Reactivation of p53 by RITA Induces Apoptosis in Human Oral Squamous Cell Carcinoma Cells.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is one of the most common tumors of the head and neck region. The tumor suppressor gene p53 (TP53) is the most frequently mutated gene in OSCC and TP53 mutations are associated with decreased survival and resistance to chemotherapy in patients with OSCC. Therefore, therapeutic strategies targeting TP53 reactivation are required to effectively treat OSCC. In this study, we investigated the effect of various p53-reactivating small molecules (RITA, PRIMA-1, and CP-31398) on the proliferation of human OSCC cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) derived from human oral tissues bearing a mutant TP53 gene. MATERIALS AND METHODS: Apoptosis induction by RITA was assessed by measuring Annexin V and propidium iodide (PI)-positive cells using flow cytometry. p53 and murine double minute 2 (MDM2) phosphorylation and Bax expression were detected in the lysates of RITA-treated Ca9-22 cells using western blotting. RESULTS: RITA markedly inhibited the growth of Ca9-22, HSC-2, HSC-3, and HSC-4 cells. In Ca9-22 cells, RITA induced apoptosis and inhibited cell proliferation while increasing p53 phosphorylation and Bax expression; however, RITA did not induce MDM2 phosphorylation. CONCLUSION: The inhibitory effect of RITA on human OSCC cell proliferation is mediated by apoptosis induction through p53 and Bax.

Comparing the Efficacy of a Microperforated Titanium Membrane for Guided Bone Regeneration with an Existing Mesh Retainer in Dog Mandibles.

Acute-type lateral ridge defects (25 mm × 6 mm × 5 mm) were bilaterally created in the mandibles of four dogs (two defects per animal). The defects were reconstructed with particulate autologous bone and covered with a microperforated titanium membrane (Ti-honeycomb membrane, TiHM) or an existing conventional titanium mesh as control. The samples were dissected after 16 weeks postoperatively and processed for radiographic, histologic, and histomorphometric analyses. Regenerated tissue and bone volume were significantly larger in the TiHM group than in the control group (p = 0.05; p = 0.049). In contrast, bone mineral density was similar between the two groups. Histomorphometric analysis revealed that the regenerated bone area and calcific osseous area were larger in the TiHM group than in the control group; however, the differences were not significant. The efficacy of TiHM was generally satisfactory with the potential to become a standard tool for the GBR procedure; however, early membrane exposure will be a major problem to overcome.

Anti-VEGFR therapy is one of the healing inhibitors of antiresorptive-related osteonecrosis of the jaw.

INTRODUCTION: Antiresorptive-related osteonecrosis of the jaw (ARONJ) is a rare but serious adverse event associated with bone-modifying agents (BMAs) and affects patients in the terminal stages of cancer. Molecular targeting drugs (MTDs), anti-vascular endothelial growth factor receptor (VEGFR), and anti-epidermal growth factor receptor (EGFR) drugs are essential in various cancer treatments, although MTDs are risk factors for ARONJ. However, the mechanism through which MTDs affect treatment prognosis of ARONJ remains unclear. Therefore, we investigated the potential inhibitory factors for healing in the conservative therapy of ARONJ with a focus on MTDs. MATERIALS AND METHODS: Sixty patients who were administered BMAs for the treatment of malignancies and who underwent conservative treatment for ARONJ were assessed. The healing rate of ARONJ for each risk factor was retrospectively evaluated. RESULTS: Among the 60 patients, 27 were male and 33 were female. The median age was 67 years, and the median follow-up period was 292 (range 91-1758) days. The healing rate was lower in those treated with both zoledronic acid (Za) and denosumab (Dmab) than in those treated with Za or Dmab alone (0% vs. 28.8%, p = 0.03). Regarding the administration of MTDs, the treatment rate with anti-VEGFR drugs was 7.1% (p = 0.04), anti-EGFR drugs was 12.5% (p = 0.18), and without MTDs was 36.8%. CONCLUSION: In the conservative treatment of ARONJ, the administration of several BMAs and anti-VEGFR drugs was the factor contributing to the inhibition of healing.

Evaluation of a Newly Designed Microperforated Titanium Membrane with Beta-Tricalcium Phosphate for Guided Bone Regeneration in Dog Mandibles.

PURPOSE: This study evaluated the efficacy of newly designed, laser-perforated pure titanium membranes for guided bone regeneration using beta-tricalcium phosphate (ß-TCP), and compared them with the existing membrane. MATERIALS AND METHODS: Bilateral acute lateral ridge defects were created in the mandibles of 12 dogs (four defects per animal), which were then randomly divided into two groups (six dogs each). The twenty-four bone defects in each group were then further divided into five groups. The groups were as follows: (1) F001M0, a prototype membrane without a frame plus ß-TCP (n = 5); (2) F001M1, a prototype membrane with a frame plus ß-TCP (n = 5); (3) FBS, an existing control membrane plus ß-TCP (n = 5); (4) control 1, ß-TCP without membrane and with covering flap only (n = 5); and (5) control 2, no treatment (no ß-TCP and no membrane) (n = 4). In all groups where ß-TCP was used, it was mixed with peripheral blood. The animals were necropsied at 6 or 12 weeks postoperatively (six dogs each), and samples were collected and processed for radiographic, histologic, and histomorphometric analyses. RESULTS: Among the three membrane groups, regenerated tissue and bone volume was greatest in the F001M1 group at both 6 and 12 weeks postoperatively, although differences among groups were not statistically significant. Bone mineral density was similar among the membrane groups. Histologic analysis revealed that immature fibroblasts were present on the laser-perforated portion at 6 weeks, which induced vascularization. In addition, more calcified bone was replaced beneath the prototypes than beneath the FBS membrane at 12 weeks. Histomorphometric analyses revealed that the calcific osseous areas at 12 weeks after surgery were significantly greater in the F001M1 and F001M0 groups than in the FBS group (P = .021, P = .032). Furthermore, the fibrous tissue areas beneath the membrane at 12 weeks postoperatively were significantly smaller in the prototype groups than in the FBS group (P = .02, P = .02). CONCLUSION: The efficacies of both prototype membranes were not inferior to that of the FBS membrane, indicating that they may facilitate bone regeneration and maturation when ß-TCP mixed with autologous blood is employed.

Evaluation of a Newly Designed Microperforated Pure Titanium Membrane for Guided Bone Regeneration.

PURPOSE: This study aimed to evaluate the safety and efficacy of newly designed, laser-perforated pure titanium membranes for guided bone regeneration and to compare them with an existing product, the FRIOS BoneShield (FBS). MATERIALS AND METHODS: Acute-type lateral ridge defects were bilaterally created in the mandibles of 13 dogs (two defects per animal). The defects were randomly divided into three groups and were reconstructed with particulate autologous bone (PAB) in combination with three different titanium membranes: (1) F001M0 (prototype without a frame), (2) F001M1 (prototype with a frame), and (3) FBS as a standard membrane. All animals were observed periodically and sacrificed 26 or 27 weeks postoperatively. At 26 weeks, approximately half of the dogs in each group underwent membrane removal to examine the postoperative condition of the titanium membranes. The samples were dissected and processed for radiographic, histologic, and histomorphometric analyses. RESULTS: Membrane exposure was not found in the F001M0 or F001M1 groups, and their membranes were removed easily without adhesion to the surrounding tissue. Regenerated bone tissue volume was largest in the F001M1 group, followed by the F001M0 and FBS groups. A significant difference was observed only between the F001M1 and FBS groups (P = .047). In contrast, bone mineral density was similar among the three groups. Histologically, a layer of fibrous tissue was present underneath the titanium membrane, overlying the regenerated cortical bone in all the groups. Notably, the tissue was highly vascular in the F001M1 and F001M0 groups compared with the FBS group. In addition, there was little difference in the semiquantitative soft tissue evaluation and histologic findings of bone regeneration among the three groups. The histomorphometric analysis revealed that the regenerated bone area was larger in the F001M1 and F001M0 groups than in the FBS group, and a significant difference was observed only between the F001M1 and FBS groups (P = .045). Calcific osseous area was similar among the three groups. CONCLUSION: The safety and efficacy of both F001M0 and F001M1 were equivalent to or greater than those of FBS, thereby indicating their potential for future clinical applications.

Temporal and Spatial Site Sharing during Spawning in Snappers Symphorichthys spilurus and Lutjanus bohar (Pisces: Perciformes: Lutjanidae) in Waters around Peleliu Island, Palau.

Jiro Sakaue, Hiroshi Akino, Manabu Endo, Hitoshi Ida, and Takashi Asahida (2016) Two species of Lutjanidae, Symphorichthys spilurus and Lutjanus bohar, form spawning aggregation, a large school specifically formed for reproduction. Although they share the same spawning site at the southernmost reef in Peleliu Island, Palau, timing of spawning and their behaviors in the spawning and resting sites differ. Although the spawning behaviors have reported previously, long term and integrated observations documenting the size of the aggregation, exact spawning duration and timing, detailed behavioral profiles, as well as oceanic conditions upon spawning have never been reported. Here, we conducted a comparative study for these species and found behavioral and environmental cues that might be key to differentiate their ecological characteristics. S. spilurus begun to aggregate at full moon. Aggregations of L. bohar on the other hand, started from four days before full moon. Size of the aggregation was > 50,000 in S. spilurus, but about 7,000 in L. bohar. Both species migrated from the resting area to the spawning site in a diel rhythm. S. spilurus started spawning every half- moon, between the full moon and the new moon, while L. bohar spawns on every full moon. The first spawning took place at around dawn but the time shifted. S. spilurus spawned only when the current directs toward the southeast (offshore flow), while L. bohar spawns only when the current directs toward the southwest (tidal flow). Characteristic swimming behavior was observed for S. spilurus, in that, one or few males that could successfully chase the quick-swimming female fish could fertilize the eggs. In contrast, the behavior of L. bohar, was in a manner typical of several other lutjanid fish. The comparative and long-term field observation conducted over 10 years identified clear differences in the spawning behaviors of S. spilurus and L. bohar. Key behavioral and environmental factors found here might be key determinants for the ecology of these species.

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.

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.

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.