Intra-oral administration of rebamipide liquid prevents tongue injuries induced by X-ray irradiation in rats.

PURPOSE: Oral mucositis is a common and serious side effect in patients who undergo cytotoxic cancer therapies. The purpose of this study was to investigate the preventive effects of rebamipide on radiation-induced glossitis model in rats. METHODS: Glossitis was induced by a single dose of 15 Gy of X-rays to the snouts of rats (day 0). A novel form of rebamipide liquid comprising its submicronized crystals was administered intra-orally. The preventive effect of rebamipide on tongue injuries was macroscopically evaluated on day 7 following irradiation. The pretreatment period, dosing frequency, and dose dependency of rebamipide were examined. RESULTS: Two percent rebamipide liquid, administered six times a day for 14 days from day -7 to day 6, significantly decreased the ulcer-like area. However, no significant effect was observed when rebamipide was given either from day -4 or from day -1. Four or six times daily, 2% rebamipide liquid significantly inhibited the ulcer-like injury area ratio, but not when given twice daily. Rebamipide liquid, 1, 2, and 4% six times daily significantly reduced the area ratios of total injury and ulcer-like injury in a dose-dependent manner. Gene expression and protein levels of proinflammatory cytokines and chemokines were dramatically elevated in the irradiated tongues of control rats on day 7 without rebamipide liquid treatment. They were dose-dependently and significantly suppressed in rebamipide-treated groups. CONCLUSION: Intra-oral administration of rebamipide liquid prevented oral mucositis dose-dependently accompanied by the suppression of inflammatory expression in the radiation-induced rats' glossitis model.

Rebamipide does not interfere with the antitumor effect of radiotherapy or chemotherapy in human oral tumor-bearing nude mice.

Recent studies have shown that rebamipide, which suppresses reactive oxygen species, prevents chemoradiotherapy-induced oral mucositis in patients with head and neck cancers. However, anticancer action of radiotherapy and chemotherapy is believed to be partially associated with generation of reactive oxygen species. The aim of this study was to determine whether rebamipide interferes with the antitumor action of radiotherapy and chemotherapy. The effect of rebamipide on tumor cell growth was investigated using a human oral squamous carcinoma cell line, HSC-2, in vitro and in vivo. Rebamipide showed no significant effect on cell or tumor growth in HSC-2 tumor-bearing nude mice. Influences of rebamipide on the antitumor action of radiotherapy and of chemotherapy with cisplatin or docetaxel were investigated using the same animal model. In radiotherapy, the tumor was treated with 2.5 Gy of X-rays for 5 days, and rebamipide (300 mg/kg p.o.) was administered during irradiation periods. In chemotherapy, tumor-bearing mice were treated once with cisplatin (8 mg/kg, i.v.) or docetaxel (15 mg/kg i.v.) and rebamipide (300 mg/kg p.o.) was administered for 5 days following the antitumor drug treatment. Rebamipide did not interfere with the antitumor action of radiotherapy and chemotherapy.

Rebamipide protects small intestinal mucosal injuries caused by indomethacin by modulating intestinal microbiota and the gene expression in intestinal mucosa in a rat model.

The effect of rebamipide, a mucosal protective drug, on small intestinal mucosal injury caused by indomethacin was examined using a rat model. Indomethacin administration (10 mg/kg, p.o.) induced intestinal mucosal injury was accompanied by an increase in the numbers of intestinal bacteria particularly Enterobacteriaceae in the jejunum and ileum. Rebamipide (30 and 100 mg/kg, p.o., given 5 times) was shown to inhibit the indomethacin-induced small intestinal mucosal injury and decreased the number of Enterococcaceae and Enterobacteriaceae in the jejunal mucosa to normal levels. It was also shown that the detection rate of segmented filamentous bacteria was increased by rebamipide. PCR array analysis of genes related to inflammation, oxidative stress and wound healing showed that indomethacin induced upregulation and downregulation of 14 and 3 genes, respectively in the rat jejunal mucosa by more than 5-fold compared to that of normal rats. Rebamipide suppressed the upregulated gene expression of TNFα and Duox2 in a dose-dependent manner. In conclusion, our study confirmed that disturbance of intestinal microbiota plays a crucial role in indomethacin-induced small intestinal mucosal injury, and suggests that rebamipide could be used as prophylaxis against non-steroidal anti-inflammatory drugs -induced gastrointestinal mucosal injury, by modulating microbiota and suppressing mucosal inflammation in the small intestine.

Novel submicronized rebamipide liquid with moderate viscosity: significant effects on oral mucositis in animal models.

This study aimed at developing a novel rebamipide liquid for an effective treatment of oral mucositis. The healing effects of a variety of liquids comprising submicronized rebamipide crystals were investigated using a rat cauterization-induced oral ulcer model. Whereas 2% rebamipide liquid comprising micro-crystals did not exhibit significant curative effect, 2% rebamipide liquids comprising submicronized crystals with moderate viscosities exhibited healing effects following intra-oral administration. The 2% and 4% optimized rebamipide liquids showed significant healing effects in the rat oral ulcer model (p<0.01). In addition, in the rat radiation-induced glossitis model, whereby the injury was caused to the tongue by exposing only around the rat's snout to a 15 Gy of X-irradiation, the 2% optimized rebamipide liquid significantly reduced the percent area of ulcerated injury (p<0.05). In conclusion, the submicronized rebamipide liquid with moderate viscosity following intra-oral administration showed better both healing effect in the rat oral ulcer model and preventive effect in the rat irradiation-induced glossitis model.

Establishment of an X-ray irradiation-induced glossitis model in rats: biphasic elevation of proinflammatory cytokines and chemokines.

Oral mucositis is a frequent and serious side effect in patients who receive radiotherapy for head and neck cancer. The purpose of this study was to develop a noninvasive and quantitative model of oral mucositis in rats, investigate the pathophysiology, and evaluate the efficacy of pharmacological interventions. Rats received a single dose of 15 Gy of X-rays to the snout after shielding of the remainder of the rat body with lead plates to protect the body from irradiation (day 0). After irradiation, the macroscopic area of tongue injury gradually increased. The total area of injury and the ulcer-like area reached a maximum on day 7 and then gradually decreased until disappearance on day 28. Expression of proinflammatory cytokines and chemokines occurred transiently within 1-4 hours after irradiation and returned to a normal level at 24 hours. This expression was again observed from days 3 to 5 and increased significantly on day 7, which approximately coincided with the histologic severity of tissue damage. Subcutaneous administration of palifermin at 3 mg/kg per day for 3 consecutive days before irradiation completely prevented ulcer formation in this model. In conclusion, we established a novel model of glossitis in rats, induced by X-ray irradiation, in which biphasic elevations of expression of proinflammatory cytokines and chemokines could be monitored. This model is considered useful to investigate the pathophysiology of oral mucositis and evaluate the preventive effect of pharmacological interventions on oral mucositis induced by X-ray irradiation.

The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends.

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PK(CS) at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK(CS) autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK(CS) autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts.

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.

Ku recruits XLF to DNA double-strand breaks.

XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.

The DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK(CS) recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK(CS) accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK(CS) influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK(CS) at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK(CS) influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK(CS) phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK(CS) with the DNA ends.

Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA double strand break.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated at the Thr-2609 cluster and Ser-2056 upon ionizing radiation (IR). Furthermore, DNA-PKcs phosphorylation at both regions is critical for its role in DNA double strand break (DSB) repair as well as cellular resistance to radiation. IR-induced DNA-PKcs phosphorylation at Thr-2609 and Ser-2056, however, exhibits distinct kinetics indicating that they are differentially regulated. Although DNA-PKcs autophosphorylates itself at Ser-2056 after IR, we have reported here that ATM mediates DNA-PKcs phosphorylation at Thr-2609 as well as at the adjacent (S/T)Q motifs within the Thr-2609 cluster. In addition, our data suggest that DNA-PKcs- and ATM-mediated DNA-PKcs phosphorylations are cooperative and required for the full activation of DNA-PKcs and the subsequent DSB repair. Elimination of DNA-PKcs phosphorylation at both regions severely compromises radioresistance and DSB repair. Finally, our result provides a possible mechanism for the direct involvement of ATM in non-homologous end joining-mediated DSB repair.

Rapid inhibition of MAPK signaling and anti-proliferation effect via JAK/STAT signaling by interferon-alpha in hepatocellular carcinoma cell lines.

The potential anti-proliferation effect of interferon-alpha (IFN-alpha) against hepatocellular carcinoma (HCC) and its growth inhibitory mechanisms remain unclear. We examined four human HCC cell lines and every cell line had the anti-proliferative effect of IFN-alpha. The PLC/PRF/5 cell line, which expressed the IFN receptor most abundantly, responded most effectively to IFN-alpha stimulation. Here, we delineate the anti-proliferative effect of IFN-alpha via the MAPK pathway in human HCC cell lines. IFN-alpha retarded G1/S transition with no evidence of apoptosis and inhibited cell proliferation. IFN-alpha diminished the phosphorylation of both extracellular signal-regulated kinase (ERK) and mitogen-activated ERK-regulating kinase (MEK), but not Raf, within 5 min. Knockdown of signal transducers of activation and transcription1 (STAT1) or Janus kinase1 (JAK1) suppressed the reduction of phosphorylation both of ERK and MEK and diminished the growth inhibition by IFN-alpha. These results suggest that IFN-alpha induces anti-proliferative signaling via the JAK/STAT pathway downstream of IFN-alpha receptors and may reduce the growth stimulation signaling by cross-talk with the MEK/ERK pathway without IFN-alpha-induced transcription.

Alterations of DNA copy number and expression in genes involved in cell cycle regulation and apoptosis signal pathways in gamma-radiation-sensitive SX9 cells and -resistant SR-1 cells.

In the present study, genomic differences related to sensitivity to radiation were examined by comparative genomic hybridization and GeneChip 45K microarray in SX9 cells (radiation-sensitive) and their parental line, SR-1 (radiation-resistant). SX9 cells have defective DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity. DNA-PKcs is a DNA double-strand break repair protein that maintains chromosomal stability through nonhomologous end joining. However, the molecular basis of the radiation sensitivity of SX9 cells is unclear. Flow cytometry analysis showed that SR-1 and SX9 cells had a larger G2/M-phase population at 12 h after 4 Gy gamma irradiation, while only SR-1 cells progressed to G1/S at 24-36 h. SX9 and SR-1 cells had similar patterns of DNA copy number alteration, but the gains were observed on chromosome 9 (cent-E2), 11 (cent-A3), and 12 (C1-E) only in SX9 cells. Expression of genes located on those regions is higher in SX-9 cells than in SR1 cells, and the regions include genes associated with apoptosis and cell cycle regulation. Time-course data for gene expression at 0, 1, 3, 6 and 12 h after 4 Gy gamma irradiation revealed that the genes whose expression was altered in SX9 cells but not in SR-1 cells are in 16 clusters. Three of these clusters included genes for cell cycle regulation: JNK, PKC (PRKC) and ceramide cascade protein. These results suggest that amplification and altered expression of genes associated with cell cycle and apoptosis regulators in DNA-PK-deficient SX9 cells affect the differences in response to gamma radiation between SX9 and SR-1 cells.

Gene expression profiles correlate with the morphology and metastasis characteristics of renal cell carcinoma cells.

Renal cell carcinoma (RCC) shows various clinical behaviors, and currently surgical modalities are the only effective therapy against this cancer. To discern the genomic background affecting clinical characteristics such as metastasis, we analyzed the gene expression profiles of the RCCs by DNA microarray using cell lines originating from primary or metastatic lesions. RNA was extracted from ten SKRC RCC cell lines and gene expression profiling was performed using a cDNA microarray of nearly 4,000 human cDNA clones. We compared the gene expression profiles between these SKRC cell lines and the normal renal proximal tubular cell line and among SKRC cell lines that showed different characteristics. The clustering of the selected 62 genes revealed similar profiling patterns between SKRC-17 and SKRC-29 cells that were derived from metastatic RCC lesions. Another cell line from a metastatic lesion, SKRC-52, with a unique spindle-shaped morphology, had a different pattern of expression profiling from the other cell lines. We found several genes up-regulated in the cell lines from metastatic lesions; transgelin, which is reportedly involved in cell proliferation and migration, was up-regulated in SKRC-52. The unique profiling pattern of gene expression clearly correlated with cell morphology and metastatic potential. Such profiling might contribute to identifying the genes involved in the clinical characteristics of RCC.

Gene expression profiling identifies platelet-derived growth factor as a diagnostic molecular marker for papillary thyroid carcinoma.

PURPOSE: Cancer diagnostics and therapeutics are often based on clinically relevant markers that are expressed specifically in a malignant tissue at levels higher than in normal tissue. We examined potential markers for papillary thyroid carcinoma (PTC) by monitoring PTC-specific gene expression using cDNA microarray. EXPERIMENTAL DESIGN: Gene expression profiles for PTC tissue, normal thyroid tissue, and healthy peripheral blood cells were compared by use of a human 4000-gene cDNA microarray. Protein expressions of the up-regulated genes in PTC were examined in thyroid tissues by immunohistochemistry. RESULTS: Sixty-four genes were overexpressed in PTC tissue relative to normal thyroid tissue and healthy peripheral blood cells. The genes that were up-regulated in PTC were involved in cell cycle regulation, DNA damage response, angiogenesis, and oncogenesis. Among these genes, basic fibroblast growth factor and platelet-derived growth factor were identified by immunochemical methods as proteins that are specifically expressed at high levels in thyroid neoplasms. Basic fibroblast growth factor, which has been identified as a biomarker for PTC, was overexpressed in 54% of PTC cases, 67% of follicular thyroid carcinomas, and 36% of benign thyroid neoplasms. Platelet-derived growth factor was overexpressed in 81% of PTC cases and 100% of follicular carcinomas, but was immunonegative in normal thyroid tissues and benign thyroid neoplasms. CONCLUSIONS: Platelet-derived growth factor may be a potential biomarker for PTC and follicular carcinoma. Expression profile analysis using a microarray followed by immunohistochemical study can be used to facilitate the development of molecular biomarkers for cancer.

Overexpression of poly(ADP-ribose) polymerase disrupts organization of cytoskeletal F-actin and tissue polarity in Drosophila.

Poly(ADP-ribose) polymerase (PARP) may play important roles in nuclear events such as cell cycle, cell proliferation, and maintenance of chromosomal stability. However, the exact biological role played by PARP or how PARP is involved in these cellular functions is still unclear. To elucidate the biological functions of PARP in vivo, we have constructed transgenic flies that overexpress Drosophila PARP in the developing eye primordia. These flies showed mild roughening of the normally smooth ommatidial lattice and tissue polarity disruption caused by improper rotation and chirality of the ommatidia. To clarify how this phenotypical change was induced, here we analyzed transgenic flies overexpressing PARP in the developing eye, embryo, and adult in detail. PARP mRNA level and the phenotype were enhanced in flies carrying more copies of the transgene. Developing eyes from third instar larvae were analyzed by using the neural cell marker to examine the involvement of PARP in cell fate. Morphological disorder of non-neuronal accessory cells was observed in PARP transgenic flies. Interestingly, overexpression of PARP did not interfere with the cell cycle or apoptosis, but it did disrupt the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupted organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Because these phenotypes closely resembled mutants or transgenic flies of the tissue polarity genes, genetic interaction of PARP with known tissue polarity genes was examined. Transgenic flies expressing either PARP or RhoA GTPase in the eye were crossed, and co-expression of PARP suppressed the effect of RhoA GTPase. Our results indicate that PARP may play a role in cytoskeletal or cytoplasmic events in developmental processes of Drosophila.