysaccharide (LPS) or 50 ng/ml tumor necrosis factor α (TNF-α) for 24 hours. Soluble macrophage inflammatory protein-2γ (MIP-2γ) in culture supernatants was determined using a sandwich ELISA. The MIP-2γ effect on the expression of GLT-1 was measured by quantitative RT-PCR, flow cytometric analysis or western blot assay. Detergent-resistant membranes from astrocytes were isolated on the basis of their ability to float in density gradients. Raft-containing fractions were tracked by the enrichment of caveolin-1 and the dendritic lipid raft marker, flotillin-1. Cell viability was determined by measuring either the leakage of lactate dehydrogenase or the reduction of 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide by viable cells and confirmed by visual inspection.ResultsThe production of the chemokine MIP-2γ by mouse cortical astrocytes increased significantly after stimulation with LPS or TNF-α in vitro. Astrocytes over-expressing MIP-2γ down-regulated the expression of GLT-1 at the mRNA and protein level and caused redistribution of GLT-1 out of the lipid rafts that mediate glutamate uptake. We used pharmacological inhibitors to identify the downstream signaling pathways underlying MIP-2γ activity. We also found complementary results by knocking down MIP-2γ activity in astrocytes with MIP-2γ small interfering RNA (siRNA). MIP-2γ overexpression in astrocytes enhanced the neuronal toxicity of glutamate by decreasing GLT-1 activity, but MIP-2γ itself was not toxic to neurons.ConclusionsThese results suggest that MIP-2γ mediates the pathogenesis of central nervous system disorders associated with neutrophil infiltration in the brain and decreased GLT-1 activity.

Serum decoy receptor 3, a potential new biomarker for sepsis

BACKGROUNDnSepsis, a common deadly systemic infection caused by a variety of pathogens, has some clinical symptoms similar to the systemic inflammatory response syndrome (SIRS), a whole-body non-infectious inflammatory reaction to severe insults, such as burn, trauma, hypotensive shock and so on. Treatment of sepsis depends mainly on anti-microbial, while remedy for SIRS might require steroids that could possibly enhance the spread of microbes. Unfortunately, it is very difficult to distinguish these two completely different serious conditions without blood culture, which takes days to grow and identify causative pathogens. We examined a biomarker, serum decoy receptor 3 (DcR3), was evaluated for its utility in the differential diagnosis between sepsis and SIRS.nnnMETHODSnSerum DcR3 level in 118 healthy controls, 24 sepsis patients and 43 SIRS patients, was quantitatively measured by enzyme-linked immunosorbent assay (ELISA).nnnRESULTSnThe serum DcR3 was significantly increased in sepsis patients compared with SIRS patients and healthy controls (6.11±2.58 ng/ml vs 2.62±1.46 ng/ml, and 0.91±0.56 ng/ml, respectively, p<0.001). The areas under the receiver operating characteristic curve of DcR3 for the normal vs. SIRS, normal vs. sepsis and SIRS vs. sepsis were 0.910 (0.870-0.950), 0.992 (0.984-1.000) and 0.896 (0.820-0.973), respectively. In addition, the DcR3 exhibited a positive correlation coefficient with APACHE II score, a most commonly used index for the severity of sepsis (r=0.556, p=0.005).nnnCONCLUSIONnThe serum DcR3 has a potential to serve as a new biomarker for sepsis with its high specificity and sensitivity.

In Vitro Sirius Red Collagen Assay Measures the Pattern Shift from Soluble to Deposited Collagen

In this study, we compared two in vitro collagen production assays ([(3)H]-proline incorporation and Sirius Red) for their ability to determine the pattern shift from soluble to deposited collagen. The effect of the antifibrotic agent, triptolide (TPL), on collagen production was also studied. The results showed that: (1) 48 h after NIH 3T3 (murine embryo fibroblast) and HFL-1(human fetal lung fibroblast) were exposed to transforming growth factor-beta 1 (TGF-β), there was an increase in soluble collagen in the culture medium; (2) on day 4, soluble collagen declined, whereas deposited collagen increased; (3) Sirius Red was easier to use than [(3)H]-proline incorporation and more consistently reflected the collagen pattern shift from soluble to deposited; (4) the in vitro Sirius Red assay took less time than the in vivo assay to determine the effect of TPL. Our results suggest that: (a) the newly synthesized soluble collagen can sensitively evaluate an agents capacity for collagen production and (b) Sirius Red is more useful than [(3)H]-proline because it is easier to use, more convenient, less time consuming, and does not require radioactive material.

Fibroblast Growth Factor-Peptide Promotes Bone Marrow Recovery After Irradiation

Various members of the fibroblast growth factor (FGF) family mitigate radiation-induced damage. We designed and synthesized the binding domain peptide of FGF-2 (FGF-P) with a dimer form resistant to peptidase and examined its mitigatory effect on murine bone marrow cells. NIH Swiss mice were exposed to different doses of total body irradiation (TBI) and treated with ten doses of 5 mg/kg FGF-P. We achieved the following results: (1) FGF-P stimulated the growth of bone marrow cells harvested from mice exposed to 3 Gy; (2) on day 25 after 6 Gy TBI, the number of leukocytes and granulocytes was higher in the FGF-P group than in the vehicle-alone group; (3) FGF-P significantly increased the number of pro-B and pre-B cells; and (4) FGF-P treatment in vivo increased the long-term hematopoietic stem cells (LT-HSC) in bone marrow. These data reveal the underlying mechanism by which FGF-P rescued a significant percentage of the exposed mice. The increase of LT-HSC in bone marrow leads to a concomitant increase of pro-B and pre-B cells followed by leukocytes and granulocytes, which in turn enhance immunity against infection.

Potential toxicity of quercetin: The repression of mitochondrial copy number via decreased POLG expression and excessive TFAM expression in irradiated murine bone marrow

The cytotoxicity of quercetin is not well understood. Using an ICR murine model, we unexpectedly found that mice exposed to 7 Gy total body irradiation (TBI) exhibited general in vivo toxicity after receiving quercetin (100 mg/kg PO), whereas this result was not observed in mice that received TBI only. In order to understand the involvement of alterations in mitochondrial biogenesis, we used a real-time qPCR to analyze the mitochondrial DNA copy number (mtDNAcn) by amplifying the MTRNR1 (12S rRNA) gene in murine bone marrow. We also utilized reverse transcription qPCR to determine the mRNA amounts transcribed from the polymerase gamma (POLG), POLG2, and mammalian mitochondrial transcription factor A (TFAM) genes in the tissue. In the mice exposed to TBI combined with quercetin, we found: (1) the radiation-induced increase of mtDNAcn was inhibited with a concurrent significant decrease in POLG expression; (2) TFAM expression was significantly increased; and (3) the expression of POLG2 was not influenced by the treatments. These data suggest that the overall toxicity was in part associated with the decrease in mtDNAcn, an effect apparently caused by the inhibition of POLG expression and overexpression of TFAM; unaltered POLG2 expression did not seem to contribute to toxicity.

SVα-MSH, a novel α-melanocyte stimulating hormone analog, ameliorates autoimmune encephalomyelitis through inhibiting autoreactive CD4+ T cells activation

Alpha-melanocyte stimulating hormone (α-MSH) plays a crucial role in the regulation of immune and inflammatory reactions. Here we report that SVα-MSH, a novel α-MSH analog, could ameliorate the clinical severity of experimental autoimmune encephalomyelitis (EAE) in a preventive and therapeutic manner. SVα-MSH treatment induced the production of regulatory T (Treg) cells and reduced the Th17 cells in the CNS of EAE mice. SVα-MSH-treated PLP peptide 139-151-specific T cells showed a down-regulation of T cell activation markers CD69 and CD134. SVα-MSH did not induce apoptosis but blocked the G1/S phase transition, reduced the expression of cyclin E, Cdk2 and the activity of NFAT and AP-1 transcription factors. Thus, SVα-MSH acts as a novel immunotherapeutic approach in the treatment of autoimmune attack on the CNS.

Transition pattern and mechanism of B-lymphocyte precursors in regenerated mouse bone marrow after subtotal body irradiation.

Little is known about the effects of ionizing radiation on the transition and the related signal transduction of progenitor B cells in the bone marrow. Thus, using an NIH Swiss mouse model, we explored the impact of ionizing radiation on the early stage of B-cell development via an examination of the transition of CLP to pro-B to pre-B cells within bone marrow as a function of radiation doses and times. Our results showed that while the total number of bone marrow lymphoid cells at different stages were greatly reduced by subtotal body irradiation (sub-TBI), the surviving cells continued to transition from common lymphoid progenitors to pro-B and then to pre-B in a reproducible temporal pattern. The rearrangement of the immunoglobulin heavy chain increased significantly 1–2 weeks after irradiation, but no change occurred after 3–4 weeks. The rearrangement of the immunoglobulin light chain decreased significantly 1–2 weeks after sub-TBI but increased dramatically after 3–4 weeks. In addition, several key transcription factors and signaling pathways were involved in B-precursor transitions after sub-TBI. The data indicate that week 2 after irradiation is a critical time for the transition from pro-B cells to pre-B cells, reflecting that the functional processes for different B-cell stages are well preserved even after high-dose irradiation.

Triptolide mitigates radiation-induced pulmonary fibrosis via inhibition of axis of alveolar macrophages-NOXes-ROS-myofibroblasts.

ABSTRACT Purpose: IR-induced pulmonary fibrosis is one of the most severe late complications of radiotherapy for lung cancer. It is urgently needed to discover a new drug for anti-IR lung fibrosis. Our previous studies have indicated that TPL exhibits both anti-IR lung fibrosis and anti-tumor activities. To reveal the mechanism of TPL on anti-IR lung fibrosis, alveolar macrophages (AMs) were examined for TPL effect on their axis of Nicotinamide adenine dinucleotide phosphate oxidase-reactive oxygen species (NOXes-ROS) and myofibroblast activation. Methods and Materials: The fibrosis-prone C57BL/6 mice were irradiated with 15 Gy on whole chest, then one day later, mice were treated without or with TPL (i.v. 0.25 mg/kg, qod for 1 month). The AMs were collected from bronchoalveolar lavage fluids and studied for the production of ROS and the levels of NOXes. The effect of AMs on myofibroblast activation as labeled with F4/80 or α-SMA (α-smooth muscle actin) were examined using flow cytometry, Western blotting, or immunohistochemical staining. Results: TPL effectively reduced the IR-induced lung fibrosis as evidenced by the less myofibroblasts, less collagen deposit and less ROS in the IR-lung tissues. We found that ROS which responsible for myofibroblasts activation was mainly from AMs and was NOX2 and NOX4 dependent. TPL significantly reduced the infiltrated AMs in IR-lung tissues, and in addition, down regulated the level of NOX2 and NOX4 in AMs both in vitro and in vivo. Furthermore, by inhibiting NOXes dependent ROS in AMs, TPL deprived AMs paracrine activation of myofibroblasts. Conclusions: Our work demonstrated that the anti-fibrotic effect of TPL on IR-induced pulmonary fibrosis was related to its inhibition on the axis of alveolar macrophages-NOXes-ROS-myofibroblasts.

Interleukin 11 Protects Bone Marrow Mitochondria from Radiation Damage

Interleukin 11 (IL-11) is a multifunctional cytokine isolated from bone marrow (BM)-derived stromal cells that promotes hematopoiesis and prolongs the life span of lethally irradiated animals. However, the underlying mechanism for the protective effect of IL-11 on BM is unclear. In this study, we explored the effect of IL-11 on irradiated BM cells. Freshly harvested BM cells were pretreated with 20 ng/ml of recombinant IL-11 for 30 min, irradiated with a dose of 0.5 Gy, cultured for 24 h, and then subjected to several assays. In vitro data showed that, as compared to the vehicle controls, IL-11: (1) reduced the production of reactive oxygen species; (2) reduced the alteration of mitochondrial membrane potential; (3) increased MitoTracker staining, suggesting that the number of mitochondria and their functions were better maintained; and (4) reduced apoptosis of BM cells and enhanced BM cell proliferation. In vivo studies of mice pretreated with saline or 100 μg/kg of IL-11 at 12 and 2 h before 10-Gy total body irradiation (TBI) demonstrated that G-CSF and IL-6 were significantly upregulated, whereas IL-2 and IL-4 were reduced. We found that IL-11 protects mitochondrial functions, acts with G-CSF and IL-6 to stimulate the growth of radiation-damaged BM, and reduces the immune response to radiation injury.