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The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 μM, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellular ROS production increased with the R24 dose. In summary, we found that R24 exhibits potent antiangiogenic and antiproliferative effects, induces apoptosis, and promotes ROS production.

In this study, we investigated the response of irradiated bone marrow cells to granulocyte colony-stimulating factor (G-CSF). Freshly harvested bone marrow cells were treated with either saline (vehicle control) or 20 ng/ml of G-CSF. Thereafter, cells were separated into nonirradiated (no-IR) and irradiated (IR, 0.5 Gy) groups. IR cells exhibited a higher proliferation rate in response to G-CSF, as compared to the no-IR cells. Reduced levels of reactive oxygen species indicated that G-CSF-treated IR cells produced fewer free radicals, as compared to the no-IR cells. The G-CSF-treated IR cells also had a lower apoptotic rate than their no-IR counterparts. Furthermore, G-CSF-treated IR cells exhibited less alteration of mitochondrial membrane potential, as compared to the no-IR cells. Finally, the mitochondrial number increased in the G-CSF-treated IR cells. The radiation-induced increase in plasma IL-6 in vivo could be enhanced by the administration of G-CSF. The data suggest that radiation potentiates the response of bone marrow cells to G-CSF treatment.

Amifostine is a first-line cytoprotective drug used to prevent radiotherapy-induced or chemotherapy-induced injuries. However, its mechanism of action is not well understood. In this study, freshly harvested bone marrow cells were treated with amifostine and analyzed with a series of mitochondrial indices. In vitro results showed that bone marrow cells treated with amifostine 0.5 h before irradiation (0.5 Gy) experienced several benefits, as compared to vehicle controls, including (1) reduced reactive oxygen species levels, which reduced the production of free radicals; (2) better preservation of mitochondria, as indicated by MitoTracker-positive staining and the increased intensity of staining; (3) reduced apoptosis, as demonstrated by Annexin V staining; and (4) a better proliferation rate, as illustrated by MTT assay. Our in vitro studies showed that amifostine-treated mice exhibited (1) higher ATP production; (2) reduced plasma IL-2 levels, suppressing the immune response triggered by radiotoxicity; and (3) enhanced radiation-induced production of granulocyte colony-stimulating factor. All of these processes benefit recovery from radiation-induced 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.

In this study, we compared two in vitro collagen production assays ([3H]-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 [3H]-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 agent’s capacity for collagen production and (b) Sirius Red is more useful than [3H]-proline because it is easier to use, more convenient, less time consuming, and does not require radioactive material.

Although glycoproteins possess a variety of functional and structural roles in intracellular and intercellular activities, the effect of ionizing radiation (IR) on glycosylation is largely unknown. To explore this effect, we established a sandwich assay in which PHA-L, a phytohaemagglutinin that agglutinates leukocytes, was used as a coating layer to capture glycoproteins containing complex oligosaccharides; the bound glycoproteins were then measured. C57BL/6 mice were exposed to 0, 3, 6, or 10 Gy, and the plasma was collected at 6, 12, 18, 24, 48, 72, or 168 h and then analyzed for galactose/N-acetylgalactosamine (Gal/GalNAc) containing proteins. We found that (1) the sandwich assay accurately measured the level of glycoproteins, (2) 6–12 h after IR, the amount of glycoproteins containing GalNAc increased, and (3) at 72 and 168 h, 10 Gy was associated with a decrease in Gal/GalNAc. These IR-induced alterations might relate to the release of glycoproteins into the blood and the damage of the proteins and genes that are related to the glycosylation process.

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.

Mitochondrial DNA (mtDNA) is maternally inherited and controls the oxygen-related production of adenosine-5′-triphosphate, which is transported from the mitochondria to other cellular compartments and used as energy for cellular activities. The mtDNA is physically separated from nuclear DNA (nDNA). Ionizing radiation (IR) causes the release of both mtDNA and nDNA into circulation. Our previous study demonstrated that nDNA has potential to be a biodosimeter. In this study, branched DNA technology was used to explore the alteration pattern of mtDNA after IR. C57BL/6 mice were exposed to 0, 1.5, 3, 6, 8, or 10 Gy total body irradiation; thereafter, plasma mtDNA was assessed with samples collected at 3, 6, 9, 15, 24, 48, 72, or 168 h. We found that: (1) the designed probesets were specific for mtDNA extracted from the liver, and they recognized the small amount of mtDNA mixed in the nDNA; (2) plasma mtDNA exhibited a statistically significant increase only at 6 h after 8 Gy irradiation. The alteration of mtDNA was not dose-dependent or time-dependent; hence, it is unlikely to be an effective biodosimeter.

Inflammatory molecules (IMs) play an important role in ionizing radiation (IR)-induced soft tissue damage. The alteration of IMs as a function of time was studied with a protein array containing 62 IMs in mouse cutaneous soft tissues exposed to 30 Gy. The results showed that: (1) 2 days after irradiation, the levels of TGF-β1, MIP-1γ, IL-1α, and sTNF RI increased, while IGFBP-3, CXCL16, and IL-1β decreased in IR skin as compared to control skin; (2) 21 days after IR, TGF-β1, and MIP-1 γ, IL-1α remained high, while CXCL16 and IL-1β remained low; (3) 3 months after IR, the cytokine pattern exhibited reversals. The levels of MIP-1γ decreased, while VCAM-1, IGFBP-3, and TGF-β1 production increased. The data indicated that: (a) IMs change as a function of time after soft tissue irradiation; (b) changing IM levels may reflect the altered balance of the cytokine network, leading to imbalance or homeostasis; and (c) an antibody-based protein array can be used to assess multiple IMs simultaneously, making it useful for bulk screening for changes in tissue cytokine levels.

Since Balb/c mice are sensitive to acute hematopoietic syndrome and C57BL/6 to late fibrovascular toxicity, we measured the effect of total body irradiation (TBI) on mitochondrial DNA (mtDNA) copy number in these two strains. Six months after TBI, we extracted total genomic DNA, including nuclear DNA (nDNA) and mtDNA, from various tissues. The findings included: (1) TBI had no significant effect on body weight for Balb/c or C57BL/6 when compared to aged controls. (2) For different organs in control mice, the ratio of mtDNA to nDNA ranged from 0.33 to 11.53 in Balb/c and 0.34 to 13.28 in C57BL/6. (3) Ratios of mtDNA to nDNA were generally similar to or higher in C57BL/6 compared to Balb/c. (4) In C57BL/6, TBI induced an increase in mitochondrial copy number only in the brain (p < 0.05). (5) In Balb/c, radiation induced a dose-dependent increase of mtDNA in the liver (p < 0.05 and p < 0.01) and a decrease in skeletal/muscle tissue (p < 0.01). Our findings indicate that radiation influences mtDNA copy number differently in various tissues. This may explain differences in late radiation toxicity to those organs and differences in radiation tolerance between murine strains.

Genomic background helps determine sensitivity to TBI. Since the LD50/30 (the dose that causes death in half of exposed mice within 30 days) following TBI varies between murine strains, we tested four murine strains to determine whether TBI sensitivity was associated with different levels of circulating DNA after radiation. The LD50/30 for the mice we tested – BALB/c, NIH Swiss, C3H/HeN, and C57BL/6 – is approximately 5.8 ± 0.3, 7.3 ± 0.2, 7.4 ± 0.3, and 8.5 ± 0.3 Gy, respectively. We estimated the radiation dose at which circulating DNA reached a peak level (peakGy), and mice were subjected to different doses at 1.84 Gy/min. The peakGy was 6, 7, 9, and 9 Gy for BALB/c, NIH Swiss, C3H/HeN, and C57BL/6, which corresponds to each strain’s respective LD50/30. BALB/c, the most sensitive strain, had the lowest DNA concentration at peakGy, while C57BL/6, the most resistant strain, had the highest concentration at peakGy. At 7 Gy, the plasma DNA was approximately 3,754 ± 636, 8,238 ± 2,704, 9,773 ± 2,821, and 22,733 ± 5,914 ng/ml for BALB/c, NIH Swiss, C3H/HeN, and C57BL/6, respectively. These findings support our hypothesis that plasma DNA level is associated with genomic background.