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.

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.

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.