(A and B) Invasion (A) and metastasis (B) at 8 weeks in nude mice transplanted with PANC-1 cells or P/ARK cells. The arrowheads in panel A indicate the position of the transplanted tumor focus. The arrowheads in panel B indicate the metastatic foci in the liver, stomach, spleen, and pancreas of a mouse transplanted with P/ARK cells. (C) Akt phosphorylation in tumors. Proteins were extracted from tumors (Tumor) derived from PANC-1 cells (P1) and P/ARK cells (PA), and Western blotting was performed with antibodies to Akt (phosphorylated Akt, pAkt; total Akt, tAkt) and to ARK5. PANC-1 cells (Cell, P1) exposed to (+) and not exposed to (−) 100 ng of IGF-1 (IGF)/ml were also analyzed by the Western blotting procedure.

PANC-1 cells (closed bars) and P/ARK cells (open bars) were transplanted into nude mice (n = 15), and the mice were observed for 12 weeks after transplantation. (A) Tumor volume; (B) photos of tumor-bearing mice. The tumor volumes are the means of 15 data, and the bars represent the standard errors. (C) At 20 weeks after transplantation, the tumor foci formed by the PANC-1 cells and P/ARK cells were extracted and stained with hematoxylin and eosin. The necrotic area is indicated by N. (D) The necrotic area of each tumor focus was measured, the ratios of the necrotic areas are shown as the means of five data, and the bars represent the standard errors. In this study, tumor tissues were used. (E) Immunohistochemical analysis of tumors with anti-mouse CD31 antibody. The numbers on the photographs are the calculated microvessel densities.

Tumor invasion assay of colon cancer cell lines with a Matrigel-coated invasion chamber. (A) Expression of ARK5 in cell extracts from DLD-1, D/ARK, and SW480 cells was assessed. (B) One microgram of double-stranded RNA with (+) or without (−) ARK5i was exposed to the D/ARK (DA) or the SW480 (SW) cell lines, and Western blotting was performed. (C) Phosphorylation of Akt in cell lines exposed or not exposed to 100 ng of IGF-1/ml for 2 h was also assessed by Western blotting. (D and E) The invasion activity of SW480 (D), DLD-1 (E) (open bars), and D/ARK (E) (closed bars) cell lines exposed to 20 μM LY294002, transiently transfected with 1 μg of DN-Akt1, DN-ARK5, control RNAi (CNTi), or ARK5i, in the presence or absence (Cont) of 100 ng of IGF-1/ml for 48 h was assessed. The numbers of invading cells are the means of data from three experiments, and bars represent the standard errors.

Secretion and expression of MMPs. (A) PANC-1 and P/ARK cell lines were cultured for 48 h with serum-free medium on a six-well plate in the presence [IGF(+)] or absence of 100 ng of IGF-1/ml. After culture, the culture media were concentrated to assess MMP-2 and MMP-9 secretion, and cell extracts were collected to assess MT1-MMP expression. pro, proenzyme; act, activated form. (B) Proteins or mRNAs were extracted from P/ARK cells exposed or not exposed (None) to 100 nM rapamycin (Rapa) or 1 μg of ARK5i, and RT-PCR (upper panels) and Western blotting (lower panels) wereperformed to detect MT1-MMP mRNA or protein. RT-PCR and Western blotting were also performed on PANC-1, and Western blotting of actin and RT-PCR of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed as controls. (C) P/ARK cells were transfected with or without (None) 1 μg of DN-ARK5, DN-Akt1, control RNAi (CNTi), or ARK5i or exposed to 20 μM LY294002, and then the cells were treated with [IGF-1 (+)] or without [IGF-1 (−)] 100 ng of IGF-1/ml for 2 h. After treatment of cells, proteins were extracted and Western blotting using antibody to mTOR (phosphorylated, pmTOR; total, tmTOR) and FKHRL1 (phosphorylated, pFKHRL; total, tFKHRL) was performed.

Tumor cell invasion activity of P/ARK. (A) Expression of ARK5 in PANC-1 (P1) or P/ARK (PA) cell lines was assessed by Western blotting with antibody to ARK5 (top, ARK5) or FLAG (middle, FLG-A5). (B) One microgram of ARK5i was transfected into P/ARK cells, and Western blotting was performed. (C) P/ARK cells were exposed or not exposed (dimethyl sulfoxide [DMSO]) to 20 μM LY294002 or 20 μM U0126 for 2 h in the presence of 100 ng of IGF-1/ml, cell extracts and immunoprecipitates with anti-FLAG were collected, and Western blotting was performed with antibody to phosphorylated Akt (pAkt, cell extracts); Akt substrate, which detects protein containing RXRXXpS (pARK5, immunoprecipitates); or ARK5 (tARK5). (D) P/ARK cells were transiently transfected with or without 1 μg of DN-ARK5, DN-Akt1, CA-Akt1, control RNAi (CNTi), or ARK5i and cultured for 48 h in the presence or absence of 20 μM LY294002, 20 μM U0126, or 100 ng of IGF-1/ml. After incubation, cells that had translocated to the bottom of the lower chamber were counted. The numbers of invading cells are the means of data from three experiments, and the bars represent standard errors.

Tumor cell invasion activity of PANC-1. (A) PANC-1 cells were transiently transfected with or without 1 μg of ARK5, DN-ARK5, DN-Akt1, CA-Akt1, control RNAi (CNTi), or ARK5i and cultured in the presence and absence of 20 μM LY294002, 20 μM U0126, or 100 ng of IGF-1/ml for 48 h. After incubation, cells that had translocated to the bottom of the lower chamber were counted. The numbers of invading cells are the means of data from three experiments, and the bars represent standard errors. (B) Expression vector (1 μg) for ARK5, DN-ARK5, DN-Akt1, or CA-Akt1 was transfected into PANC-1 cells, and 48 h after transfection, Western blotting of cell extracts was performed with antibody to ARK5 (ARK5 and DN-ARK5) or myc tag (DN-Akt1 and CA-Akt1). Western blotting of actin was performed as an internal control. (C) PANC-1 cells were exposed to 100 ng of IGF-1/ml for 2 h with or without 20 μM LY294002, and Western blotting was performed with antibody to phosphorylated (pAkt) or total (tAkt) Akt. (D) PANC-1 cells were transiently transfected with 1 μg of FLAG-ARK5 together with either 1 μg of DN-Akt1 (+DN-Akt1) or empty vector [IGF-1 (−), None, and +LY294002], and then cells were treated with [IGF-1 (+)] or without [IGF-1 (−)] 100 ng of IGF-1/ml for 2 h in the presence (+LY294002) or absence of 20 μM LY294002 at 48 h after transfection. After treatment of cells, kinase activity of ARK5 was measured as previously described (56). FLAG-ARK5 was collected with anti-FLAG antibody, and phosphorylation of GST-SAMS was measured (pSAMS, GST-SAMS phosphorylation by autoradiography; tSAMS, total GST-SAMS by Western blotting using anti-GST antibody). (E) (Left panel) Expression vector containing either wild-type FLAG-ARK5 (ARK5), DN-ARK5, or empty vector (Empty) was introduced into PANC-1 cells, and then cells were treated with 100 ng of IGF-1/ml for 2 h. After treatment, immunoprecipitates with anti-FLAG antibody were collected and Western blotting was performed using antibody to FLAG (tARK5) or Akt substrate (pARK5). (Right panel) Expression vectors containing wild-type FLAG-ARK5 (2 μg of DNA) and DN-ARK5 (0, 1, or 2 μg of DNA) were introduced into PANC-1 cells, and then IGF-1 treatment (100 ng/ml for 2 h) was performed. After treatment, cell extracts (for Akt) and immunoprecipitates by anti-FLAG antibody (for ARK5) were collected. Western blotting was performed with antibody to either FLAG (tARK5), Akt substrate (pARK5), Akt (tAkt), or phosphorylated Akt (Ser473, pAkt). (F) Cell extracts were collected from PANC-1 cells transfected with 1 μg of CA-Akt1 or treated with 100 ng of IGF-1/ml, and then Western blotting was performed with antibody to phosphorylated (pGSK) or total (tGSK) GSK-3β to assess the activity of CA-Akt1.