Failure of Indomethacin and Radiation to Prevent Blast-induced Heterotopic Ossification in a Sprague-Dawley Rat Model.

BACKGROUND: Although use of nonsteroidal antiinflammatory drugs and low-dose irradiation has demonstrated efficacy in preventing heterotopic ossification (HO) after THA and surgical treatment of acetabular fractures, these modalities have not been assessed after traumatic blast amputations where HO is a common complication that can arise in the residual limb. QUESTIONS/PURPOSES: The purpose of this study was to investigate the effectiveness of indomethacin and irradiation in preventing HO induced by high-energy blast trauma in a rat model. METHODS: Thirty-six Sprague-Dawley rats underwent hind limb blast amputation with a submerged explosive under water followed by irrigation and primary wound closure. One group (n = 12) received oral indomethacin for 10 days starting on postoperative Day 1. Another group (n = 12) received a single dose of 8 Gy irradiation to the residual limb on postoperative Day 3. A control group (n = 12) did not receive either. Wound healing and clinical course were monitored in all animals until euthanasia at 24 weeks. Serial radiographs were taken immediately postoperatively, at 10 days, and every 4 weeks thereafter to monitor the time course of ectopic bone formation until euthanasia. Five independent graders evaluated the 24-week radiographs to quantitatively assess severity and qualitatively assess the pattern of HO using a modified Potter scale from 0 to 3. Assessment of grading reproducibility yielded a Fleiss statistic of 0.41 and 0.37 for severity and type, respectively. By extrapolation from human clinical trials, a minimum clinically important difference in HO severity was empirically determined to be two full grades or progression of absolute grade to the most severe. RESULTS: We found no differences in mean HO severity scores among the three study groups (indomethacin 0.90 ± 0.46 [95% confidence interval {CI}, 0.60-1.19]; radiation 1.34 ± 0.59 [95% CI, 0.95-1.74]; control 0.95 ± 0.55 [95% CI, 0.60-1.30]; p = 0.100). For qualitative HO type scores, the radiation group had a higher HO type than both indomethacin and controls, but indomethacin was no different than controls (indomethacin 1.08 ± 0.66 [95% CI, 0.67-1.50]; radiation 1.89 ± 0.76 [95% CI, 1.38-2.40]; control 1.10 ± 0.62 [95% CI, 0.70-1.50]; p = 0.013). The lower bound of the 95% CI on mean severity in the indomethacin group and the upper bound of the radiation group barely spanned a full grade and involved only numeric grades < 2, suggesting that even if a small difference in severity could be detected, it would be less than our a priori-defined minimum clinically important difference and any differences that might be present are unlikely to be clinically meaningful. CONCLUSIONS: This work unexpectedly demonstrated that, compared with controls, indomethacin and irradiation provide no effective prophylaxis against HO in the residual limb after high-energy blast amputation in a rat model. Such an observation is contrary to the civilian experience and may be potentially explained by either a different pathogenesis for blast-induced HO or a stimulus that overwhelms conventional regimens used to prevent HO in the civilian population. CLINICAL RELEVANCE: HO in the residual limb after high-energy traumatic blast amputation will likely require novel approaches for prevention and management.

Breast cancer stem cell-like cells are more sensitive to ionizing radiation than non-stem cells: role of ATM.

There are contradictory observations about the different radiosensitivities of cancer stem cells and cancer non-stem cells. To resolve these contradictory observations, we studied radiosensitivities by employing breast cancer stem cell (CSC)-like MDA-MB231 and MDA-MB453 cells as well as their corresponding non-stem cells. CSC-like cells proliferate without differentiating and have characteristics of tumor-initiating cells [1]. These cells were exposed to γ-rays (1.25-8.75 Gy) and survival curves were determined by colony formation. A final slope, D(0), of the survival curve for each cell line was determined to measure radiosensitivity. The D(0) of CSC-like and non-stem MDA-MB-453 cells were 1.16 Gy and 1.55 Gy, respectively. Similar results were observed in MDA-MB-231 cells (0.94 Gy vs. 1.56 Gy). After determination of radiosensitivity, we investigated intrinsic cellular determinants which influence radiosensitivity including cell cycle distribution, free-radical scavengers and DNA repair. We observed that even though cell cycle status and antioxidant content may contribute to differential radiosensitivity, differential DNA repair capacity may be a greater determinant of radiosensitivity. Unlike non-stem cells, CSC-like cells have little/no sublethal damage repair, a low intracellular level of ataxia telangiectasia mutated (ATM) and delay of γ-H2AX foci removal (DNA strand break repair). These results suggest that low DNA repair capacity is responsible for the high radiosensitivity of these CSC-like cells.

5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis.

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis.

Reactive oxygen species up-regulate p53 and Puma; a possible mechanism for apoptosis during combined treatment with TRAIL and wogonin.

BACKGROUND AND PURPOSE: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptotic death in a variety of cancer cells without marked toxicity to most normal cells. We previously reported that wogonin, a potent anticancer agent from a Chinese herb, up-regulates p53 in prostate cancer cells. In this study, the effects of combinations of TRAIL and wogonin on a human prostate cancer cell line LNCaP, resistant to TRAIL, was evaluated for evidence of synergy in triggering apoptosis. EXPERIMENTAL APPROACH: Western blot assay and the 'comet' assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL-induced apoptosis in the presence of wogonin. KEY RESULTS: During combined treatment with wogonin and TRAIL, cytotoxicity, poly(ADP-ribose) polymerase cleavage and caspase activation were associated with up-regulation of p53 through DNA damage and reactive oxygen species (ROS) generation. N-acetylcysteine (NAC), an antioxidant, inhibited ROS generation and synergistic interaction between TRAIL and wogonin. Experimental results in human colon cancer HCT116 cells demonstrated that p53-dependent Puma up-regulation played an important role; deficiency in either p53 or Puma prevented wogonin-enhanced TRAIL-induced apoptosis. CONCLUSIONS AND IMPLICATIONS: The present studies suggest that wogonin enhances TRAIL-induced cytotoxicity through up-regulation of p53 and Puma, mediated by ROS.

Effects of low dose quercetin: cancer cell-specific inhibition of cell cycle progression.

Quercetin is a flavonoid present in many vegetables, fruits, and beverages. Due to its anti-oxidant, anti-tumor, and anti-inflammatory activity, quercetin has been studied extensively as a chemoprevention agent in several cancer models. Since most of these studies used higher doses of quercetin than clinically achievable, we focused on the effectiveness of physiologically relevant doses of quercetin. A low dose of quercetin exerted cancer cell-specific inhibition of proliferation and this inhibition resulted from cell cycle arrest at the G(1) phase. Quercetin induced p21 CDK inhibitor with a concomitant decrease of phosphorylation of pRb, which inhibits the G(1)/S cell cycle progression by trapping E2F1. A low dose of quercetin induced mild DNA damage and Chk2 activation, which is the main regulator of p21 expression by quercetin. In addition, quercetin down-regulated the cyclin B1 and CDK1, essential components of G(2)/M cell cycle progression. Inhibition of the recruitment of key transcription factor NF-Y to cyclin B1 gene promoter by quercetin led to transcriptional inhibition. This study proved that the chemo-preventive efficacy of a physiologically relevant dose of quercetin can be achievable through the inhibition of cell cycle progression.

In vitro determination of radiation sensitivity parameters for DU-145 prostate cancer cells.

PURPOSE: The prolonged delivery times associated with intensity modulated radiation therapy (IMRT) may reduce treatment effectiveness of radiation therapy for cancers with short repair half-times. In this study, in vitro radiation experiments with DU-145 prostate cancer cells were designed to quantify the half-time of sublethal damage repair. METHOD AND MATERIALS: A series of single-fraction and split-dose clonogenic survival experiments were performed and analyzed using the linear-quadratic (LQ) survival model with mono-/two-component exponential and reciprocal-time repair kinetic models. RESULTS: Our data indicate that DU-145 cells are very radiosensitive (alpha = 0.44 Gy(-1), standard CI: 0.41-0.49 Gy(-1)) and are relatively insensitive to dose fractionation (alpha/beta = 16 Gy, standard CI: 12-34 Gy). The estimated repair half-time is 23 min (standard CI: 10-97 min) with some evidence that a small portion of the sublethal damage is repaired more slowly. CONCLUSION: The reported radiosensitivity parameters (alpha and alpha/beta) are larger than those derived from other in vitro experiments and clinical data. In contrast, the half-time for sublethal damage repair ( approximately 23 min) is close to the one derived from clinical data ( approximately 16 min). For such short repair half-times, the effectiveness of IMRT treatments may be substantially improved by decreasing the fraction delivery time.

Radiosensitization of head/neck squamous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein.

PURPOSE: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. EXPERIMENTAL PROCEDURES: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. RESULTS: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. CONCLUSIONS: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.

Mre11-Rad50-Nbs1 complex is activated by hypertonicity.

When exposed to hypertonic conditions, cells accumulate double-strand DNA breaks (DSBs) like they are exposed to ionizing radiation. It has been proposed that inactivation of the Mre11-Rad50-Nbs1 (MRN) complex due to nuclear exit is responsible for the accumulation of DSBs as cells fail to repair DSBs produced during normal cellular activity. In this study, we examined the MRN complex in cells switched to hypertonicity. Surprisingly, we found that the MRN complex stayed in the nucleus and remained intact in response to hypertonicity. In fact, the MRN complex was dramatically activated after 4 h of switch to hypertonicity in a dose-dependent manner as shown by formation of foci. Activation of ATM and the MRN complex by hypertonicity and bleomycin was additive as was activation of their downstream targets including gammaH2AX and Chk2 indicating that the cellular response to DSB was intact in hypertonic conditions. Activation of Chk2 in response to hypertonicity was not observed in mutant cells with functionally impaired MRN complex confirming that they are in the same pathway. After 20 h of a switch to hypertonicity, MRN foci and gammaH2AX returned to a control level, suggesting that cells adapted to hypertonicity by repairing DNA. We conclude that cells respond normally to DSB and repair the DNA damages induced by hypertonicity.

Hypoxia and low glucose differentially augments TRAIL-induced apoptotic death.

Tumor microenvironment, which is characterized by hypoxia, low-glucose concentrations, high-lactate concentrations, low-extracellular pH, can alter the therapeutic response in tumors. In this study, we investigated whether hypoxia affects TRAIL-induced apoptotic death. When human prostate adenocarcinoma DU-145 cells were treated with 50 ng/mL TRAIL or hypoxia for 4 h, the survival was 45.7 and 32.5%, respectively. The combination of TRAIL and hypoxia synergistically increased cell death. Similar results were observed in human prostate adenocarcinoma LNCaP cells. Western blot analysis showed that the hypoxia augmented TRAIL-induced PARP cleavage as well as the activation of caspase-8 and caspase-3, but not caspase-9. Unlike hypoxia, low glucose promoted caspase-9 activation during TRAIL treatment. These results suggest that hypoxia or low glucose-augmented TRAIL cytotoxicity is mediated through the mitochondria-independent pathway or -dependent pathway, respectively.

Spermine oxidation induced by Helicobacter pylori results in apoptosis and DNA damage: implications for gastric carcinogenesis.

Oxidative stress is linked to carcinogenesis due to its ability to damage DNA. The human gastric pathogen Helicobacter pylori exerts much of its pathogenicity by inducing apoptosis and DNA damage in host gastric epithelial cells. Polyamines are abundant in epithelial cells, and when oxidized by the inducible spermine oxidase SMO(PAOh1) H(2)O(2) is generated. Here, we report that H. pylori up-regulates mRNA expression, promoter activity, and enzyme activity of SMO(PAOh1) in human gastric epithelial cells, resulting in DNA damage and apoptosis. H. pylori-induced H(2)O(2) generation and apoptosis in these cells was equally attenuated by an inhibitor of SMO(PAOh1), by catalase, and by transient transfection with small interfering RNA targeting SMO(PAOh1). Conversely, SMO(PAOh1) overexpression induced apoptosis to the same levels as caused by H. pylori. Importantly, in H. pylori-infected tissues, there was increased expression of SMO(PAOh1) in both human and mouse gastritis. Laser capture microdissection of human gastric epithelial cells demonstrated expression of SMO(PAOh1) that was significantly attenuated by H. pylori eradication. These results identify a pathway for oxidative stress-induced epithelial cell apoptosis and DNA damage due to SMO(PAOh1) activation by H. pylori that may contribute to the pathogenesis of the infection and development of gastric cancer.

Caspase-1 enhances the apoptotic response of prostate cancer cells to ionizing radiation.

BACKGROUND: The significance of caspase-1 in prostate cancer has recently been documented (Cancer Res 61: 1227-1232, 2001). In this study, we investigated the role of caspase-1 in radiation-induced apoptosis in order to identify the significance of this apoptotic initiator in radiation resistance. MATERIALS AND METHODS: Caspase-1 was over-expressed in DU-145 prostate cancer cells (which have weak endogenous expression of caspase-1), via transfection-mediated gene transfer. Stable transfectants were cloned and expression of caspase-1 was established at the mRNA and protein levels by RT-PCR and Western blot, respectively. Caspase-1 overexpressing clones were characterized for their apoptotic response to ionizing irradiation (0-9 Gy) on the basis of cell viability and Hoechst staining assays and profiling of expression of key apoptosis regulators, such as caspase -3 and -9. RESULTS: Caspase-1 transfectants exhibited a greater sensitivity in response to ionizing radiation than the neomycin control transfectants, as demonstrated by a dramatic loss in cell viability, that temporally correlated with apoptosis induction. Furthermore, caspase-1 overexpression resulted in a significant decrease in clonogenic survival following treatment with ionizing radiation, while the caspase-1 inhibitor, Z-YVAD.fmk, suppressed apoptosis induction in caspase-1 transfectants (p<0.008). The apoptotic effect was associated with increased expression of the pro-enzyme form of caspase-3 in both the caspase-1 transfectants and neo controls cells, with the activated caspase-3 being detected in caspase-1 transfectants only. While this activation of caspase-3 was paralleled by an elevated caspase-9 expression at 9 h post-irradiation, there was no major induction in Apaf-1 or cytochrome c release. CONCLUSION: The present study provides an initial mechanistic insight into the functional involvement of caspase-1 in changing the apoptotic threshold of prostate cancer cells to radiation. These findings will enhance the understanding of the molecular basis of prostate tumor radioresistance and may have significant clinical relevance in improving the therapeutic index of radiotherapy in prostate cancer patients.

The ceramide analog, B13, induces apoptosis in prostate cancer cell lines and inhibits tumor growth in prostate cancer xenografts.

BACKGROUND: Apoptosis is a therapeutic target for the elimination of cancer cells. As elevations in ceramide levels induce apoptosis, there is much excitement about the use of agents that elevate ceramide levels as novel chemotherapeutic agents. Ceramidases are enzymes involved in degradation of ceramide and inhibition of ceramidase has been proposed as a mechanism to increase ceramide levels. This study provides the first insight into the effect of B13, an inhibitor of acid ceramidase, on human prostate cancer cell lines and xenografts. METHODS: Cell death was evaluated by the trypan blue assay; apoptosis by the Apo2.7 apoptosis assay; and glutathione levels by HPLC. Tumors were irradiated with a dose of 5 Gy of X-rays (250 kVp, 15 mA, 2 Gy/min) and tumor volume was measured during the course of the experiment. At the conclusion of the experiment, tumor weight was determined and the tumors were evaluated histologically. RESULTS: B13 is an inducer of cell death, by apoptosis, in cultured prostate cancer cells. LNCaP and PC3 cells have different responsiveness to the enantiomers of B13. In LNCaP cells, the R enantiomer of B13 (10 microM) was significantly more effective than the S enantiomer at inducing cell death as determined by the trypan blue assay, culminating in approximately 90% cell death at 48 hr. In contrast, the same concentration of B13S induced <20% cell death at 48 hr. In PC3 cells, the S enantiomer was a more effective inducer of cell death, culminating in approximately 30% cell death, relative to 14% for B13R in this model. Evaluation of induction of apoptosis by the Apo2.7 mitochondrial assay confirmed that this induction of cell death was by apoptosis. Concurrent with induction of apoptosis, glutathione levels drop in response to B13. Specifically, B13R caused a significant drop in glutathione levels in LNCaP cells, culminating in a reduction to 40% control values at 48 hr. In PC3 cells, in contrast, the drop in glutathione levels was more dramatic, culminating in a drop to 12% control values in response to B13S at 48 hr. The effects of B13R, however, were not significantly different from control values. In in vivo studies using a model of xenografted androgen-insensitive prostate cancer, B13 sensitized the tumors to the effects of radiation, resulting in a significant reduction in tumor volume and weight after treatment with the combination of B13 and radiation. Microscopic evaluation of the tumors indicated that apoptosis was the primary mechanism of this effect. CONCLUSIONS: Targeting ceramide pathways may be a novel treatment strategy for hormone refractory prostate cancer.

Combination radiation and adenovirus-mediated P16(INK4A) gene therapy in a murine model for head and neck cancer.

Since adenoviral P16(INK4A) gene transfer has shown limited promise in preclinical studies, we developed a strategy combining ionizing radiation (x-rays) with Ad-P16 gene therapy. Both radiation (2 Gy) and Ad-P16 alone demonstrated anti-tumor responses. The combination of radiation and Ad-P16 in vitro showed an augmented therapeutic response. Two head and neck squamous cell carcinoma (HNSCC) cell lines treated with Ad-P16 demonstrated positive staining to annexin V and terminal deoxynucleotidyl-transferase, suggesting apoptosis. When the HNSCC cell lines were grown in nude mice, the anti-tumor response by Ad-P16, radiation, and combination therapy ranged from limited benefit of the single-agent therapy to a statistically significant additive response to combination therapy. Our data suggest that Ad-P16 gene therapy may prove effective in treating human HNSCC that is refractory to radiation therapy.

Role of glutaredoxin in metabolic oxidative stress. Glutaredoxin as a sensor of oxidative stress mediated by H2O2.

Epitope-tagged glutaredoxin (GRX) was utilized to determine the role of GRX in oxidative stress-induced signaling and cytotoxicity in glucose-deprived human cancer cells (MCF-7/ADR and DU-145). GRX-overexpressing cells demonstrated resistance to glucose deprivation-induced cytotoxicity and decreased activation of c-Jun N-terminal kinase (JNK1). Deletion mutants showed the C-terminal portion of apoptosis signal-regulating kinase 1 (ASK1) bound GRX, and glucose deprivation disrupted binding. Treatment with l-buthionine-(S,R)-sulfoximine reduced glutathione content by 99% and prevented glucose deprivation-induced dissociation of GRX from ASK1. A thiol antioxidant, N-acetyl-l-cysteine, or overexpression of an H(2)O(2) scavenger, catalase, inhibited glucose deprivation-induced dissociation of GRX from ASK1. GRX active site cysteine residues (Cys(22) and Cys(25)) were required for dissociation of GRX from ASK1 during glucose deprivation. Kinase assays revealed that SEK1 and JNK1 were regulated in an ASK1-dependent fashion during glucose deprivation. Overexpression of GRX or catalase inhibited activation of ASK1-SEK1-JNK1 signaling during glucose deprivation. These results demonstrate that GRX is a negative regulator of ASK1 and dissociation of GRX from ASK1 activates ASK1-SEK1-JNK1 signaling leading to cytotoxicity during glucose deprivation. These results support the hypothesis that the GRX-ASK1 interaction is redox sensitive and regulated in a glutathione-dependent fashion by H(2)O(2).