The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma.

Background: Oxidative stress plays key roles in the progression of lung adenocarcinoma. Recently, we reported that peroxiredoxin 4 (PRDX4), an antioxidant enzyme, can be a prognostic marker of lung adenocarcinoma (LUAD). In the present study, we aimed to further investigate the relationship among the PRDX4 expression, epidermal growth factor receptor (EGFR) mutations and cell proliferation in LUAD. Methods: The expression of PRDX4 was immunohistochemically analyzed and the EGFR mutation status was examined in 127 paraffin-embedded human surgical specimens from patients with stage I LUAD. The PRDX4 expression was considered to be high when >40% of the adenocarcinoma cells were positively stained. In vitro, using plasmid transfection methods, PRDX4 plasmid DNAs were transfected into human lung adenocarcinoma cell lines, A549 (EGFR-wild) or PC-9 (EGFR mutant). The viability of these cells was analyzed using a Cell Counting Kit-8 kit. Results: The number of cases with high PRDX4 expression levels among patients with LUAD with EGFR mutations was significantly larger than that in patients with EGFR wild-type. The combination of the PRDX4 expression level with the EGFR mutation status was closely associated with the prognosis of patients with stage I LUAD. Viability assays showed that the proliferation of A549 cells was significantly suppressed after PRDX4 plasmid transfection, while the overexpression of PRDX4 had no effect on the proliferation of EGFR-mutant PC-9 cells. Conclusions: The PRDX4 expression and EGFR mutation status were significantly associated with the prognosis of patients with stage I LUAD, and EGFR mutations affected the role of PRDX4 in the proliferation of LUAD cells.

Corticosterone inhibits the expression of cannabinoid receptor-1 and cannabinoid receptor agonist-induced decrease in cell viability in glioblastoma cells.

The endocannabinoid system regulates physiological and pathological conditions, including inflammation and cancer. Recently, emotional and physical stressors were observed to be involved in impairing the endocannabinoid system, which was concomitant with an increase in serum corticosteroids. However, the influence of corticosteroids on the endocannabinoid system has yet to be completely elucidated. The present study investigated the effects of corticosterone, one of the corticosteroids, on the endocannabinoid system in malignant glioblastoma cells in vitro. U-87 MG cells derived from malignant glioblastoma were subjected to corticosterone stimulation and their viability, signal transduction, and endocannabinoid-related gene expression were examined. Corticosterone decreased the mRNA and protein expressions of cyclooxygenase-2. Of note, although endocannabinoids decreased cell viability, corticosterone inhibited the cannabinoid receptor agonist-induced decrease in cell viability by downregulating the mRNA and protein expressions of cannabinoid receptor 1 (CB1) in glioblastoma cells. These results suggest that corticosteroids modify the endocannabinoid system in glioblastoma cells, and a reduction in the beneficial anti-tumor effects of endocannabinoids through downregulation of the CB1 receptor by corticosterone may promote the malignant phenotype of glioblastoma.

The Association of Peroxiredoxin 4 with the Initiation and Progression of Hepatocellular Carcinoma.

AIMS: Peroxiredoxin 4 (PRDX4) is a member of the peroxiredoxin family of antioxidant enzymes. Previously, we reported that PRDX4 can restrain the initiation and progression of nonalcoholic steatohepatitis by reducing local and systemic reactive oxygen species (ROS) levels. Oxidative stress is recognized as a key factor in hepatocarcinogenesis, and a high ROS level has also been found in hepatocellular carcinoma (HCC). Here, our aim is to investigate roles of PRDX4 in the initiation and progression of HCC. RESULTS: In this study, for hepatocarcinogenesis, wild-type (WT), PRDX4 knockout (PRDX4-/y), and human PRDX4 transgenic (hPRDX4+/+) mice were given a weekly intraperitoneal injection of diethylnitrosamine for 25 weeks. The HCC incidence was higher in PRDX4-/y mice than in WT or hPRDX4+/+ mice. Intrahepatic and circulating oxidative stress and inflammatory cell infiltration in the liver were obviously decreased in hPRDX4+/+ mice, compared with WT mice. Furthermore, in our cohort study, human HCC specimens with low expression of PRDX4 had higher ROS levels and a highly malignant phenotype, which was associated with a reduced overall survival, compared with those with high PRDX4 expression. However, in human HCC cell lines, PRDX4 knockdown led to a rapidly increased intracellular ROS level and suppressed cell proliferation, inducing cell death. Innovation and Conclusion: Our results clearly indicate that PRDX4 has an inhibitory effect in the initiation of HCC, but a dual (inhibitory or promoting) role in the progression of HCC, suggesting the potential utility of PRDX4 activators or inhibitors as therapy for different stages and phenotypes of HCC.

Hypoxia-induced inhibition of the endocannabinoid system in glioblastoma cells.

The endocannabinoid system plays an important role in the regulation of physiological and pathological conditions, including inflammation and cancer. Hypoxia is a fundamental phenomenon for the establishment and maintenance of the microenvironments in various physiological and pathological conditions. However, the influence of hypoxia on the endocannabinoid system is not fully understood. In the present study, we investigated the effects of hypoxia on the endocannabinoid system in malignant brain tumors. We subjected U-87 MG cells, derived from malignant glioblastoma, to hypoxia (1.5% O2) for 3 days, and evaluated their viability and expression of endocannabinoid-related genes. Hypoxia decreased the expression of cannabinoid receptor 1 and the astrocyte marker glial fibrillary acidic protein, and increased the expression of vascular endothelial growth factor and cyclooxygenase-2, the enzyme responsible for the metabolism of endocannabinoids, in U-87 MG cells. Although cannabinoid receptor (CB) engagement induces cell death in U-87 MG cells in normoxic conditions, CB agonist-induced death was attenuated in hypoxic conditions. These results suggest that hypoxia modifies the endocannabinoid system in glioblastoma cells. Hypoxia-induced inhibition of the endocannabinoid system may aid the development of glioblastoma.

Surgical treatment of comminuted mandibular fractures using a low-profile locking mandibular reconstruction plate system.

OBJECTIVE: The treatment of comminuted mandibular fractures is challenging due to the severity of associated injuries and the need for a careful diagnosis with adequate treatment planning. Recently, open reduction and stable internal fixation (OR-IF) with a load-bearing reconstruction plate have been advocated for reliable clinical outcomes with minimal complications. This clinical prospective study evaluated OR-IF in the surgical management of comminuted mandibular fractures with a new low-profile, thin, mandibular locking reconstruction plate. MATERIALS AND METHODS: We prospectively assessed OR-IF of comminuted mandibular fractures with a low-profile locking mandibular reconstruction plate in 12 patients (nine men, three women; mean age 32.2 [range 16-71] years) between April 2010 and December 2011. The clinical characteristics and associated clinical parameters of patients were evaluated over a minimum follow-up period of 12 months. RESULTS: Traffic accidents caused 50% of the fractures, followed by falls (25%). Four patients (33.3%) had associated midfacial maxillofacial fractures, while five patients had other mandibular fractures. Seven patients (58.3%) needed emergency surgery, mostly for airway management. Anatomical reduction of the comminuted segments re-established the mandibular skeleton in stable occlusion with rigid IF via extraoral (33.3%), intraoral (50%), or combined (16.7%) approaches. Immediate functional recovery was achieved. Sound bone healing was confirmed in all patients, with no complications such as malocclusion, surgical site infection, or malunion with a mean follow-up of 16.3 (range 12-24) months. CONCLUSIONS: OR-IF using a low-profile reconstruction plate system is a reliable treatment for comminuted mandibular fractures, enabling immediate functional recovery with good clinical results.

Upregulation of aquaporin expression in the salivary glands of heat-acclimated rats.

It is known that aquaporin (AQP) 5 expression in the apical membrane of acinar cells in salivary glands is important for the secretion of saliva in rodents and humans. Although heat acclimation enhances saliva secretion in rodents, the molecular mechanism of how heat induces saliva secretion has not been determined. Here, we found that heat acclimation enhanced the expression of AQP5 and AQP1 in rat submandibular glands concomitant with the promotion of the HIF-1α pathway, leading to VEGF induction and CD31-positive angiogenesis. The apical membrane distribution of AQP5 in serous acinar cells enhanced after heat acclimation, while AQP1 expression was restricted to the endothelial cells in the submandibular glands. A network of AQPs may be involved in heat-acclimated regulation in saliva secretion. Because AQPs probably plays a crucial role in saliva secretion in humans, these findings may lead to a novel strategy for treating saliva hyposecretion.

High-dose argatroban therapy for stroke: novel treatment for delayed treatment and the recanalization mechanism.

BACKGROUND: There has been little effective treatment in patients with cerebral infarction at >24 hours after onset. We assessed the effects of high-dose argatroban therapy in delayed administration, and investigated the mechanism based on our clinical findings. METHODS: Argatroban 30 mg was first administered for 15 minutes intravenously, and then 90 mg for 60 minutes followed by 60 mg for 60 minutes were infused continuously. The change of vascular obstruction caused by the treatment was assessed with magnetic resonance angiography. RESULTS: In 4 patients studied, high-dose argatroban resulted in 100% recanalization of occluded vessels (5/5), even though argatroban was administrated >24 hours after onset. On the other hand, when an inadequate dose of argatroban was administered, a hemorrhage was identified. This supports our hypothesis that high-dose argatroban promotes recanalization by deactivating thrombin and exerting an anticoagulant effect on the vascular endothelium. CONCLUSIONS: High-dose argatroban is an effective treatment for cerebral infarction and offers a novel therapeutic approach for delayed hospitalized patients at >24 hours after onset. Additional studies are necessary to identify the cellular and molecular mechanisms and determine the adequate dose in order to reduce risks of complication.

Maxillary verrucous carcinoma coincident with cervical lymph node metastasis of colon adenocarcinoma.

Cervical lymph node metastasis is an extremely rare event in oral verrucous carcinoma. Isolated cervical lymph node metastasis of colon cancer is also rare. This article describes a case of maxillary verrucous carcinoma accompanied by colon adenocarcinoma that metastasized to a cervical lymph node in a 69-year-old Japanese woman. During preoperative evaluation for maxillary verrucous carcinoma, enlarged cervical lymph nodes and colon cancer were suspected by positron emission tomography. Colonoscopy with biopsies confirmed primary colon adenocarcinoma. Left radical neck dissection, partial maxillectomy, and full-thickness skin graft to the mucosa of the upper lip were performed before treatment of colon adenocarcinoma. Cervical lymph nodes showed metastasis from colon adenocarcinoma, and right hemicolectomy was performed. This is the first case report of synchronous oral verrucous carcinoma and colon adenocarcinoma with cervical lymph node metastasis.

Simultaneous sinus lifting and alveolar distraction of a severely atrophic posterior maxilla for oral rehabilitation with dental implants.

We retrospectively reviewed a new preimplantation regenerative augmentation technique for a severely atrophic posterior maxilla using sinus lifting with simultaneous alveolar distraction, together with long-term oral rehabilitation with implants. We also analyzed the regenerated bone histomorphologically. This study included 25 maxillary sinus sites in 17 patients. The technique consisted of alveolar osteotomy combined with simultaneous sinus lifting. After sufficient sinus lifting, a track-type vertical alveolar distractor was placed. Following a latent period, patient self-distraction was started. After the required augmentation was achieved, the distractor was left in place to allow consolidation. The distractor was then removed, and osseointegrated implants (average of 3.2 implants per sinus site, 80 implants) were placed. Bone for histomorphometric analysis was sampled from six patients and compared with samples collected after sinus lifting alone as controls (n = 4). A sufficient alveolus was regenerated, and all patients achieved stable oral rehabilitation. The implant survival rate was 96.3% (77/80) after an average postloading followup of 47.5 months. Good bone regeneration was observed in a morphological study, with no significant difference in the rate of bone formation compared with control samples. This new regenerative technique could be a useful option for a severely atrophic maxilla requiring implant rehabilitation.

Transfection of hypoxia-inducible factor-1 decoy oligodeoxynucleotides suppresses expression of vascular endothelial growth factor in oral squamous cell carcinoma cells.

Vasculature development is thought to be an important aspect in the growth and metastasis of solid tumors. Among the many angiogenic factors produced by tumor cells, vascular endothelial growth factor (VEGF) is considered to play a key role in angiogenic processes. VEGF synthesis is modulated by hypoxia-inducible factor-1 (HIF-1) function within the hypoxic microenvironment of growing cancer tissue. To inhibit HIF-1 activation, oligodeoxynucleotides (ODNs) were synthesized and transferred with either the consensus sequence for HIF-1 binding or a mutated form of this sequence. If we could transfer a large number of ODNs into the cancer cell nucleus, activated HIF-1 might bind to the ODNs, resulting in inhibition of hypoxia-induced VEGF synthesis. We transferred these ODNs into cultured oral squamous cell carcinoma cells (SAS cells) using the hemagglutinating virus of Japan (HVJ)-liposome method. Hypoxia-mediated expression of VEGF by cancer cells was suppressed by transfection of HIF-1 decoy ODNs, but not by mutated HIF-1 decoy ODNs. HIF-1 decoy ODN transfection also inhibited VEGF protein synthesis. These results suggest that transfection with HIF-1 decoy ODNs is effective for regulating tumor growth by reducing VEGF.

Oral symptoms including dental erosion in gastroesophageal reflux disease are associated with decreased salivary flow volume and swallowing function.

BACKGROUND: This preliminary clinical study aimed to evaluate the effects of salivary flow volume and swallowing function on oral symptoms including dental erosion in gastroesophageal reflux disease (GERD). METHODS: The subjects were 40 GERD patients and 30 (15 younger, 15 older) healthy controls. Detailed medical, dietary, and dental histories were obtained to identify individual behavioral habits potentially associated with dental erosion. Oral examination evaluated dental erosion and determined scores for the decayed, missing, filled (DMF) index, the papillary, marginal, attached (PMA) index for gingivitis, and the Simplified Oral Hygiene Index (OHI-S). Salivary flow volume and swallowing function were evaluated by the Saxon test and repetitive saliva swallowing test, respectively. RESULTS: The DMF index and OHI-S scores differed significantly between all 3 groups. The PMA index was significantly different between the GERD group and the two control groups. The prevalence of dental erosion was 24.3% in the GERD group (0% in the control groups). No specific relationship was found between the incidence of dental erosion and dietary history or behavioral habits. The Saxon test results were significantly lower in the GERD group than in both the control groups. Frequency of swallowing was significantly lower and time to first swallow was significantly longer in the GERD group than in the two control groups. CONCLUSIONS: Oral symptoms in GERD are likely to be associated with impaired salivary flow volume or swallowing function. Treatment for the oral dryness induced by reduced salivary flow volume and rehabilitation for swallowing function could be indicated in patients with GERD.

Autocrine loop between vascular endothelial growth factor (VEGF)-C and VEGF receptor-3 positively regulates tumor-associated lymphangiogenesis in oral squamoid cancer cells.

Numerous past studies have suggested a critical role of the paracrine effect between tumor vascular endothelial growth factor (VEGF)-C and lymphatic FLT-4 in solid tumor-associated lymphangiogenesis. In contrast, the pathophysiological role of tumor cell-associated FLT-4 in tumor progression remains to be elucidated. Here, we investigated this role using a tumor implantation model. SAS cells, an oral squamous carcinoma cell line expressing both VEGF-C and FLT-4 but neither FLK-1/KDR nor VEGF-D were adopted for experiments. Stable transformants of dominant-negative (dn) SAS cells were established in which the cytoplasmic domain-deleted FLT-4 was exogenously overexpressed, which can lead to inactivation of endogenous FLT-4 through competitive antagonism and is associated with down-activation of endogenous FLT-4-related intracellular signals. In vitro and in vivo proliferation assays showed lower proliferative activity of dn-SAS cells. An immunohistochemical study revealed that the tumor lymphangiogenesis was significantly suppressed, and the level of human VEGF-C mRNA was significantly lower in dn-SAS cell-derived tumor tissues. Moreover, in vitro studies demonstrated that the significant suppression of VEGF-C and VEGF-A expression was evident in dn-SAS cells or wild-type SAS cells treated with either the FLT-4 kinase inhibitor MAZ51 or the inhibitor of FLT-4-related signals. These findings together suggested that the VEGF-C/FLT-4 autocrine loop in tumor cells was a potential enhancer system to promote cancer progression, and FLT-4 in tumor tissue might become an effective target for cancer therapy.

The association between the computed tomography findings, histologic features, and outcome of osteosarcoma of the jaw.

PURPOSE: The study goal was to clarify the association between computed tomography (CT) findings, histologic features, and outcome of osteosarcoma of the jaw (OSJ). MATERIALS AND METHODS: The CT findings and histologic features of 10 patients with OSJ were retrospectively analyzed. The patients were histopathologically diagnosed to have OSJ. The points analyzed on the CT included the patterns of osteogenesis and any signs of bone destruction. The histologic types were classified according to the 1993 World Health Organization histologic classification of bone tumors. Furthermore, the histologic subtype was classified into the following 3 types: osteoblastic, chondroblastic, and fibroblastic. The grade of the tumor tissue was classified from I to IV. These were compared with the affected jaw site and the outcome of the patients with OSJ. RESULTS: All tumors were classified into the conventional central osteosarcoma histologically. Eight cases were osteoblastic, and 2 cases were chondroblastic. Grade I or II (4 of 5 patients are alive without disease) dominated in the maxilla, but in contrast, grade III or IV (4 of 5 patients are dead of disease or alive with disease) dominated in the mandible. A significant association was seen between the osteogenesis found on the CT images and the outcome, between the grade and the outcome, and between the outcome and the affected jaw site ( P = .02), respectively, in OSJ in this study. However, no significant association was observed between the CT findings and the histologic features in OSJ in this series. CONCLUSIONS: The osteogenesis on the CT, grade, and affected jaw site were considered to be prognostic factors in OSJ in this limited series.

Fibroblast growth factor-2 gene transfer can stimulate hepatocyte growth factor expression irrespective of hypoxia-mediated downregulation in ischemic limbs.

Hepatocyte growth factor (HGF) is a potent angiogenic polypeptide that stimulates angiogenesis. Transcriptional regulation of HGF, however, has not been fully defined, with the exception of the hypoxia-mediated downregulation in cultured cells. In the present study, we report that angiogenic growth factors, including HGF, were upregulated in a murine model of critical limb ischemia in vivo, a finding that was in conflict with previous in vitro data. Mice deficient in basic fibroblast growth factor-2 (FGF-2) showed reduced induction of HGF protein in ischemic muscles, and overexpression of FGF-2 via gene transfer stimulated endogenous HGF, irrespective of the presence of ischemia. In culture, FGF-2 rapidly stimulated HGF mRNA, and a sustained expression was evident in the time course in vascular smooth muscle cells and fibroblasts. FGF-2-mediated induction of HGF was fully dependent on the mitogen-activated protein kinase pathway yet was not affected by either hypoxia or a protein kinase A inhibitor. In the early expression, FGF-2 directly stimulated HGF mRNA without the requirement of new protein synthesis, whereas sustained induction of HGF in the later phase was partly mediated by platelet-derived growth factor-AA. Furthermore, in vivo overexpression of FGF-2 significantly improved the blood perfusion, and the effect was abolished by systemic blockade of HGF in ischemic limbs. This is the first demonstration of a regulational mechanism of HGF expression via FGF-2 that was independent of the presence of hypoxia. The harmonized therapeutic effects of FGF-2, accompanied with the activity of endogenous HGF, may provide a beneficial effect for the treatment of limb ischemia.

Inhibition of epidermal growth factor-induced invasion by dexamethasone and AP-1 decoy in human squamous cell carcinoma cell lines.

Invasive squamous cell carcinoma (SCC) cells degrade extracellular matrix (ECM) via an extracellular protease cascade that includes urokinase-type plasminogen activator (uPA), plasmin, and the metalloprotease (MMP) family of collagenases. In this study, treatment of oral SCC cells with epidermal growth factor (EGF) stimulated the cells to invade Matrigel (constructive basement membrane (BM) protein). EGF-induced cell invasion was inhibited by antibodies to uPA and by synthetic uPA inhibitors. EGF also induced increased expression of uPA and uPA receptor (uPAR) proteins and mRNA, as well as transcription factor activator protein-1 (AP-1)-DNA binding. These EGF-induced changes were inhibited by treatment with dexamethasone (DEX). DEX treatment also stimulated the production of plasminogen activator inhibitor type 1. Moreover, transfection of SCC cells with AP-1 decoy oligodeoxynucleotides (ODNs) resulted in the suppression of EGF-induced uPA and uPAR expression and Matrigel invasion. These results suggest that oral SCC cells invade Matrigel mainly through the uPA-plasmin cascade, which is mediated by the transcription factor AP-1. The uPA-uPAR interaction is essential for augmenting proteolytic activity and uPAR-mediated signaling, which ultimately induce motility and invasion. Since DEX inhibits the expression of both uPA and uPAR, it may be a useful treatment for oral SCC.