Localization of Parotid Gland Tumors in Relation to the Intraparotid Facial Nerve on 3D Double-Echo Steady-State with Water Excitation Sequence.

BACKGROUND AND PURPOSE: Reliable preoperative facial nerve mapping may help avoid or minimize facial nerve injury during parotid tumor resection. The purpose of this study was to investigate the diagnostic performance of the 3D double-echo steady-state with water excitation sequence in localizing parotid gland tumors through direct visualization of the intraparotid facial nerve in comparison with indirect methods of estimating the facial nerve location. MATERIALS AND METHODS: We retrospectively reviewed 91 parotid gland tumors in 90 patients who underwent surgical resection and preoperative MR imaging, including the 3D double-echo steady-state with water excitation sequence. The tumor locations were categorized as deep or superficial on the basis of direct and 3 indirect methods: the facial nerve line, retromandibular vein, and Utrecht line. Surgical localization was considered the criterion standard. The diagnostic performance for localizing deep lobe lesions using direct and indirect methods was calculated and compared using the McNemar test. RESULTS: Surgical localization confirmed 75 superficial lesions and 16 deep lesions. The interobserver variability of the 3D double-echo steady-state with water excitation sequence was excellent (κ = 0.870). The diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for localizing deep lobe lesions using the 3D double-echo steady-state with water excitation method were 97.8%, 87.5%, 100%, 100%, and 97.4%, respectively. These findings were significantly higher than the facial nerve line in sensitivity, the retromandibular vein in sensitivity, and the Utrecht line in accuracy and specificity (P < .05). Overall, the direct method was the most accurate, sensitive, and specific in localizing parotid gland tumors. CONCLUSIONS: We can achieve higher diagnostic performance in localizing parotid gland tumors by directly visualizing the intraparotid facial nerve using the 3D double-echo steady-state with water excitation sequence compared with indirect methods.

Synaptotagmin XIII expression and peritoneal metastasis in gastric cancer.

BACKGROUND: Peritoneal metastasis is a frequent cause of death in patients with gastric cancer. The aim of this study was to identify molecules responsible for mediating peritoneal metastasis of gastric cancer. METHODS: Transcriptome and bioinformatics analyses were conducted to identify molecules associated with peritoneal metastasis. The therapeutic effects of intraperitoneally administered small interfering (si) RNA were evaluated using mouse xenograft models. Expression of mRNA and protein was determined in gastric tissues from patients with gastric cancer. RESULTS: Synaptotagmin XIII (SYT13) was expressed at significantly higher levels in patients with peritoneal recurrence, but not in those with hepatic or distant lymph node recurrence. Inhibition of SYT13 expression in a gastric cancer cell line transfected with SYT13-specific siRNA (siSYT13) was associated with decreased invasion and migration ability of the cells, but not with proliferation and apoptosis. Intraperitoneal administration of siSYT13 significantly inhibited the growth of peritoneal nodules and prolonged survival in mice. In an analysis of 200 patients with gastric cancer, SYT13 expression in primary gastric cancer tissues was significantly greater in patients with peritoneal recurrence or metastasis. A high level of SYT13 expression in primary gastric cancer tissues was an independent risk factor for peritoneal recurrence. CONCLUSION: SYT13 expression in gastric cancer is associated with perioneal metatases and is a potential target for treatment.

Atp1a3-deficient heterozygous mice show lower rank in the hierarchy and altered social behavior.

Atp1a3 is the Na-pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3-deficient heterozygous mice (Atp1a3+/- ) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3+/- and compared the rank and hierarchy structure between Atp1a3+/- and wild-type mice within a housing cage using the round-robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3+/- than wild-type mice, and Atp1a3+/- approached Atp1a3+/- mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3+/- group than in the wild-type group, while no difference was observed in the mixed-genotype housing condition. (3) Hierarchy formation was not different between Atp1a3+/- and wild type. (4) Atp1a3+/- showed a lower rank in the mixed-genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3+/- showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy.

Streptozotocin induces neurite outgrowth via PI3K-Akt and glycogen synthase kinase 3ß in Neuro2a cells.

Streptozotocin (STZ), a naturally occurring chemical, is toxic to the various kinds of cells such as insulin-producing beta cells. However, the beneficial effect of STZ on neuronal cells such as neurite outgrowth-inducing activity has been unknown. In this study, we examined the effect of STZ on neurite outgrowth in mouse neuronal Neuro2a cells. STZ (0.01 mM~5 mM) exerted remarkable neurite outgrowth-inducing activity in Neuro2a cells in a concentration dependent manner. STZ also had the same neurite outgrowth-inducing activity as that of retinoic acid (RA), which is well known neurite outgrowth inducer. As with the result of RA treatment, STZ administration increased MAP2-positive cells. The MAP2-positive cells reflect neurite outgrowth-induced cells. STZ (0.01 mM~5 mM) did not induce cell death, but significantly decreased cell proliferation. The serine/threonine kinase Akt, a downstream target of phosphatidylinositol-3 kinase (PI3K), was transiently phosphorylated at Ser473 and at Thr303 by STZ (5 mM) administration. Glycogen synthase kinase 3ß (GSK3ß), which has been reported to be inactivated by Akt, was also transiently phosphorylated at Ser9 by STZ (5 mM) administration. In addition, a blocker of PI3K, LY294002 (10 µM), significantly attenuated STZ-induced neurite outgrowth. These results suggest that STZ induces neurite outgrowth via activation of PI3K-Akt signaling pathway and GSK3ß inhibition.

Improvement of gemcitabine sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells by RUNX2 depletion-mediated augmentation of TAp73-dependent cell death.

Pancreatic cancer exhibits the worst prognostic outcome among human cancers. Recently, we have described that depletion of RUNX2 enhances gemcitabine (GEM) sensitivity of p53-deficient pancreatic cancer AsPC-1 cells through the activation of TAp63-mediated cell death pathway. These findings raised a question whether RUNX2 silencing could also improve GEM efficacy on pancreatic cancer cells bearing p53 mutation. In the present study, we have extended our study to p53-mutated pancreatic cancer MiaPaCa-2 cells. Based on our current results, MiaPaCa-2 cells were much more resistant to GEM as compared with p53-proficient pancreatic cancer SW1990 cells, and there existed a clear inverse relationship between the expression levels of TAp73 and RUNX2 in response to GEM. Forced expression of TAp73α in MiaPaCa-2 cells significantly promoted cell cycle arrest and/or cell death, indicating that a large amount of TAp73 might induce cell death even in the presence of mutant p53. Consistent with this notion, overexpression of TAp73α stimulated luciferase activity driven by p53/TAp73-target gene promoters in MiaPaCa-2 cells. Similar to AsPC-1 cells, small interfering RNA-mediated knockdown of RUNX2 remarkably enhanced GEM sensitivity of MiPaCa-2 cells. Under our experimental conditions, TAp73 further accumulated in RUNX2-depleted MiaPaCa-2 cells exposed to GEM relative to GEM-treated non-silencing control cells. As expected, silencing of p73 reduced GEM sensitivity of MiPaCa-2 cells. Moreover, GEM-mediated Tyr phosphorylation level of TAp73 was much more elevated in RUNX2-depleted MiaPaCa-2 cells. Collectively, our present findings strongly suggest that knockdown of RUNX2 contributes to a prominent enhancement of GEM sensitivity of p53-mutated pancreatic cancer cells through the activation of TAp73-mediated cell death pathway, and also provides a promising strategy for the treatment of patients with pancreatic cancer bearing p53 mutation.

Silicon nanocrystal-noble metal hybrid nanoparticles.

We report a novel and facile self-limiting synthesis route of silicon nanocrystal (Si NC)-based colloidally stable semiconductor-metal (gold, silver and platinum) hybrid nanoparticles (NPs). For the formation of hybrid NPs, we employ ligand-free colloidal Si NCs with heavily boron (B) and phosphorus (P) doped shells. By simply mixing B and P codoped colloidal Si NCs with metal salts, hybrid NPs consisting of metal cores and Si NC shells are spontaneously formed. We demonstrate the synthesis of highly uniform and size controllable hybrid NPs. It is shown that codoped Si NCs act as a reducing agent for metal salts and also as a protecting layer to stop metal NP growth. The process is thus self-limiting. The development of a variety of Si NC-based hybrid NPs is a promising first step for the design of biocompatible multifunctional NPs with broad material choices for biosensing, bioimaging and solar energy conversion.

Salvage pharyngolaryngectomy with total esophagectomy following definitive chemoradiotherapy.

Historically, total pharyngolaryngectomy with total esophagectomy has been the standard radical surgical treatment for synchronous cancer of the thoracoabdominal esophagus and pharyngolaryngeal region, and for cancer of the cervical esophagus that has invaded as far as the thoracic esophagus. Although definitive chemoradiotherapy that enables preservation of the larynx has often been the first choice of treatment for cancers involving the cervical esophagus, total pharyngolaryngectomy with total esophagectomy is required as a salvage therapy for cases involving failure of complete remission or locoregional recurrence after chemoradiotherapy. However, salvage esophageal surgery after definitive high-dose chemoradiotherapy is generally associated with high morbidity and mortality. The aim of this study was to examine the short-term outcome of salvage total pharyngolaryngectomy with total esophagectomy. From 2001 to 2014, nine patients underwent salvage total pharyngolaryngectomy with total esophagectomy at the Department of Gastroenterological Surgery, Nagoya University. The mortality and morbidity rates were high at 22% and 89%, respectively. Four patients (44%) developed tracheal necrosis, which in two patients eventually led to lethal hemorrhage. Salvage total pharyngolaryngectomy with total esophagectomy is an uncommon and highly demanding surgical procedure that should be carefully planned and conducted in selected centers of excellence. Measures must be taken to preserve the tracheal blood supply, thus avoiding fatal complications.

Detection of serum melanoma-associated antigen D4 in patients with squamous cell carcinoma of the esophagus.

Despite improvements in surgical techniques, perioperative management, and multidisciplinary therapy, treatment outcomes of patients with esophageal squamous cell carcinoma (ESCC) remain poor. Therefore, development of novel molecular biomarkers, which either predict patient survival or become therapeutic targets, is urgently required. In the present study, to facilitate early detection of ESCC and predict its clinical course, we investigated the relationship of the serum level of melanoma-associated antigen (MAGE)-D4 to patients' clinicopathological characteristics. Using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assays, we determined the levels of MAGE-D4 mRNA and protein in cell lysates and conditioned medium of cultures, respectively, of nine ESCC cell lines. Further, we determined MAGE-D4 levels in serum samples collected from 44 patients with ESCC who underwent radical esophagectomy without neoadjuvant therapy as well as from 40 healthy volunteers. Samples of conditioned medium and cell lysates contained comparable levels of MAGE-D4 that correlated closely with the levels of MAGE-D4 mRNA. Preoperative MAGE-D4 levels in the sera of 44 patients with ESCC, which varied from 0 to 2,354 pg/mL (314 ± 505 pg/mL, mean ± standard deviation), were significantly higher compared with those of healthy volunteers. By setting the cutoff at the highest value for healthy volunteers (50 pg/mL), the MAGE-D4-positive group of patients was more likely to have shorter disease-specific and disease-free survival compared with those of the MAGE-D4-negative group, although the differences were not statistically significant. Our results indicate that the elevation of preoperative serum MAGE-D4 levels in some patients with ESCC was possibly caused by excess production of MAGE-D4 by tumor cells followed by its release into the circulation. Clinical implications of serum MAGE-D4 levels should be validated in a large population of patients with ESCC.

Visualization of the Peripheral Branches of the Mandibular Division of the Trigeminal Nerve on 3D Double-Echo Steady-State with Water Excitation Sequence.

BACKGROUND AND PURPOSE: Although visualization of the extracranial branches of the cranial nerves has improved with advances in MR imaging, only limited studies have assessed the detection of extracranial branches of the mandibular nerve (V3). We investigated the detectability of the branches of V3 on a 3D double-echo steady-state with water excitation sequence. MATERIALS AND METHODS: We retrospectively evaluated the detectability of the 6 branches of the V3, the masseteric, buccal, auriculotemporal, lingual, inferior alveolar, and mylohyoid nerves, by using a 5-point scale (4, excellent; 3, good; 2, fair; 1, poor; and 0, none) in 86 consecutive patients who underwent MR imaging with the 3D double-echo steady-state with water excitation sequence. Weighted κ analysis was used to calculate interobserver variability among the 3 readers. RESULTS: The detection of the lingual and inferior alveolar nerves was the most successful, with excellent average scores of 3.80 and 3.99, respectively. The detection of the masseteric, the buccal, and the auriculotemporal nerves was good, with average scores of 3.31, 2.67, and 3.11, respectively. The mylohyoid nerve was difficult to detect with poor average scores of 0.62. All nerves had excellent interobserver variability across the 3 readers (average weighted κ value, 0.95-1.00). CONCLUSIONS: The 3D double-echo steady-state with water excitation sequence demonstrated excellent visualization of the extracranial branches of V3 in most patients. The 3D double-echo steady-state with water excitation sequence has the potential for diagnosing V3 pathologies and preoperatively identifying peripheral cranial nerves to prevent surgical complications.

Overexpression of melanoma-associated antigen D4 is an independent prognostic factor in squamous cell carcinoma of the esophagus.

To pursue an urgently needed treatment target for esophageal cancer (EC), we investigated the function of the recently discovered melanoma-associated antigen (MAGE)-D4 in squamous cell EC. MAGE-D4 messenger RNA (mRNA) expression was analyzed in nine EC cell lines using quantitative reverse transcription polymerase chain reaction. In 65 surgical specimens of squamous cell EC with no prior neoadjuvant therapy, MAGE-D4 mRNA expression in EC tissues and corresponding normal tissues was analyzed and compared, and evaluated in terms of clinicopathological factors. In representative cases, MAGE-D4 protein distribution was analyzed immunohistochemically. The heterogeneity of MAGE-D4 mRNA expression was confirmed in EC cell lines by quantitative reverse transcription polymerase chain reaction. In surgical specimens, MAGE-D4 mRNA expression was significantly higher in EC tissues than in corresponding normal tissues (P < 0.001). Patients with the highest MAGE-D4 mRNA expression in EC tissues (top quartile, n = 17) had significantly shorter overall survival than patients with low expression (2-year survival: 44% and 73%, respectively, P = 0.006). Univariate analysis identified age (≥65 years), lymphatic involvement, and high MAGE-D4 mRNA expression as significant prognostic factors; high MAGE-D4 mRNA expression was also an independent prognostic factor in multivariable analysis (hazard ratio: 2.194; P = 0.039) and was significantly associated with Brinkman index (P = 0.008) and preoperative carcinoembryonic antigen level (P = 0.002). Immunohistochemical MAGE-D4b expression was consistent with MAGE-D4 mRNA profiling. Our results suggest that MAGE-D4 overexpression influences tumor progression, and MADE-D4 can be a prognostic marker and a potential molecular target in squamous cell EC.

Silencing of RUNX2 enhances gemcitabine sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the stimulation of TAp63-mediated cell death.

Runt-related transcription factor 2 (RUNX2) has been considered to be one of master regulators for osteoblast differentiation and bone formation. Recently, we have described that RUNX2 attenuates p53/TAp73-dependent cell death of human osteosarcoma U2OS cells bearing wild-type p53 in response to adriamycin. In this study, we have asked whether RUNX2 silencing could enhance gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells. Under our experimental conditions, GEM treatment increased the expression level of p53 family TAp63, whereas RUNX2 was reduced following GEM exposure, indicating that there exists an inverse relationship between the expression level of TAp63 and RUNX2 following GEM exposure. To assess whether TAp63 could be involved in the regulation of GEM sensitivity of AsPC-1 cells, small interfering RNA-mediated knockdown of TAp63 was performed. As expected, silencing of TAp63 significantly prohibited GEM-dependent cell death as compared with GEM-treated non-silencing cells. As TAp63 was negatively regulated by RUNX2, we sought to examine whether RUNX2 knockdown could enhance the sensitivity to GEM. Expression analysis demonstrated that depletion of RUNX2 apparently stimulates the expression of TAp63, as well as proteolytic cleavage of poly ADP ribose polymerase (PARP) after GEM exposure, and further augmented GEM-mediated induction of p53/TAp63-target genes, such as p21 (WAF1) , PUMA and NOXA, relative to GEM-treated control-transfected cells, implying that RUNX2 has a critical role in the regulation of GEM resistance through the downregulation of TAp63. Notably, ablation of TAp63 gave a decrease in number of γH2AX-positive cells in response to GEM relative to control-transfected cells following GEM exposure. Consistently, GEM-dependent phosphorylation of ataxia telangiectasia-mutated protein was remarkably impaired in TAp63 knockdown cells. Collectively, our present findings strongly suggest that RUNX2-mediated repression of TAp63 contributes at least in part to GEM resistance of AsPC-1 cells, and thus silencing of RUNX2 may be a novel strategy to enhance the efficacy of GEM in p53-deficient pancreatic cancer cells.

Value of peritoneal cytology in potentially resectable pancreatic cancer.

BACKGROUND: Peritoneal lavage cytology (CY) is used in the diagnosis and staging of various cancers. The clinical significance of positive cytology results in patients with pancreatic cancer is yet to be determined. METHODS: Peritoneal washing samples were collected from consecutive patients with pancreatic cancer between July 1991 and December 2012. The correlations between cytology results, clinicopathological parameters and recurrence patterns were evaluated. The prognostic impact of CY status, regarding resectability and the effectiveness of adjuvant chemotherapy, were analysed. RESULTS: Of 523 included patients, 390 underwent resection. Patients with tumours at least 2 cm in diameter were more likely to have CY+ status than patients with tumours smaller than 2 cm (48 of 312 versus 3 of 78 respectively; P = 0·005) and there was a significant correlation between CY+ status and tumour invasion of the anterior pancreatic capsule (43 of 276 versus 8 of 113 with no invasion of the capsule; P = 0·030). Although the overall survival of patients with resected CY+ tumours was worse than that of patients with resected CY- tumours, it was significantly better than the survival of unresected patients regardless of CY status. Multivariable analysis of all patients who had pancreatectomy did not identify CY+ as an independent prognostic factor. Patients with CY+ tumours tended to develop peritoneal metastasis more often than those with CY- tumours, although not significantly so. The median survival time of 34 patients with resected CY+ tumours who received adjuvant chemotherapy was better than that of 17 patients who had surgery alone, although this was not statistically significant (15·3 versus 10·0 months; P = 0·057). CONCLUSION: CY+ status is not clinically equivalent to gross peritoneal metastasis in patients with pancreatic cancer. Curative resection is still recommended regardless of CY status.

Runt-related transcription factor 2 (RUNX2) inhibits p53-dependent apoptosis through the collaboration with HDAC6 in response to DNA damage.

Runt-related transcription factor 2 (RUNX2) is the best known as an essential protein for osteoblast differentiation. In this study, we have found for the first time that RUNX2 acts as a negative regulator for p53 in response to DNA damage. On DNA damage mediated by adriamycin (ADR) exposure, p53 as well as RUNX2 was induced at protein and mRNA level in human osteosarcoma-derived U2OS cells in association with a significant upregulation of various p53-target genes. Indirect immunostaining and co-immunoprecipitation experiments demonstrated that RUNX2 colocalizes with p53 in cell nucleus and forms a complex with p53 following ADR treatment. Chromatin immunoprecipitation assays revealed that RUNX2/p53 complex is efficiently recruited onto p53-target promoters in response to ADR, suggesting that RUNX2 might be involved in the regulation of transcriptional activation mediated by p53. Indeed, forced expression of RUNX2 resulted in a remarkable downregulation of p53-target genes. Consistent with these observations, knockdown of RUNX2 enhanced ADR-mediated apoptosis and also elevated p53-target gene expression in response to ADR. On the other hand, depletion of RUNX2 in p53-deficient human lung carcinoma-derived H1299 cells had an undetectable effect on p53-target gene expression regardless of ADR treatment, indicating that RUNX2-mediated downregulation of p53-target genes is dependent on p53. Furthermore, RUNX2/p53 complex included histone deacetylase 6 (HDAC6) and HDAC6 was also recruited onto p53-target promoters following ADR exposure. Of note, HDAC6-specific chemical inhibitor tubacin treatment enhanced ADR-mediated upregulation of p53-target gene expression, indicating that deacetylase activity of HDAC6 is required for RUNX2-mediated downregulation of p53-target gene. Taken together, our present findings strongly suggest that RUNX2 inhibits DNA damage-induced transcriptional as well as pro-apoptotic activity of p53 through the functional collaboration with HDAC6 and therefore might be an attractive therapeutic target for cancer treatment.

Growth and development of rabbit oocytes in vitro: effect of fetal bovine serum concentration on culture medium.

The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 µm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P < 0.1). The rate of development to the blastocyst stage was also higher in the medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P < 0.05). Next, using oocytes recovered from follicles 200 to 399 µm in diameter which were cultured in 0.05% FBS, oxygen consumption and the number of cells were analyzed. Blastocysts from oocytes grown in vitro with 0.05% FBS had reduced oxygen consumption and number of cells compared with those from ovulated oocytes (21.66 ± 4.54 × 10(14) vs. 50.19 ± 4.61 × 10(14) mol/sec, 244 ± 25 vs. 398 ± 24, P < 0.05). Rabbit oocytes grown in vitro with 0.05% FBS achieved pregnancy, but pregnancies were not maintained to term. In conclusion, the addition of 0.05% FBS to the culture medium for IVG improved developmental competence of rabbit oocytes grown in vitro.

Antitumor efficacy of oncolytic herpes simplex virus adsorbed onto antigen-specific lymphocytes.

Although several studies have reported that locally administering oncolytic viruses effectively targets malignancies, the efficacy of systemically administered oncolytic viruses is restricted. Recently, however, it was reported that systemic administration of oncolytic vesicular stomatitis virus adsorbed onto antigen-specific lymphocytes was effective against malignancies. We hypothesized that intravenously administering such virus might have significant potential in treatment of the malignant tumors. We adsorbed oncolytic herpes simplex virus-1 mutant R3616 onto lymphocytes harvested from mice with acquired antitumor immunity. We administered adsorbed R3616 to peritoneally disseminated tumors and analyzed the efficacy of this treatment. Mice administered adsorbed R3616 survived significantly longer than mice administered R3616 adsorbed onto non-specific lymphocytes, or mice administered either virus or tumor antigen-specific lymphocytes alone. In this context, herpes oncolytic virus is a promising treatment not only for primary lesions, but also for multiple metastasizing lesions. This treatment strategy may become one of the most effective methods for systemic virus delivery.

Impact of novel oncolytic virus HF10 on cellular components of the tumor microenviroment in patients with recurrent breast cancer.

Oncolytic viruses are a promising method of cancer therapy, even for advanced malignancies. HF10, a spontaneously mutated herpes simplex type 1, is a potent oncolytic agent. The interaction of oncolytic herpes viruses with the tumor microenvironment has not been well characterized. We injected HF10 into tumors of patients with recurrent breast carcinoma, and sought to determine its effects on the tumor microenvironment. Six patients with recurrent breast cancer were recruited to the study. Tumors were divided into two groups: saline-injected (control) and HF10-injected (treatment). We investigated several parameters including neovascularization (CD31) and tumor lymphocyte infiltration (CD8, CD4), determined by immunohistochemistry, and apoptosis, determined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Median apoptotic cell count was lower in the treatment group (P=0.016). Angiogenesis was significantly higher in treatment group (P=0.032). Count of CD8-positive lymphocytes infiltrating the tumors was higher in the treatment group (P=0.008). We were unable to determine CD4-positive lymphocyte infiltration. An effective oncolytic viral agent must replicate efficiently in tumor cells, leading to higher viral counts, in order to aid viral penetration. HF10 seems to meet this criterion; furthermore, it induces potent antitumor immunity. The increase in angiogenesis may be due to either viral replication or the inflammatory response.

Identification of the A kinase anchor protein 12 (AKAP12) gene as a candidate tumor suppressor of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major health problem, and identification of new tumor-related genes is an urgent task. METHODS: To detect tumor-related genes effectively, we performed double-combination array analysis, which consisted of an expression array and a single nucleotide polymorphism (SNP) array of a single surgical HCC specimen. RESULTS: Expression array analysis identified AKAP12 as one of the genes with reduced expression in HCC tissues when compared with non-cancerous adjacent hepatic tissues. In addition, AKAP12 expression levels in tumor tissues from 48 HCC samples were significantly lower (P < 0.001) than those in normal tissues, and the downregulation was significantly correlated with poor overall survival rate (P = 0.003). However, SNP array analysis revealed that locus 6q24-q25 where AKAP12 was located did not show chromosomal deletion. In contrast, hypermethylation in the AKAP12 promoter regions was observed in 41 of 48 HCC samples. We then confirmed that AKAP12 gene re-expression occurs after 5-aza-2'-deoxycytidine (5-aza-dC) treatment through direct sequence analysis of the AKAP12 promoter region in HCC cell lines. CONCLUSIONS: The current data suggest that AKAP12 is downregulated in cancer tissues through promoter hypermethylation, and may have a role as a candidate tumor suppressor gene for HCC.