Carcinoid tumors are rare neuroendocrine tumors with a predilection for the

Carcinoid tumors are rare neuroendocrine tumors with a predilection for the gastrointestinal tract. PKD2-kinase dead (PKD2KD) or pcDNA3 (control). Similarly, inhibition of PKD2 activity with small interfering RNA (siRNA) significantly decreased proliferation and invasion compared to cells transfected with non-targeting control (NTC) siRNA. These data support an important role for PKD2 in carcinoid tumor progression. Targeted inhibition of the PKD family may prove to be a novel treatment option for patients with carcinoid tumors. and carcinoid tumor model. BON cells display morphological and physiological characteristics consistent with the carcinoid phenotype, including the presence of numerous dense core granules and the expression and secretion of chromogranin A, serotonin, pancreastatin, and other peptides. The BON cell line is especially useful in the delineation of mechanisms underlying tumor hormone secretion, growth, and invasion, and serves as a novel model for carcinoid behavior [5, 9C11]. We have previously utilized the cell line to study the effects of various agents on tumor buy 63659-18-7 growth for 30 min at 4C) and protein concentrations determined using the method of Bradford [18]. Briefly, total protein (60 g) was resolved on a 10% Nu-PAGE Bis-Tris gel and transferred to PVDF membranes. Filters were incubated overnight at 4C in blotting solution (Tris-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20), followed by a 1 h incubation with primary antibodies. Filters were buy 63659-18-7 washed three times in a blocking solution and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. After three additional washes, the immune complexes were visualized by ECL detection. MTT assay MTT assay was performed as previously described [19]. Briefly, after treatment or transfection, cells were washed with PBS, counted, and diluted to 50,000 cells/ml. Cells (5,000 cells/well) were plated in duplicate in 96 well plates. After 24 h of incubation in 5% CO2 at 37C, MTT buy 63659-18-7 labeling reagent buy 63659-18-7 (10 l) was added, followed by the addition of 100 l ofsolubilization solution 4 h later. The production of blue formazan produced by viable cells was measured on a microplate reader at an absorbance of 570 nm (against a reference of 650 nm) per the manufacturers protocol. Experiments were performed in triplicate. Invasion assay Matrigel invasion assay was performed as previously described [20]. Matrigel inserts (3 per cell line) were rehydrated by adding 0.5 ml of DMEM/F12 50/50 media without FBS to the insert and well and incubating in 5% CO2 at 37C for 2 h. Media was removed, and 0.75 ml DMEM/F12 containing 5% FBS was added to each well; IGF-1 (10ng/ml) was added to media for invasion assay of siRNA-transfected cells. Cell suspensions in FBS-free DMEM/F12 culture media containing 50,000 cells/ml were prepared, and 0.5 ml was added to each insert. Chambers were then placed in 5% CO2 at 37C for 24 h. Non-invading cells were removed by scrubbing with a cotton tipped swab, the membrane was placed in 500 l of methanol at room temperature for 10 min, and then transferred to 500 l of 1% crystal violet for 15 min to stain. Total number of cells invading each membrane was then counted. Statistical analysis Both absorbance from BON-MTT assay and the number of cells invading Matrigel membrane were analyzed using one-way classification analysis of variance. Groups were assessed ALR at the 0.05 of significance. Fishers least significant difference procedure was used for multiple comparisons with Bonferroni adjustment for the number of comparisons. All statistical computations were conducted using the SAS? system, Release 9.1 [21]. RESULTS Expression of PKD2WT increases proliferation of BON cells Since the role of the PKD2 isoform in proliferation and invasion of BON cells remains unclear, we first determined the effect of PKD2 overexpression on BON cell proliferation. BON cells were co-transfected with pcDNA3, GST-PKD2WT, or GST-PKD2KD by electroporation, and stably transfected clones were selected in medium containing G418. Expression of endogenous PKD2 and GST-PKD2 was assessed by Western blotting (Fig. 1A). After plating the cells in duplicate, MTT assay was then performed, and cell proliferation determined at 24 h by measurement of blue formazan production.