Glucose and glutamine metabolism in oral squamous cell carcinoma: insight from a quantitative metabolomic approach.

OBJECTIVE: To characterize the metabolic system of oral squamous cell carcinoma (OSCC) by metabolome analysis. STUDY DESIGN: The metabolome profiles, including the Embden-Meyerhof-Parnas pathway (EMPP), the pentose phosphate pathway, the tricarboxylic acid cycle (TCAC), and amino acids, were obtained from OSCC and its surrounding normal tissues (32 patients) using capillary electrophoresis and a time-of-flight mass spectrometer. RESULTS: Enhancement of glucose consumption and lactate production (Warburg effect) was observed in OSCC tissues. The decrease of glucose along with the decrease of the downstream intermediates in the EMPP suggests that incorporated glucose is mainly consumed for biosynthesis. Glutamine consumption with the increase of the intermediates in the last half of the TCAC suggests the involvement of glutaminolysis, in which glutamine is converted to lactate via the last half of the TCAC. CONCLUSIONS: It is suggested that OSCC tissues show the Warburg effect, which stems from the combined enhancement of glucose consumption and glutaminolysis.

Immunohistochemical assessment of ATG7, LC3, and p62 in ameloblastomas.

BACKGROUND: To investigate the roles of autophagy in tumorigenesis, cytodifferentiation, and prognosis of odontogenic tumors, we analyzed the immunohistochemical expression of ATG7, LC3, and p62 in odontogenic tissues. METHODS: Tissue specimens of nine dental follicles and 69 ameloblastomas were immunohistochemically examined with antibodies against ATG7, LC3, and p62. RESULTS: Immunohistochemical reactivity for ATG7, LC3, and p62 was detected in many odontogenic epithelial cells and several endothelial cells and fibroblasts in dental follicles and ameloblastomas. ATG7 reactivity in ameloblatomas was significantly higher than that in dental follicles. Expression of ATG7, LC3, and p62 was found markedly in neoplastic cells near the basement membrane rather than central polyhedral cells in ameloblastomas. Reactivity for these molecules was significantly higher in unicystic ameloblastomas than in solid ameloblastomas. Granular cells in granular cell ameloblastomas showed obvious reactivity for the autophagy- related molecules, and LC3 reactivity in granular cell ameloblastomas was significantly higher than in other ameloblastoma variations. Recurrent ameloblastomas showed significantly lower reactivity of LC3 and p62 than primary ameloblastomas. CONCLUSIONS: Expression of ATG7, LC3, and p62 in dental follicles and ameloblastomas suggests that autophagy regulation might be affected by microenvironment alterations during tumorigenesis. The molecular machinery for autophagy is possibly involved in tissue architecture, neoplastic cell differentiation, and prognosis of the benign epithelial odontogenic tumor.

Targeting of tumor endothelial cells combining 2 Gy/day of X-ray with Everolimus is the effective modality for overcoming clinically relevant radioresistant tumors.

Radiotherapy is widely used to treat cancer because it has the advantage of physically and functionally conserving the affected organ. To improve radiotherapy and investigate the molecular mechanisms of cellular radioresistance, we established a clinically relevant radioresistant (CRR) cell line, SAS-R, from SAS cells. SAS-R cells continue to proliferate when exposed to fractionated radiation (FR) of 2 Gy/day for more than 30 days in vitro. A xenograft tumor model of SAS-R was also resistant to 2 Gy/day of X-rays for 30 days. The density of blood vessels in SAS-R tumors was higher than in SAS tumors. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, sensitized microvascular endothelial cells to radiation, but failed to radiosensitize SAS and SAS-R cells in vitro. Everolimus with FR markedly reduced SAS and SAS-R tumor volumes. Additionally, the apoptosis of endothelial cells (ECs) increased in SAS-R tumor tissues when both Everolimus and radiation were administered. Both CD34-positive and tomato lectin-positive blood vessel densities in SAS-R tumor tissues decreased remarkably after the Everolimus and radiation treatment. Everolimus-induced apoptosis of vascular ECs in response to radiation was also followed by thrombus formation that leads to tumor necrosis. We conclude that FR combined with Everolimus may be an effective modality to overcome radioresistant tumors via targeting tumor ECs.

The regenerated bone quality by implantation of octacalcium phosphate collagen composites in a canine alveolar cleft model.

OBJECTIVE: Synthetic octacalcium phosphate and porcine atelocollagen composites significantly enhanced bone regeneration more than ß-tricalcium phosphate collagen composite and hydroxyapatite collagen composite in a rat cranial defect model. However, the long-term stability and quality of octacalcium phosphate collagen (OCP/Col) composites-derived regenerated bone, when implanted in a canine alveolar cleft model, have yet to be elucidated. The present study investigated the longterm stability and quality of bone regenerated by OCP/Col. DESIGN: Disks of OCP/Col or collagen were implanted in a canine alveolar-cleft model (n = 6). Then, bone regeneration in the implanted areas was investigated macroscopically, radiographically, and histologically at 10 months after implantation. In addition, three-dimensional quantitative images of regenerated bone were analyzed by microcomputed tomography. RESULTS: Macroscopically, the OCP/Col treated alveolus was clearly augmented, and radio-opacity in the OCP/Col implanted area was comparable to that of the original alveolus bone. On histological analysis, the area was mostly filled with newly formed bone, and a few granules of implanted OCP/Col were enclosed in it. In the microcomputed tomography analysis, the regenerated bone volume in the OCP/Col group was larger than that in the collagen group. OCP/Col-derived bone consisted of outer cortical and inner cancellous structure with dense trabeculae and seemed like the original bone structure. CONCLUSIONS: OCP/Co composites could be a useful bone regenerative material to substitute for autogenous bone because their implantation could elicit high bone regeneration and active structural reconstitution.

First clinical application of octacalcium phosphate collagen composite in human bone defect.

We have previously demonstrated that octacalcium phosphate (OCP) collagen composite (OCP/collagen) promotes bone regeneration in a critical-sized bone defect of a rodent or canine model. This study was designed to investigate the bone regeneration of OCP/collagen in human bone defect as a first clinical trial. Two patients who had a radicular cyst or apical periodontitis consented to participate in our clinical study, and OCP/collagen was implanted into the defects after operation. Radiographic examination showed effective bone healing in each bone defect at 3 or 6 months. Likewise, computed tomography value significantly increased after implantation. Postoperative wound healing was uneventful, and neither infection nor allergic reaction against OCP/collagen was observed for the entire period. This study demonstrated that OCP/collagen would be safely used and enhanced bone regeneration in human bone defects. To reinforce the efficacy of OCP/collagen as a bone substitute material, it should be compared with other suitable comparators in the future.

Comparison of bone regeneration between octacalcium phosphate/collagen composite and ß-tricalcium phosphate in canine calvarial defect.

OBJECTIVE: The present study evaluated the efficacy of bone regeneration between synthetic octacalcium phosphate (OCP) granules combined with porcine atelocollagen (OCP/Col) and ß-tricalcium phosphate (ß-TCP). STUDY DESIGN: A disk of OCP/Col (20 mm diameter, 2.5 mm thick) or commercially available sintered porous ß-TCP was implanted into a critical-sized calvarial defect (20 mm diameter) of adult male canines (n = 10). The newly formed bone in the defect was analyzed radiographically, crystallographically, histologically, and histomorphometrically at 6 months after implantation. RESULTS: Histomorphometry showed that there was significantly more newly formed bone in OCP/Col-treated defects than for ß-TCP (P < .05). X-Ray diffraction patterns of implanted OCP/Col were similar to those of original bone and different from those of implanted ß-TCP. CONCLUSIONS: These results suggest that OCP/Col implantation in canine critical-sized defect enhanced bone regeneration more than ß-TCP, which is the most commonly used synthetic bone substitutes.

Unilateral atrophy of the masticatory muscles and mandibular ramus due to pure trigeminal motor neuropathy: a case report.

Pure trigeminal motor neuropathy is a very unusual disease that is characterized by trigeminal motor paralysis without trigeminal sensory disturbances and without the involvement of the other cranial nerves. We report a case of pure trigeminal motor neuropathy in a 70-year-old woman. The diagnosis was based on the results of clinical, electromyographic, and radiologic examinations. Only the motor branch of the left trigeminal nerve was damaged. Atrophy of the left-side masticatory muscles and jaw resulted in facial asymmetry. Magnetic resonance imaging (MRI) of the head and face did not detect any pathologic lesion, with the exception of atrophy and fatty infiltration of the muscles innervated by the left trigeminal motor nerve. The etiology of the patient's pure trigeminal motor neuropathy was undetermined. Patients with suspected trigeminal motor neuropathy should undergo MRI of the head and face to evaluate the sequelae of denervation and to detect an intracranial or extracranial lesion.

Granule size-dependent bone regenerative capacity of octacalcium phosphate in collagen matrix.

The present study was designed to determine whether the osteoconductivity of octacalcium phosphate-collagen (OCP/Col) composite can be improved by controlling the granule size of OCP. The granules of synthetic OCP, with diameters in the range of 53 to 300, 300 to 500, and 500 to 1000 µm, were used as an inorganic source of composite materials mixed with atelo-Col. After vacuum dehydrothemal treatment, OCP/Col disks were implanted into critical-sized calvaria defects in Wistar rats for 4, 8, and 12 weeks and examined radiographically, histologically, histomorphometrically, and histochemically. The materials were characterized according to mercury intrusion porosimetry and scanning electron microscopy. X-ray diffraction was performed before and after implantation. The dissolution of OCP crystals in a Col matrix was determined by immersing OCP/Col disks in a culture medium. OCP/Col had a constant pore size (~30 µm) regardless of OCP granule size. OCP in the Col matrix tended to convert to hydroxyapatite (HA) during the implantation. OCP/Col with the smallest granules of OCP enhances both bone regeneration and biodegradation the most through tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cellular resorption of OCP granules. The smallest OCP granules in the Col matrix showed the highest dissolution and had the greatest potential to form HA. The results indicated that the size of the included OCP granules can controll the osteoconductivity of OCP/Col. The overall results suggest that the physicochemical property of OCP crystals is a factor that determines the bone regenerative capacity of OCP/Col in critical-sized calvaria large bone defects in rats.

Involvement of sensory neurons in bone defect repair in rats.

We investigated bone repair in sensory-denervated rats, compared with controls, to elucidate the involvement of sensory neurons. Nine-week-old male Wistar rats received subcutaneous injections of capsaicin to denervate sensory neurons. Rats treated with the same amount of vehicle served as controls. A standardized bone defect was created on the parietal bone. We measured the amount of repaired bone with quantitative radiographic analysis and the mRNA expressions of osteocalcin and cathepsin K with real-time polymerase chain reaction (PCR). Quantitative radiographic analysis showed that the standard deviations and coefficients of variation for the amount of repaired bone were much higher in the capsaicin-treated group than in the control group at any time point, which means that larger individual differences in the amount of repaired bone were found in capsaicin-treated rats than controls. Furthermore, radiographs showed radiolucency in pre-existing bone surrounding the standardized defect only in the capsaicin-treated group, and histological observation demonstrated some multinuclear cells corresponding to the radiolucent area. Real-time PCR indicated that there was no significant difference in the mRNA expression levels of osteocalcin and cathepsin K between the control group and the capsaicin-treated group. These results suggest that capsaicin-induced sensory denervation affects the bone defect repair.

The effect of synthetic octacalcium phosphate in a collagen scaffold on the osteogenicity of mesenchymal stem cells.

Although the efficacy of the in vivo osteogenic capabilities of synthetic octacalcium phosphate (OCP) crystal implantation can be explained through its stimulatory capacity for the differentiation of the host osteoblastic cell lineage, direct evidence that OCP supports bone regeneration by osteogenic cells in vivo has not been shown. Mesenchymal stem cells (MSCs) isolated from 4-week-old male Wistar rat long bones were pre-incubated in osteogenic or maintenance medium in the presence or absence of basic fibroblast growth factor (bFGF). OCP/Collagen (OCP/Col) or collagen disks were seeded with MSCs that had been pre-incubated in osteogenic medium containing bFGF, which exhibited the highest differentiation induction, and then incubated for an additional day. The disks were implanted in critical-sized calvaria defects of 12-week-old male Wistar rats and the specimens were analysed radiographically, histologically, histomorphometrically, and by micro-computed tomography (CT) imaging at 4 and 8 weeks after the implantation. The OCP/Col·MSCs group rapidly induced more bone regeneration, even within 4 weeks, compared to the OCP/Col group without MSCs. The bone mineral density of the OCP/Col·MSCs group was also greater than the OCP/Col group. The Col·MSCs group did not exhibit prominent osteogenicity. These results indicate that OCP crystals in a collagen matrix efficiently promote exogenously introduced osteogenic cells to initiate bone regeneration if the cells are pre-treated in a suitable differentiation condition.

Reconstruction of critical-sized bone defect in dog skull by octacalcium phosphate combined with collagen.

PURPOSE: The present study was designed to investigate whether synthetic octacalcium phosphate (OCP) combined with collagen (OCP/collagen) can repair a critical-sized defect in dog skull. OCP/collagen has been shown to biodegrade and to tend to be replaced by newly formed bone if implanted in rat calvaria defects. MATERIALS AND METHODS: An OCP/collagen disk was prepared from pepsin-digested atelocollagen isolated from porcine dermis and synthetic OCP. Two critical-sized defects (20 mm in diameter) were made in a dog skull. Ten disks of OCP/collagen or collagen (control) were implanted in the bone defects and resected with surrounding tissues at 3, 6, or 12 months after the implantation. The specimens were analyzed radiographically, crystallographically, histologically, and histomorphometrically. RESULTS: X-ray diffraction and FTIR analyses showed that OCP tended to convert to a poorly crystallized hydroxyapatite, similar to that of biological apatite, by 3 months. Radiographic and histologic analyses showed that the implantation of OCP/collagen disks initiated new bone formation in the defects at 3 months after implantation. However, there was no promotion of bone formation by control collagen disks even with prolonged implantation up to 12 months. Histomorphometric analysis revealed that the percentage of newly formed bone in the defect implanted with OCP/collagen increased significantly, from 30.91 ± 6.65 at 3 months to 51.22 ± 5.99 at 12 months, although the value tended to reach a plateau at 6 months (44.49 ± 3.34). On the other hand, the percentage of remaining OCP was estimated at approximately 10% at 3 months and remained nearly unchanged thereafter. CONCLUSION: The results suggest that bone regeneration of a critical-sized bone defect of dog calvaria by OCP/collagen can be enhanced for 3 to 6 months and that OCP/collagen holds potential as a bone substitute material.

Rapamycin suppresses ROS-dependent apoptosis caused by selenomethionine in A549 lung carcinoma cells.

PURPOSE: Although selenium compounds possess chemotherapeutic features by inducing apoptosis in cancer cells with trivial side effects on normal cells, the mechanisms underlying its anti-cancer activity are insufficiently understood at the present. In this study, we investigated the effects of rapamycin on apoptosis induced by seleno-L-methionine (SeMet) or selenite in A549 cells. METHODS: The effects of Se compounds, SeMet and selenite, on cell proliferation, apoptosis and its signaling pathway were investigated in established human adenocarcinoma cell line (A549). Cancer cells were treated with each Se during different periods. Cell apoptosis and signaling molecules were analyzed by flow cytometry (TUNEL method) or immunoblotting, respectively. RESULTS: SeMet induces reactive oxygen species generation associated with the induction of apoptosis, because pretreatment of cells with N-acetyl-L-cysteine completely blocked SeMet-induced apoptosis. We also found that rapamycin completely suppressed the apoptosis of cells treated by SeMet, but not selenite. SeMet-induced apoptosis is significantly downregulated in combination with PI3 K family inhibitors (LY294002, wortmannin, PI-103, and 3-methyladenine). In addition, ROS generation was included in downstream signaling events associated with the phosphorylation of mTOR, because pretreatment of cells with rapamycin inhibited ROS generation. CONCLUSION: These results suggest that SeMet-induced apoptosis is affected by the Akt/mTOR/ROS pathway in A549 cells. Akt serves an anti-survival function in the system of SeMet-treated lung cancer cells, but autophagic signaling remained unsolved.

Effects of mevastatin on grafted bone in MRL/MpJ mice.

Statins, lipid-lowering drugs, have been reported to influence bone metabolism. However, the available information about their effects on bone formation and resorption in vivo is still limited. The present study was undertaken to determine whether the topical administration of mevastatin could increase the bone mass of isografted bone. The tibiae were bilaterally dissected from a donor MRL/MpJ mouse and transplanted subcutaneously in the dorsal region of a recipient mouse. One grafted tibia was topically infused for either 1, 2, 3, or 4 weeks with mevastatin, using an osmotic minipump at a dose of 2.5 pmol/hr. The other tibia was infused with 0.9% NaCl (control). Our three results were: (1) Topical mevastatin stimulated bone formation and numerous cuboidal osteoblasts appeared on the surface of newly formed bone. Bone mineral density and bone area in mevastatin-treated bone were significantly increased. (2) Topical mevastatin increased the number of osteoclasts. (3) The expression of bone morphogenetic protein-2 (BMP-2) mRNA and receptor activator of NF-kB ligand (RANKL) mRNA were upregulated in mevastatin-treated bone. These results suggest that the topical infusion of mevastatin increases bone mass of isografted bone by increasing bone turnover and, at least in part, by promoting the expression of BMP-2 and RANKL mRNA.

Differential apoptotic response of human cancer cells to organoselenium compounds.

PURPOSE: Selenium (Se) compounds are well known to inhibit cell proliferation and induce cell death in human cancer cells. Respective chemical forms of Se are intracellularly metabolized via complicated pathways, which target distinct molecules and exhibit varying degrees of anti-carcinogenicity in different cancer types; however, the precise mechanisms by which Se activates apoptosis remain poorly understood. METHODS: The effects of Se compounds, Se-methylselenocysteine (MSC), selenomethionine (SeMet), and selenite on cell proliferation, apoptosis and its pathway in established human carcinoma cell lines (HSC-3, -4, A549, and MCF-7) were investigated. Cancer cells were treated with each Se compound during different periods. Cell apoptosis, caspase activity and ER stress markers were analyzed by flow cytometric or immunoblotting analysis, respectively. RESULTS: We examined four cell lines for their sensitivity to MSC and SeMet in comparison with selenite. SeMet increased apoptotic cells in p53-positive A549 cells, whereas MSC increased apoptotic cells in p53-mutated HSC-3 cells. High activities of caspase-3, -8 and -9 were observed during apoptosis, and a pan-caspase inhibitor, z-VAD-fmk, rescued the cell viability of HSC-3 cells exposed to MSC. In addition, the occurrence of endoplasmic reticulum (ER) stress was suggested by the observation that levels of phosphorylated eIF2alpha and caspase-12 activity are increased in Se-treated cells. Selenite and MSC were accompanied with the concurrent reduction of phosphorylated Akt levels, and the inhibitory effects of these Se compounds on vascular endothelial growth factor expression were observed with identical patterns. CONCLUSION: The present findings demonstrate that Se-induced apoptosis in carcinoma cells is basically a caspase-dependent process involving complicated mechanisms. Activation of both the intrinsic apoptotic pathway and ER stress pathway plays a major and concurrent role, while p53 activation seems to have only a functional role in SeMet.

Mechanical stress-related calvaria bone augmentation by onlayed octacalcium phosphate-collagen implant.

Previous studies have suggested that the biodegradability of octacalcium phosphate-collagen (OCP/Col) composite by osteoclasts is accelerated in association with mechanical stress suffered by the host tissue around the implant. The present study was designed to investigate whether alleviation of mechanical stress restores the bone regenerative properties of OCP/Col, as previously shown in nonload-bearing sites. OCP/Col discs supported with a polytetrafluoroethylene (PTFE) ring, which has a higher modulus than OCP/Col, were implanted in a rat subperiosteal pocket for up to 12 weeks. The structural features of the implant and biological responses were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, histomorphometry, histochemistry, and tissue mRNA expression around the implants. The effect of compression was analyzed using mouse stromal ST-2 cells by deforming the cell-seeded OCP/Col discs in vitro with or without a PTFE ring. The results clearly indicated the restoration of bone formation by the alleviation of mechanical stress and the upregulation of osteoblast-related genes, such as osterix on the other hand, the implantation of OCP/Col on calvaria or in an in vitro test without PTFE support resulted in the upregulation of osteoclast-related genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, in the tissues or receptor activator of the nuclear factor-kappaB ligand (RANKL) in ST-2 cells. The results confirmed that calvaria augmentation is enhanced by implanting OCP/Col if suitable conditions regarding mechanical stress are provided.

A mechanism for the suppression of interleukin-1-induced nuclear factor kappaB activation by protein phosphatase 2Ceta-2.

IL-1 (interleukin-1) is a pro-inflammatory cytokine that has a variety of effects during the process of inflammation. Stimulating cells with IL-1 initiates a signalling cascade that includes the activation of NF-kappaB (nuclear factor kappaB), and subsequently induces a variety of inflammatory genes. Although the molecular mechanism for the IL-1-induced activation of NF-kappaB has been well documented, much less is known about the mechanism by which protein phosphatases down-regulate this pathway. Here we show that mouse PP2Ceta-2 (protein serine/threonine phosphatase 2Ceta-2), a novel member of the protein serine/threonine phosphatase 2C family, inhibits the IL-1-NF-kappaB signalling pathway. Ectopic expression of PP2Ceta-2 in human embryonic kidney HEK293IL-1RI cells inhibited the IL-1-induced activation of NF-kappaB. TAK1 (transforming-growth-factor-beta-activated kinase 1) mediates the IL-1 signalling pathway to NF-kappaB, and we observed that the TAK1-induced activation of NF-kappaB was suppressed by PP2Ceta-2 expression. Expression of IKKbeta [IkappaB (inhibitory kappaB) kinase beta], which lies downstream of TAK1, activates NF-kappaB, and this activation was also readily reversed by PP2Ceta-2 co-expression. Additionally, PP2Ceta-2 knockdown with small interfering RNA further stimulated the IL-1-enhanced phosphorylation of IKKbeta and destabilization of IkappaBalpha in HeLa cells. PP2Ceta-2 knockdown also increased the IL-1-induced expression of IL-6 mRNA. Furthermore, IKKbeta was readily dephosphorylated by PP2Ceta-2 in vitro. These results suggest that PP2Ceta-2 inhibits the IL-1-NF-kappaB signalling pathway by selectively dephosphorylating IKKbeta.

Enhancement of cisplatin cytotoxicity by SAHA involves endoplasmic reticulum stress-mediated apoptosis in oral squamous cell carcinoma cells.

PURPOSE: The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances cisplatin [cis-diammine dichloroplatinum (II)] (CDDP)-induced apoptosis in the oral squamous cell carcinoma (OSCC) cell line by complex, multifunctional mechanisms. We investigated the role of endoplasmic reticulum (ER) stress in the enhancing effect of SAHA on CDDP, compared with the ER stressor thapsigargin. METHODS: We chose OSCC cell line HSC-3 to ascertain the mechanism of SAHA-enhanced cytotoxicity among various cell lines. HSC-3 cells were incubated with CDDP/SAHA for 48 h, followed by the assessment of cell chemosensitivity to CDDP with MTT and TUNEL assays. Western blot analysis was used to detect the expressions of ER-related molecules, and flow cytometry was used to monitor caspase activity. RESULTS: Treatment with CDDP/SAHA potently induced apoptosis in HSC-3 cells with a significant increase in caspase-4 and -12 functions. For example, 60% of cells became apoptotic after 48 h of treatment with CDDP/SAHA. In addition, SAHA alone rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)alpha, which is up-regulated during ER stress. Inhibition of ER stress by salubrinal, an inhibitor of eIF2alpha dephosphorylation, abrogated SAHA's enhancement of CDDP cytotoxicity. Levels of phospho-Akt are decreased in SAHA-treated cells, and this is in turn associated with increased activity of protein phosphatase 1 (PP1) by SAHA, the phosphatase upstream of Akt. CONCLUSION: These data indicate that up-regulation of specific-ER stress-associated events is an integral part of the mechanism by which SAHA enhances CDDP-induced apoptosis, and PP1 up-regulation followed by Akt dephosphorylation plays an important role in SAHA-enhanced CDDP apoptosis.

Zebularine suppresses the apoptotic potential of 5-fluorouracil via cAMP/PKA/CREB pathway against human oral squamous cell carcinoma cells.

PURPOSE: During tumorigenesis, tumor suppressor and tumor-related genes are commonly silenced by aberrant DNA methylation in their promoter regions, which is one of the important determinants of susceptibility to 5-fluorouracil (5-FU) in oral squamous cell carcinoma (OSCC) cells. Here, we examine the chemotherapeutic efficacy of epigenetic agents on 5-FU cytotoxicity. METHOD: We investigated the effect of a DNA methyltransferase (DNMT) inhibitor, zebularine (Zeb), on the chemosensitivity of 5-FU and cisplatin (CDDP) by MTT and TUNEL methods, and compared the molecular mechanism of action with those of a GSK3beta inhibitor, LiCl, and an Hsp90 inhibitor, 17-AAG. RESULTS: A significant apoptotic effect by a combination of Zeb or 17-AAG was found in CDDP treatment; however, considerable suppression of 5-FU-induced apoptosis was observed after incubation with Zeb, 17-AAG, or LiCl. Zeb's suppressive effects were associated with activation of the cAMP/PKA/CREB pathway, differing from mechanisms of 17-AAG and LiCl. Suppression of 5-FU-induced apoptosis by Zeb was not associated with increased Bcl-2 and Bcl-xL expressions dependent on transcription factor CREB, and with the expression level of thymidylate synthase. CONCLUSIONS: In the present study, we identified a more detailed mechanism of action by which Zeb suppresses 5-FU-induced apoptosis. These results indicate that combination therapies have to be carefully investigated due to potential harmful effects in the clinical application of DNMT inhibitors.

Synthetic octacalcium phosphate augments bone regeneration correlated with its content in collagen scaffold.

Previous studies have shown that synthetic octacalcium phosphate (OCP) facilitates in vitro osteoblastic cell differentiation in an OCP dose-dependent manner and that a complex of OCP and collagen (OCP/collagen) enhances critical-sized rat calvaria defects more than OCP alone. The present study was designed to investigate whether the bone regenerative properties of OCP/collagen are augmented in an OCP dose-dependent manner, thereby establishing a suitable composition of this composite as a bone substitute material. OCP/collagens with a wide range of mixing ratios from 23:77 to 83:17, including the previously examined composition (77:23), were prepared by blending granules of OCP with atelocollagen and molded into a disk as an implant. A critical-sized defect was made in rat calvaria, and each disk was implanted into the defect for 4 or 12 weeks and then examined radiographically, histologically, and histomorphometrically. Mouse bone marrow-derived stromal ST-2 cells were cultured in dishes pre-coated with OCP/collagen or OCP alone with different OCP contents to determine the capacity of cell attachment and proliferation up to 14 days. Histological and radiographic examinations showed that newly formed bone was observed in relation to OCP granules within the collagen matrix. Histomorphometric analysis confirmed that increasing the amount of OCP in collagen matrices resulted in progressive enhancement of bone regeneration and that the ratio 83:17 generated the maximum repair level of approximately 64% of the defect at 12 weeks. OCP/collagen promoted the proliferation and attachment of ST-2 cells more than OCP alone regardless of OCP content. Fourier transform infrared spectroscopy analysis of the coatings after the incubation indicated that OCP tended to convert to apatite regardless of the presence of collagen. The present study demonstrated that the osteoconductive characteristics of OCP/collagen can be displayed in an OCP dose-dependent manner. The results suggest that collagen promotes the proliferation and attachment of host osteoblastic cells on OCP/collagen composite implants.