CAPE suppresses migration and invasion of prostate cancer cells via activation of non-canonical Wnt signaling.

Prostate cancer (PCa) was the fifth most common cancer overall in the world. More than 80% of patients died from PCa developed bone metastases. Caffeic acid phenethyl ester (CAPE) is a main bioactive component of honeybee hive propolis. Transwell and wound healing assays demonstrated that CAPE treatment suppressed the migration and invasion of PC-3 and DU-145 PCa cells. Gelatin zymography and Western blotting indicated that CAPE treatment reduced the abundance and activity of MMP-9 and MMP-2. Analysis using Micro-Western Array (MWA), a high-throughput antibody-based proteomics platform with 264 antibodies detecting signaling proteins involved in important pathways indicated that CAPE treatment induced receptor tyrosine kinase-like orphan receptor 2 (ROR2) in non-canonical Wnt signaling pathway but suppressed abundance of ß-catenin, NF-κB activity, PI3K-Akt signaling, and epithelial-mesenchymal transition (EMT). Overexpression or knockdown of ROR2 suppressed or enhanced cell migration of PC-3 cells, respectively. TCF-LEF promoter binding assay revealed that CAPE treatment reduced canonical Wnt signaling. Intraperitoneal injection of CAPE reduced the metastasis of PC-3 xenografts in tail vein injection nude mice model. Immunohistochemical staining demonstrated that CAPE treatment increased abundance of ROR2 and Wnt5a but decreased protein expression of Ki67, Frizzle 4, NF-κB p65, MMP-9, Snail, ß-catenin, and phosphorylation of IκBα. Clinical evidences suggested that genes affected by CAPE treatment (CTNNB1, RELA, FZD5, DVL3, MAPK9, SNAl1, ROR2, SMAD4, NFKBIA, DUSP6, and PLCB3) correlate with the aggressiveness of PCa. Our study suggested that CAPE may be a potential therapeutic agent for patients with advanced PCa.

Cholestane-3ß, 5α, 6ß-triol suppresses proliferation, migration, and invasion of human prostate cancer cells.

Oxysterols are oxidation products of cholesterol. Cholestane-3ß, 5α, 6ß-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10-40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20-40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27(Kip). Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of ß-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.

A sesquiterpenol extract potently suppresses inflammation in macrophages and mice skin and prevents chronic liver damage in mice through JNK-dependent HO-1 expression.

This study aimed to elucidate the anti-inflammatory and hepatoprotective bioactivities of a sesquiterpenol, (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one (BSL), isolated from Cryptomeria japonica (Taxodiaceae) wood extract. BSL markedly suppressed TNF-α and IL-6 secretion, PGE(2) production, and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated mouse macrophages. BSL also potently inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced protein levels of nitrotyrosine and COX-2 in mouse skin with dermatitis. Conversely, the stress protein heme oxygenase-1 (HO-1) was found upregulated in the same BSL-treated macrophages, probably through activation of the JNK-dependent pathway. LPS-induced activation of NF-κB and mitogen-activated protein kinase signaling pathways, however, was not responsive to BSL treatment. A BSL-enriched extract (BSL-E; 10mg/kg) significantly prevented CCl(4)-induced chronic liver injury, lipid accumulation, and cell necrosis and inhibited aminotransferase activities and iNOS and COX-2 overexpression in mice liver tissues, an effect comparable with that of silymarin, a hepatoprotective drug.

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [(3)H]8-OH-DPAT and other serotonergic ligands.

[(3)H]8-OH-DPAT is a selective ligand for labeling 5-HT(1A) receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [(3)H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [(125)I]RTI-55 and [(3)H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [(3)H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [(3)H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [(125)I]cyanopindolol, [(3)H]ketanserin/[(3)H]mesulergine, [(3)H]GR-65630, [(3)H]GR-113808 and [(3)H]LSD) that specifically labeled 5-HT(1), 5-HT(2), 5-HT(3), 5-HT(4) and 5-HT(5-7) receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.

Discovery and characterization of [3H]8-OH-DPAT binding to HeLaS3 cells.

Some G protein-coupled receptors (GPCRs) have functional links to cancer biology, yet the manifestation of GPCRs in tumor types is little studied to date. Using a battery of radioligand binding assays, we sought to characterize GPCR recognition binding sites on HeLaS3 tumor cells. High levels of binding of the selective serotonin 5-HT(1A) receptor agonist [3H]8-OH-DPAT were observed in these cells. Saturation and homologous competition experiments indicated that [3H]8-OH-DPAT bound different populations of high- and low-affinity sites. In competition experiments, several serotonergic compounds displaced [3H]8-OH-DPAT binding with low potency from its high-affinity binding sites, suggesting that low-affinity binding is the predominant mode of binding. A variety of drugs targeting different classes of receptors did not affect [3H]8-OH-DPAT binding. These observations may help elucidate the pathophysiological and functional relevance of 5-HT receptors in tumor cells and link GPCRs and tumorigenic mechanisms to pharmacological and chemotherapeutic paradigms.

Proline-directed protein kinase FA is a powerful and independent prognostic predictor for progression and patient survival of hepatocellular carcinoma.

PURPOSE: Molecular, cellular, and animal studies have established that overexpressed proline-directed protein kinase F(A) (PDPK F(A)) is essential for the development of tumorigenesis, invasion, and metastasis of human cancer cells. However, the prognostic role of PDPK F(A) in cancer patients remains largely unknown. In this study, association of PDPK F(A) expression with poor prognosis of hepatocellular carcinoma (HCC) patients was examined. PATIENTS AND METHODS: PDPK F(A) expression in the resected tumors of 134 HCC patients (112 men and 22 women) with ages ranging from 33 to 83 years (mean, 55 years) was analyzed by immunohistochemistry. Highly condensed cytoplasmic and nuclear PDPK F(A) associated with tumor cells was used as the major scoring parameter for positive PDPK F(A) expression. RESULTS: Approximately 68% of the patients (91 of 134) exhibited positive PDPK F(A) expression. Patients with positive PDPK F(A) showed poorer disease-free survival and overall survival (P < .001). Cox multivariate regression analysis further established PDPK F(A) as the strongest independent prognosticator for progression and patient survival of HCC (hazard ratio [HR], 2.878; 95% CI, 1.634 to 5.067 for disease-free survival; and HR, 5.035; 95% CI, 2.137 to 11.866 for overall survival; P < .001). CONCLUSION: Consistent with PDPK F(A)'s essential role in the development of highly malignant phenotypes, the present study establishes the potential prognostic role of PDPK F(A) in progression and patient survival of surgically resected primary HCC. Taken together, PDPK F(A) represents a new modifiable signal-transducing target for prognostic prediction and adjuvant treatment of patients with aggressive HCC after hepatic resection.

Biological effect of far-infrared therapy on increasing skin microcirculation in rats.

BACKGROUND/PURPOSE: Insufficient microcirculation of skin leads to acute and chronic tissue ischemia in cases of trauma, reconstructive surgery, diabetes mellitus and peripheral arterial occlusive disease. The autonomic nervous system and nitric oxide (NO) play important roles in maintaining blood perfusion of the skin. Far-infrared (FIR) therapy provides low energy of light emitted from an artificial radiator and has been used to treat many vascular-related disorders. Nevertheless, the mechanisms through which FIR works remain unclear. The present study aims to test the hypothesis that the effect of FIR is through increasing skin microcirculation by a mechanism other than its thermal effect. METHODS: Sixty rats were used in the present study. A WS TY301 FIR emitter was placed 20 cm above the rats. Skin temperature and blood flow were continuously measured by a K-type thermocouple. Under laboratory control, the abdominal skin temperature steadily increased from 38-39 degrees C, and was kept at constant temperature. Skin microcirculation was measured with a continuous laser Doppler flowmeter. RESULTS: There was no significant change of skin blood flow during FIR treatment. Skin blood flow increased significantly soon after the removal of the FIR emitter. The stimulating effect on skin blood flow was more significant in the rats treated with FIR for 45 min and could be sustained as long as 60 min. These findings suggested a non-thermic biological effect of FIR on skin microcirculation. The promotive effect of FIR on increasing skin blood flow was not influenced by pretreatment of APP (atropine, propranolol and phentolamine), but was suppressed by pretreatment with NG-nitro-L-arginine methyl ester (an endothelial nitric oxide synthase inhibitor). CONCLUSION: In conclusion, FIR therapy exerts a NO-related biological effect to increase skin microcirculation in rats. This might bring into perspective the clinical application of FIR to treat ischemic disease by augmenting L-arginine/NO pathway.

Proline-directed protein kinase FA as a new signal transducing target for lethal cancer treatment.

Proline-directed protein kinase F(A) (PDPK F(A)) has been established as a multisubstrate/multifunctional PDPK essential for the development of highly malignant phenotypes and rapid progression of lethal cancers. The recent immunohistochemical, immunocytochemical and clinicopathologic studies combined demonstrate that overexpressed PDPK F(A) is dynamically and closely associated with the most aggressive malignant cells disseminating from primary tumors to peripheral blood, ascites, pleural effusions and second metastatic tumors of various types of cancer patients with poor prognosis. The antisense gene therapeutic studies further demonstrate that overexpressed PDPK F(A) is essential for the development of all aspects of neoplasia including highly metastatic spread, peritoneal dissemination, splenomegaly and chemoradioresistances. The inhibition of cancer progression together with the simultaneous enhancement of chemoradiosensitivities through the suppression of overexpressed PDPK F(A) by specific drug design may work synergistically with surgery and chemoradiotherapy to improve the poor survival rate and life quality of the patients with lethal malignancies. PDPK F(A), therefore, may represent the heel of Achilles and a new promising target for the strategic development of more efficacious treatment for lethal cancers

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion injury in rats.

Hyperbaric oxygen (HBO) therapy is an effective adjunct in treating ischemia-reperfusion (I/R) injury of brain, small intestine, testis, and crushing extremities. This study was designed to test the hypotheses that preconditioning the rats with HBO could protect the liver against subsequent I/R injury. Daily treatment with one-dose HBO (90 min, 2.5 ATA) was brought about for male Sprague Dawley rats for 1 to 3 days before an I/R injury of liver. Hepatic expression of heat-shock protein 70 (Hsp70), total concentration of glutathione (GSH), activity of catalase, superoxide dismutase (SOD), and serum AST and ALT were estimated before and after HBO, as well as after I/R injury. The results showed that activity of hepatic catalase was decreased by one dose, but not three doses, of HBO as compared with baseline data. However, hepatic Hsp70 expression fluctuated insignificantly. AST and ALT increase less in rats preconditioned with one-dose HBO as compared with those without HBO or with three-dose HBO. Our results showed preconditioning by one-dose HBO protects rat liver against subsequent ischemia-reperfusion injury.

Proline-directed protein kinase FA as a potential target for diagnosis and therapy of human cancers.

Proline-directed protein kinase FA (PDPK FA) was originally identified as a phosphatase activating factor (FA) but has subsequently been characterized as a multisubstrate/multifunctional PDPK possibly associated with human cancers. In recent years, the immunohistochemical study revealed that PDPK FA was highly expressed in tumor mass and preferentially overexpressed in the invasive lesions of the resected tissue sections obtained from various types of cancer patients. The clinicopathologic study further revealed a close correlation of the overexpression of PDPK FA with poor prognosis of the cancer patients. The antisense gene therapy study also confirmed that due to its multisubstrate/multifunctional PDPK nature, the overexpression of PDPK FA is essential for the development of malignant growth, tumorigenesis, invasion, metastasis, anti-differentiation, anti-apoptosis and chemoresistance in human cancers. From immunohistochemical, clinicopathologic and antisense gene therapeutic studies combined together, PDPK FA has emerged as a key regulator of all aspects of neoplasia. In this way, nature provides prima facie evidence of a particular protein kinase's pivotal importance to the neoplastic state. PDPK FA therefore represents a newly-described, previously-undiscovered novel signal transducing target for diagnosis, disease monitoring, drug screening and therapy of human cancers.

Suppression of proline-directed protein kinase F(A) inhibits the malignant growth of human pancreatic ductal adenocarcinoma.

BACKGROUND: Proline-directed protein kinase F(A) (PDPK F(A)) was originally identified as a specific phosphatase activating factor, but has subsequently been demonstrated as a multisubstrate PDPK possibly involved in the regulation of diverse malignant characteristics of various types of human cancers including prostate, leukemia, bladder and colon cancers. However, the role of this PDPK in a lethal carcinoma, such as pancreatic ductal adenocarcinoma, remains to be established. MATERIALS AND METHODS: The stable antisense clones with specific suppression of overexpressed PDPK F(A) of human pancreatic ductal adenocarcinoma cells (MIA PaCa-2) were first selected and subsequently characterized for the in vitro and in vivo growth studies. RESULTS: The molecular and cellular studies revealed that the antisense clones of MIA PaCa-2 cells with specific suppression of overexpressed PDPK F(A) potentially exhibited cell growth retardation, decreased serum independence, poor clonogenic growth and loss of anchorage-independent growth. The in vivo study further confirmed that the SCID mice injected with the antisense clones with low-level PDPK F(A) did not develop any detectable tumors even after 7-week observation. In sharp contrast, the parental or control-transfected clones developed very large tumors (>5 cm3) under identical conditions. CONCLUSION: The molecular, cellular and animal results taken together demonstrate that overexpressed PDPK F(A) is essential for the malignant growth of human pancreatic ductal adenocarcinoma.

Association of proline-directed protein kinase FA with tumorigenesis, invasion, and poor prognosis of human colon carcinoma.

BACKGROUND: Initial studies revealed that the multisubstrate proline-directed protein kinase F(A) (PDPK F(A)) is overexpressed in various types of human carcinomas relative to normal controls. Suppression of overexpressed PDPK F(A) inhibits the growth of cancer cells, suggesting a role of this PDPK in human malignancy. In this study, we combine immunohistologic, molecular, cellular, animal, and clinicopathologic studies to demonstrate an essential and critical role of PDPK F(A) in progression and poor prognosis of human colon carcinoma. METHODS: The stable antisense clones of human colon carcinoma cells with specific suppression of PDPK F(A) were established for tumorigenesis and invasion studies. In immunohistologic and clinicopathologic studies, the expression and localization of PDPK F(A) were analyzed by immunohistochemical staining of the specimens obtained from human colon carcinoma patients with Dukes Stage B/C. RESULTS: Initial molecular and cellular studies revealed that the antisense clone of colon carcinoma cells (COLO-205) with specific suppression of PDPK F(A) dramatically lost capabilities of adhesion, chemotaxis, and invasion when compared with the parental or control-transfected colon carcinoma cells. This is the first indication of an association of overexpressed PDPK F(A) with colon carcinoma progression. In agreement with this notion, the in vivo study also revealed that the mice injected with the antisense clone with low-level PDPK F(A) only developed very small tumors (< 0.5 cm(3)) even after a 6-week observation. This is in contrast to the parental or control-transfected cells that developed large tumors (> 5 cm(3)) under identical conditions. Pathologic evaluation revealed invasion to the muscle layer in all tumors formed by the parental cells. In contrast, there was no sign of invasion in mice injected with the antisense clone, confirming an essential role of PDPK F(A) in colon carcinoma progression. Clinicopathologic study also revealed that PDPK F(A) is preferentially overexpressed in the invasive area of colon carcinomatous tissues and overexpression of PDPK F(A) is statistically and closely correlated with venous/lymphatic infiltration, lymph node metastasis, and poor prognosis of colon carcinoma patients with Dukes Stage B/C. CONCLUSIONS: The results demonstrate an essential and critical role of overexpressed PDPK F(A) in progression and poor prognosis of colon carcinoma patients. Suppression of overexpressed PDPK F(A) may provide a new powerful adjuvant approach to prevent human colon carcinoma progression and poor prognosis after surgery and chemotherapy.

Association of overexpressed proline-directed protein kinase F(A) with chemoresistance, invasion, and recurrence in patients with bladder carcinoma.

BACKGROUND: It has been shown previously that proline-directed protein kinase F(A) (PDPK F(A)) is overexpressed in various human malignancies compared with its expression in normal controls, and the suppression of overexpressed PDPK F(A) is capable of inhibiting the growth of various types of human carcinoma cells, suggesting a role for this PDPK in human malignancies. In this report, the authors combine immunohistologic, molecular, cellular, and clinicopathologic studies to demonstrate further an essential critical role for overexpressed PDPK F(A) in bladder carcinoma invasion, chemoresistance, and poor prognosis. METHODS: The expression and localization of PDPK F(A) were analyzed by the immunohistochemical staining of specimens obtained from patients with primary transitional cell carcinoma (TCC) of the bladder. The stable antisense clones of human bladder carcinoma cells with specific suppression of overexpressed PDPK F(A) were established for invasion and chemosensitivity studies. RESULTS: The immunohistochemical study revealed that PDPK F(A) was overexpressed preferentially in the invasive bladder carcinoma tissues. It was found that the stable antisense clones with specific suppression of overexpressed PDPK F(A) to approximately 40% of the parental control level were capable of inhibiting the invasive activity and simultaneously enhancing the chemosensitivity of bladder carcinoma cells to various therapeutic drugs, such as vinblastine, vincristine, paclitaxel, and bleomycin. Clinicopathologic studies also revealed a correlation between overexpressed PDPK F(A) and disease recurrence/survival in patients with primary TCC (P < 0.05). CONCLUSIONS: Taken together, the results demonstrate an essential critical role of overexpressed PDPK F(A) in invasion, chemoresistance, and poor prognosis. Suppression of overexpressed PDPK F(A) may provide a new potential target for therapeutic intervention aimed at preventing chemoresistance, disease progression, and recurrence in patients with bladder carcinoma.