SV40 microRNA miR-S1-3p Downregulates the Expression of T Antigens to Control Viral DNA Replication, and TNFα and IL-17F Expression.

SV40-encoded microRNA (miRNA), miR-S1, downregulates the large and small T antigens (LTag and STag), which promote viral replication and cellular transformation, thereby presumably impairing LTag and STag functions essential for the viral life cycle. To explore the functional significance of miR-S1-mediated downregulation of LTag and STag as well as the functional roles of miR-S1, we evaluated viral DNA replication and proinflammatory cytokine induction in cells transfected with simian virus 40 (SV40) genome plasmid and its mutated form lacking miR-S1 expression. The SV40 genome encodes two mature miR-S1s, miR-S1-3p and miR-S1-5p, of which miR-S1-3p is the predominantly expressed form. MiR-S1-3p exerted strong repressive effects on a reporter containing full-length sequence complementarity, but only marginal effect on one harboring a sequence complementary to its seed sequence. Consistently, miR-S1-3p downregulated LTag and STag transcripts with complete sequence complementarity through miR-S1-3p-Ago2-mediated mRNA decay. Transfection of SV40 plasmid induced higher DNA replication and lower LTag and STag transcripts in most of the examined cells compared to that miR-S1-deficient SV40 plasmid. However, miR-S1 itself did not affect DNA replication without the downregulation of LTag transcripts. Both LTag and STag induced the expression of tumor necrosis factor α (TNFα) and interleukin (IL)-17F, which was slightly reduced by miR-S1 due to miR-S1-mediated downregulation of LTag and STag. Forced miR-S1 expression did not affect TNFα expression, but increased IL-17F expression. Overall, our findings suggest that miR-S1-3p is a latent modifier of LTag and STag functions, ensuring efficient viral replication and attenuating cytokine expression detrimental to the viral life cycle.

Inhibition of EP2/EP4 prostanoid receptor-mediated signaling suppresses IGF-1-induced proliferation of pancreatic cancer BxPC-3 cells via upregulating γ-glutamyl cyclotransferase expression.

Inhibition of prostaglandin E2 signaling via EP2/EP4 prostanoid receptors suppresses Insulin-like growth factor (IGF)-1-induced proliferation of pancreatic cancer BxPC-3 cells. To better understand the mechanism of EP2/EP4 signaling for controlling cell proliferation, we performed metabolome analyses in BxPC-3 cells treated with IGF-1 alone or IGF-1 plus EP2/EP4 inhibitors. These analyses revealed increased g-aminobutyric acid and 5-oxoproline production following the addition of EP2/EP4 inhibitors to IGF-1-treated cells. The expression of a 5-oxoproline-catalyzing enzyme, γ-glutamylcyclotransferase (GGCT), was also upregulated by IGF-1 treatment and further enhanced by the addition of EP2/EP4 inhibitors. Knockdown of GGCT expression resulted in the loss of suppressive effects of EP2/EP4 inhibitors on IGF-1-induced BxPC-3 cell proliferation, whereas GGCT overexpression repressed the basal proliferation of BxPC-3 cells but did not affect the suppressive effects of EP2/EP4 inhibitors. To summarize, we propose a role for EP2/EP4 signaling in regulating IGF-1-induced cell proliferation, in which EP2/EP4 signaling represses IGF-1-induced GGCT expression, which mediates and whose amount controls a branch of IGF-1 signaling to promote cell proliferation via extracellular signal-regulated kinase phosphorylation.

[Coronary Artery Bypass Surgery for the Patient with a History of Anaphylaxis due to Neuromuscular Blocking Agent ; Report of a Case].

We present herein a patient with rocuronium anaphylaxis, which had been identified using skin test, underwent conventional coronary artery bypass surgery without any neuromuscular blocking agent. Immobility was achieved with sedatives and analgesics.

Modifications of azoxymethane-induced carcinogenesis and 90-day oral toxicities of 2-tetradecylcyclobutanone as a radiolytic product of stearic acid in F344 rats.

A 90-day oral toxicity test in rats was performed to evaluate the toxicity of 2-tetradecylcyclobutanone (2-tDCB), a unique radiolytic product of stearic acid. Six-week-old male and female F344 rats (n=15/group) were given 2-tDCB at concentrations of 0, 12, 60 and 300 ppm in a powder diet for 13 weeks. Slight dose-dependent increases in serum total protein and albumin in male rats were found, but these changes were not considered to be a toxic effect. The fasting, but not non-fasting, blood glucose levels of the male rats in the 300 ppm group and female rats in the 60 and 300 ppm groups were lower than those of the controls. Gas chromatography-mass spectrometry analysis showed dose-dependent accumulation of 2-tDCB in adipose tissue, notably in males. Next, we performed an azoxymethane (AOM)-induced two-stage carcinogenesis study. After injection of 6-week-old male F344 rats (n=30/group) once a week for 3 weeks, the animals received 2-tDCB at concentrations of 0, 10, 50 and 250 ppm in a powder diet for 25 weeks. The incidences of colon tumors for the 2-tDCB dosages were 34%, 45%, 40% and 37%, respectively, and were not statistically significant. These data suggest that 2-tDCB shows no toxic or tumor-modifying effects under the present conditions, and that the no-observed-adverse-effect level for 2-tDCB is 300 ppm in both sexes, equivalent to 15.5 mg/kg b.w./day in males and 16.5 mg/kg b.w./day in females.

Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link.

Serum/glucose starvation strikingly reduces heterogeneous nuclear ribonucleoprotein A1 protein and its target, cyclin D1.

Our investigation to explore cellular alterations related to undernutrition in cancer cells revealed that the protein level of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is drastically decreased by serum/glucose starvation. Its loss was reversible, serum/glucose starvation-specific and universal throughout cell types and species. The hnRNP A1 mRNA level and hnRNP A1 mRNA/protein stability were not altered under this condition. CCND1 mRNA, which we newly identified as the binding target of hnRNP A1, was decreased by serum/glucose starvation. Under similar conditions, CCND1 protein was reduced in vitro and in vivo, whereas hnRNP A1 mRNA level and CCND1 mRNA level revealed no correlation in most clinical samples. Functional analyses revealed that CCND1 mRNA stability is certainly dependent on hnRNP A1 protein level and that RNA recognition motif-1 (RRM1) in hnRNP A1 plays a central role in maintaining CCND1 mRNA stability and subsequent protein expression. The injection of RRM1-deleted hnRNP A1-expressing cancer cells in the mouse xenograft model did not form any tumours, and that of hnRNP A1-expressing cancer cells retained CCND1 expression at the lesion adjacent to necrosis with a slight increase in tumour volume. Furthermore, RRM1 deletion caused growth suppression with the induction of apoptosis and autophagy, whereas CCND1 restoration completely recovered it. Our results indicate that serum/glucose starvation triggers entire hnRNP A1 protein loss, and its loss may play a role in CCND1 mRNA destabilization and CCND1-mediated cellular event inhibition, i.e. growth promotion, apoptosis induction and autophagosome formation.

Autopsy case of amebic granulomatous meningoencephalitis caused by Balamuthia mandrillaris in Japan.

Balamuthia mandrillaris is a free-living ameba that causes amebic encephalitis. Herein, we report an autopsy case of Balamuthia encephalitis proven with polymerase chain reaction (PCR) and immunohistochemistry from paraffin-embedded brain biopsy specimens. A 68-year-old Japanese male presented at a hospital with progressive right hemiparesis approximately 3 months before his death. An open-brain biopsy specimen showed diffuse meningitis with massive coagulative necrosis. The perivascular spaces contained numerous lymphocytes, histiocytes and giant cells, although the etiology was not determined. The patient deteriorated into coma and died from cerebral herniation. Autopsy revealed abundant trophozoites and cysts in the subarachnoid and Virchow-Robin's spaces. Electron-micrographs of the amebic cysts showed a characteristic triple-walled envelope. The amebas were identified as Balamuthia mandrillaris based on immunohistochemical analysis from the autopsy and biopsy specimens. Primer sets designed to amplify approximately 200 bp bands of mitochondrial 16S rRNA gene of Balamuthia by PCR produced positive results from the biopsy specimens but negative results from the autopsy specimens. In summary, PCR to amplify shorter segments of DNA may be of diagnostic value in detecting suspected cases of balamuthiasis in formalin-fixed, paraffin-embedded specimens. Increased awareness and timely diagnosis of Balamuthia encephalitis might lead to earlier initiation of therapy and improved outcome.

SV40 miR-S1 and Cellular miR-1266 Sequester Each Other from Their Targets, Enhancing Telomerase Activity and Viral Replication.

Virus-encoded microRNAs (miRNAs) target viral and host mRNAs to repress protein production from viral and host genes, and regulate viral persistence, cell transformation, and evasion of the immune system. The present study demonstrated that simian virus 40 (SV40)-encoded miRNA miR-S1 targets a cellular miRNA miR-1266 to derepress their respective target proteins, namely, T antigens (Tags) and telomerase reverse transcriptase (TERT). An in silico search for cellular miRNAs to interact with viral miR-S1 yielded nine potential miRNAs, five of which, including miR-1266, were found to interact with miR-S1 in dual-luciferase tests employing reporter plasmids containing the miRNA sequences with miR-S1. Intracellular bindings of miR-1266 to miR-S1 were also verified by the pull-down assay. These miRNAs were recruited into the Ago2-associated RNA-induced silencing complex. Intracellular coexpression of miR-S1 with miR-1266 abrogated the downregulation of TERT and decrease in telomerase activity induced by miR-1266. These effects of miR-S1 were also observed in miR-1266-expressing A549 cells infected with SV40. Moreover, the infected cells contained more Tag, replicated more viral DNA, and released more viral particles than control A549 cells infected with SV40, indicating that miR-S1-induced Tag downregulation was antagonized by miR-1266. Collectively, the present results revealed an interplay of viral and cellular miRNAs to sequester each other from their respective targets. This is a novel mechanism for viruses to manipulate the expression of viral and cellular proteins, contributing to not only viral lytic and latent replication but also cell transformation observed in viral infectious diseases including oncogenesis.

Inhibitory effect of soluble platelet-derived growth factor receptor ß on intraosseous growth of breast cancer cells in nude mice.

Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor ß (PDGFRß) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRß signaling using soluble PDGFRß (sPDGFRß) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRß. A stable transfectant (TNBCT/Bo-sPDGFRß) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRß cells. Intratibial injection of TNBCT/Bo-sPDGFRß cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P < 0.01). This attenuated growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRß is useful for the treatment of breast cancer bone metastasis.

[Comparison of urinary output during general anesthesia, between patients administered with remifentanil and those without remifentanil administration].

BACKGROUND: Transient renal failure during surgery is caused by increasing secretion of stress hormone such as ADH and renin. We suspected that urinary output varies according to administration of remifentanil with potent analgesic effects. Consequently, we studied intraoperative urinary output of two groups, patients administered with remifentanil and those without remifentanil administration. METHODS: We compared urinary output during general anesthesia, of 327 patients administered with remifentanil (Group R) and 314 patients without remifentanil administration (Group NR) retrospectively. Patients were excluded if they were under the age of eighteen, receiving epidural anesthesia, or having medicine with diuretic effect. RESULTS: There were no significant difference in background of the patients in each group, in particular, age, sex, body weight, and ASA grade. We found no significant difference in intraoperative factors; operation time, total blood loss, volume of infusion, anesthesia time, and given dose of fentanyl. Urinary output of Group R was estimated as 512 +/- 435 ml, and that of Group NR was 409 +/- 405 ml (P value was 0.02). CONCLUSIONS: We found a significance difference in urinary output during anesthesia, between patients administered with remifentanil and those without remifentanil administration. We suspect that remifentanil decreases urinary output in the perioperative period.

[Anesthetic management of a child with an esophageal foreign body which was misdiagnosed as asthma attack].

We have experienced anesthesia of a child with airway stenosis caused by esophageal foreign body (coin-type lithium battery), which was misdiagnosed as asthma. The case is an 18-month-old boy who had asthma-like symptoms since 2 months before, and visited our hospital because these symptom had started to worsen 3 days before visit. His stridor was significant and we noted a coin-shaped foreign body on chest X-ray taken for admission. Therefore we removed the foreign body using upper gastrointestinal endoscope under general anesthesia. The airway management was very difficult because airway stenosis had been aggravated for delayed diagnosis, diagnosing.

[A case report of anesthesia for a child with Pompe's disease (glycogen storage disease type II)].

A case report of a child with Pompe's disease (glycogen storage disease type II), who underwent two general anesthetics, is presented. The progressive infiltration of heart and skeletal muscle with glycogen results in a severe form of cardiomyopathy and respiratory muscle weakness. Death usually occurs by 1 year of age from respiratory insufficiency or end-stage cardiomyopathy. Consequently, there are significant problems in the anesthetic management of these patients. The patient, a female child of 6 months presented signs of cardiac failure and took treatment with olprinone and diuretic. The initial surgical procedure was placement of a subcutaneous central venous catheter. Anesthesia was induced with ketamine and midazolam intravenously, and was maintained with nitrous oxide (50%) and intravenous ketamine. The dosage of the dopamine and olprinone was necessary to maintain circulation. For tracheotomy, the second procedure, anesthesia was induced and maintained with sevoflurane and dopamine. Recently a promising enzyme replacement therapy has resulted in improved clinical outcomes, and the number of anesthesia for infants of with Pompe's disease will increase in future.

Soluble EP2 neutralizes prostaglandin E2-induced cell signaling and inhibits osteolytic tumor growth.

Prostaglandin E2 (PGE2) plays a key role in osteolytic bone metastasis as well as roles in inflammation, cell growth, and tumor development. PGE2 exerts its effects by binding and activating E-prostanoid receptor (EP). In this study, we propose a new approach for blocking EP-mediated cell signaling using a soluble chimeric EP2 fragment. Mammalian expression vectors encoding several human EP2 cDNAs were introduced into 293 cells and the culture medium was tested for their function as a decoy receptor for PGE2. PGE2 binding assays revealed that culture medium containing the second extracellular region of EP2 (FuEP2/Ex2) had binding activity. FuEP2/Ex2 neutralized PGE2-induced cyclic AMP production, cyclic AMP-responsive element binding protein phosphorylation, and subsequent induction of cyclooxygenase-2, interleukin (IL)-1beta, and IL-6 mRNAs. In human osteoblasts, this culture medium neutralized the induction of receptor activator of nuclear factor-kappaB ligand mRNA. A stable transfectant expressing FuEP2/Ex2 was established from human prostate cancer PC-3 cells (PC3-FuEP2/Ex2). PC3-FuEP2/Ex2 cells grew at similar rates to vector control cells under normal culture conditions, although PGE2-induced growth stimulation was suppressed. Intraosseous injection of PC3-FuEP2/Ex2 cells into the tibia of athymic nude mice revealed that the degrees of tumor growth and osteolysis were decreased compared with control cell-injected mice, with decreased osteoclasts and increased apoptotic cells. Furthermore, the cyclooxygenase-2, IL-1beta, and IL-6 mRNA levels were reduced in the tumor lesions. These data suggest that FuEP2/Ex2 is useful for treating osteolytic bone metastasis and cancers that depend on EP signaling for their growth and development.

[Anesthetic management of the ex-utero intrapartum treatment (EXIT) procedure for giant epignathus and of the tumor excision].

We report an anesthetic management of the ex-utero intrapartum treatment (EXIT) procedure performed in a fetus with giant epignathus due to laryngeal atresia at 28 weeks' gestation. Anesthesia of the mother was induced with thiamylal and vecuronium, and maintained with 4% sevoflurane in 100% oxygen before delivery. Sevoflurane provided excellent uterine relaxation. To maintain the arterial pressure, the patient received acetate Ringer and ephedrine 4mg. After hysterotomy, a pulse oxymeter and an ultrasound transducer were applied to monitor fetal Sp(O2) and heart rate. No anesthetic agents were injected into the fetus in addition to transplacental sevoflurane. Tracheostomy was performed on the fetus by pediatric surgeons on placental support. The uterine tone improved soon after discontinuing sevoflurane, intramyometrial injection of oxytocin and ergometrine infusion after delivery. Excision of the tumor was performed on day 2 of life. Pediatric surgeons tried to excise it totally, but it was hard to differentiate the tumor from the normal tissue, and partial excision was performed. After the excision, the neonate weighed 944 g and excised specimen weighed 253 g. Though the neonate was immature and the tumor was very large, no perioperative complications were associated with EXIT and the tumor excision.

Tsumura-Suzuki obese diabetic mice-derived hepatic tumors closely resemble human hepatocellular carcinomas in metabolism-related genes expression and bile acid accumulation.

BACKGROUND AND AIMS: Tsumura-Suzuki obese diabetic (TSOD) is a good model of metabolic syndrome showing typical lesions found in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, and develops spontaneous hepatic tumors with a high frequency. Majority of the developing tumors overexpress glutamine synthetase (GS), which is used as a marker of hepatocellular carcinoma (HCC). The aim of this study is to assess the status of expression of metabolism-related genes and the level of bile acids in the TSOD mice-derived tumors and to determine the association with metabolic dysregulation between human HCC and TSOD mice-derived tumors. METHODS: GS-positive hepatic tumors or adjacent normal tissues from 71-week-old male TSOD mice were subjected to immunohistochemical staining, quantitative RT-PCR (qRT-PCR), quantitation of cholic acid and taurocholic acid. RESULTS: We found that downregulation of the rate-limiting enzyme for betaine synthesis (BADH), at both mRNA and protein levels in GS-positive TSOD mice-derived tumors. Furthermore, the bile acid receptor FXR and the bile acid excretion pump BSEP (Abcb11) were found to be downregulated, whereas BAAT and Akr1c14, involved in primary bile acid synthesis and bile acid conjugation, were found to be upregulated at mRNA level in GS-positive TSOD mice-derived tumors. BAAT and Akr1c14 were also overexpressed at protein levels. Total cholic acid was found to be increased in GS-positive TSOD mice-derived tumors. CONCLUSION: Our results strongly support the significance of TSOD mice as a model of spontaneously developing HCC.

Daily Coffee Intake Inhibits Pancreatic Beta Cell Damage and Nonalcoholic Steatohepatitis in a Mouse Model of Spontaneous Metabolic Syndrome, Tsumura-Suzuki Obese Diabetic Mice.

BACKGROUND: Metabolic syndrome is one of the most important health issues worldwide. Obesity causes insulin resistance, hyperlipidemia, diabetes, and various diseases throughout the body. The liver phenotype, which is called nonalcoholic steatohepatitis (NASH), frequently progresses to hepatocellular carcinoma. We recently established a new animal model, Tsumura-Suzuki obese diabetic (TSOD) mice, which spontaneously exhibit obesity, diabetes, hyperlipidemia, and NASH with liver nodules. METHODS: We examined the effects of coffee intake on various conditions of the metabolic syndrome using TSOD mice. The daily volume of coffee administered was limited so that it reflected the appropriate quantities consumed in humans. To clarify the effects of the specific components, animals were divided into two coffee-intake groups that included with and without caffeine. RESULTS: Coffee intake did not significantly affect obesity and hyperlipidemia in TSOD mice. In contrast, coffee intake caused various degrees of improvement in the pancreatic beta cell damage and steatohepatitis with liver carcinogenesis. Most of the effects were believed to be caused by a synergistic effect of caffeine with other components such as polyphenols. However, the antifibrotic effects of coffee appeared to be due to the polyphenols rather than the caffeine. CONCLUSIONS: A daily habit of drinking coffee could possibly play a role in the prevention of metabolic syndrome.

Cyclooxygenase-2 plays a suppressive role for induction of apoptosis in isoliquiritigenin-treated mouse colon cancer cells.

Cellular damage induced by chronic inflammation is a well known cause of colon carcinogenesis. Cyclooxygenase-2 (COX-2), the enzyme that converts arachidonic acid to prostanoids, is known to play an important role in inflammation. Herbal flavonoid isoliquiritigenin (ILTG) has previously been reported to be a strong suppresser of the COX-2 pathway as well as an inducer of apoptosis. Here we report that the susceptibility to apoptosis by ILTG is dependent on the level of COX-2 in mouse colon adenocarcinoma Colon 26, which spontaneously expresses COX-2. This dependency was observed to be enhanced by blockage of the lipoxigenases (LOXs)-mediated metabolic pathway and attenuated by addition of a number of prostaglandins and thromboxanes. Taken together, these findings indicate that ILTG-induced apoptosis is negatively regulated by the COX-2 expression level.

Histopathological characteristics of glutamine synthetase-positive hepatic tumor lesions in a mouse model of spontaneous metabolic syndrome (TSOD mouse).

We previously reported that Tsumura-Suzuki obese diabetic (TSOD) mice, a polygenic model of spontaneous type 2 diabetes, is a valuable model of hepatic carcinogenesis via non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). One of the characteristics of tumors in these mice is the diffuse expression of glutamine synthetase (GS), which is a diagnostic marker for hepatocellular carcinoma (HCC). In this study, we performed detailed histopathological examinations and found that GS expression was diffusely positive in >70% of the hepatic tumors from 15-month-old male TSOD mice. Translocation of ß-catenin into nuclei with enhanced membranous expression also occurred in GS-positive tumors. Small lesions (<1 mm) in GS-positive cases exhibited dysplastic nodules, with severe nuclear atypia, whereas large lesions (>3 mm) bore the characteristics of human HCC, exhibiting nuclear and structural atypia with invasive growth. By contrast, the majority of GS-negative tumors were hepatocellular adenomas with advanced fatty change and low nuclear grade. In GS-negative tumors, loss of liver fatty acid-binding protein expression was observed. These results suggest that the histological characteristics of GS-positive hepatic tumors in TSOD mice resemble human HCC; thus, this model may be a useful tool in translational research targeting the NAFLD/NASH-HCC sequence.

Inhibition of EP2/EP4 signaling abrogates IGF-1R-mediated cancer cell growth: involvement of protein kinase C-θ activation.

Associations between growth factor receptor-mediated cell signaling and cancer cell growth have been previously characterized. Receptors for prostaglandin E2, such as EP2, and EP4, play roles in cancer growth, progression and invasion. Thus, we examined the interactions between EP2/EP4- and IGF-1R-mediated cellular signaling in human pancreatic cancer cells. Selective antagonists against EP2 and EP4 abrogated IGF-1-stimulated cell growth and suppressed MEK/ERK phosphorylation. In subsequent experiments, phospho-antibody arrays indicated increased phosphorylation levels of protein kinase C-θ (PKC-θ) at the Thr538 position following the inhibition of EP2/EP4-mediated signaling. Inhibition of PKC-θ activity impaired cell viability compared with EP2/EP4-antagonized IGF-1-stimulated cells. PKC-θ kinase MAP4K3, which plays a pivotal role in PKC-θ activation, also affected growth signaling in the presence of EP2/EP4 antagonists. Administration of EP2 and EP4 antagonists significantly inhibited the growth of an orthotopic xenograft of IGF-1-secreting pancreatic cancer cells, with increased phospho-PKC-θ and decreased phospho-ERK. Clinico-pathological analyses showed that 17.4% of surgical pancreatic cancer specimens were quadruple-positive for IGF-1R, EP2 (or EP4), MAP4K3, and PKC-θ. These results indicate a novel signaling crosstalk between EP2/EP4 and IGF-1R in cancer cells, and suggest that the MAP4K3-PKC-θ axis is central and could be exploited as a molecular target for cancer therapy.

Induction of retinol-binding protein 4 and placenta-specific 8 expression in human prostate cancer cells remaining in bone following osteolytic tumor growth inhibition by osteoprotegerin.

New drugs that inhibit the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL)/RANK pathway have demonstrated efficacy for the treatment of bone metastasis. Toxicities induced by these drugs, however, including osteonecrosis of the jaw and hypocalcemia, may adversely affect therapy. The aim of this study was to identify additional therapeutic targets that can be combined with OPG/RANKL/RANK pathway inhibition in the treatment of prostate cancer bone metastasis. We established a stable transfectant that produces high levels of OPG mRNA and protein from PC-3 human prostate cancer cells (PC3-OPG). The culture medium of PC3-OPG cells significantly inhibited the differentiation of mouse monocytes into mature osteoclasts. Furthermore, when PC3-OPG cells were injected into the bones of nude mice, bone destruction and tumor-induced osteoclast formation were reduced. Injection into bone of the mixtures containing equal amounts of green fluorescent protein (GFP)-expressing PC-3 cells (PC3-GFP) and PC3-OPG cells also reduced bone destruction, compared to the control mixture. PC3-GFP cells were subsequently isolated from bone tumors and used for microarray analysis to assess changes in gene expression following osteolytic tumor growth inhibition by OPG. We selected the top 10 upregulated genes based on results from microarrays and confirmed mRNA expression of each gene by RT-PCR. The expression patterns of retinol-binding protein 4 (RBP4) and placenta-specific 8 (PLAC8) were consistent with microarray results. Expression of these genes was also increased in the bone tumors of PC3-GFP/PC3-OPG-injected mice. Knockdown of both RBP4 and PLAC8 by siRNA inhibited the growth of PC-3 cells in vitro. Thus, RBP4 and PLAC8 may become new therapeutic targets for prostate cancer bone metastasis, in combination with OPG/RANKL/RANK pathway inhibition.