Saposin C promotes survival and prevents apoptosis via PI3K/Akt-dependent pathway in prostate cancer cells

Abstract

Background: In addition to androgens, growth factors are also implicated in the development and neoplastic growth of the prostate gland. Prosaposin is a potent neurotrophic molecule. Homozygous inactivation of prosaposin in mice has led to the development of a number of abnormalities in the male reproductive system, including atrophy of the prostate gland and inactivation of mitogen-activated protein kinase (MAPK) and Akt in prostate epithelial cells. We have recently reported that prosaposin is expressed at a higher level by androgen-independent (AI) prostate cancer cells as compared to androgen-sensitive prostate cancer cells or normal prostate epithelial and stromal cells. In addition, we have demonstrated that a synthetic peptide (prosaptide TX14A), derived from the trophic sequence of the saposin C domain of prosaposin, stimulated cell proliferation, migration and invasion and activated the MAPK signaling pathway in prostate cancer cells. The biological significances of saposin C and prosaposin in prostate cancer are not known.

Results: Here, we report that saposin C, in a cell type-specific and dose-dependent manner, acts as a survival factor, activates the Akt-signaling pathway, down-modulates caspase-3, -7, and -9 expression and/or activity, and decreases the cleaved nuclear substrate of caspase-3 in prostate cancer cells under serum-starvation stress. In addition, prosaptide TX14A, saposin C, or prosaposin decreased the growth-inhibitory effect, caspase-3/7 activity, and apoptotic cell death induced by etoposide. We also discovered that saposin C activates the p42/44 MAP kinase pathway in a pertussis toxin-sensitive and phosphatidylinositol 3-kinase (PI3K) /Akt-dependent manner in prostate cancer cells. Our data also show that the anti-apoptotic activity of saposin C is at least partially mediated via PI3K/Akt signaling pathway.

Conclusion: We postulate that as a mitogenic, survival, and anti-apoptotic factor for prostate cancer cells, saposin C or prosaposin may contribute to prostate carcinogenesis at its early androgen-dependent or metastatic AI state.

Figure 1

Saposin C acts as a survival factor for prostate cancer cells. Cells were cultured in their complete media for 3 days and shifted to their basal (serum-free) media or RPMI-1% FBS (only for LNCaP) in the presence or absence of the indicated concentrations of saposin C for 2, 4, or 6 days. Tissue culture media and saposin C were refreshed every 2 days. At the end of incubation periods, cells were trypsinized and cell number was determined using a hemocytometer and trypan blue exclusion method. PC-3 and DU-145 were used as androgen-independent and LNCaP cells were used as androgen-sensitive prostate cancer cell lines. Data represent the average of three independent experiments in triplicate samples; bars, ± SEM. * indicates P < 0.05, and ** indicates P < 0.01 compared to control. Statistical significance was determined by one-way ANOVA with Bonferroni's corrections.

Figure 2

Saposin C activates Akt signaling pathway in prostate cancer cells. A, cells were cultured up to 70% confluency in their complete media, serum-deprived for 24 h, and treated with 10% FBS or saposin C at 0.1, 1, or 10 nM for 10 min. A representative culture plate was also treated with LY294002 (LY; 50 μM) before treating with saposin C (at 1 nM for LNCaP and 10 nM for PC-3 and DU-145). Fifteen μg protein per sample was subjected to SDS-PAGE under reducing conditions and immunoblotting was carried out using phospho-specific Akt antibodies against serine 473 or threonine 308. B, non-radioactive in vitro kinase assay was performed to determine the effect of saposin C on Akt kinase activity as described in details in Materials and Methods. Briefly, cells were grown as described above and Akt was selectively immunoprecipitated from 250 μg protein using 20 μl of immobilized Akt 1G1 monoclonal antibody. Immunocomplexes were pelletted and resuspended in kinase buffer in the presence of 200 μM ATP and 1 μg of Akt/PKB substrate-glycogen synthase kinase fusion protein (GSK-3α/β) and incubated for 30 min at 30°C, allowing immunoprecipitated Akt (if activated) to phosphorylate GSK-3. After terminating the kinase reaction, phosphorylated GSK-3 was detected by SDS-PAGE and immunoblotting using phospho-GSK-3α/β antibody. Control loading was evaluated with anti-Akt antibody to determine total Akt-level. Each experiment was performed in duplicate, and the assays were repeated three times.

Figure 3

Effect of saposin C on expression/activity of caspases and PARP under serum-starvation stress. Cells were cultured routinely up to 60% confluency, washed with PBS, and incubated in their respective serum-free media supplemented with or without saposin C for 48 h. Cell lysates were prepared as described in Materials and Methods and 75 μg of clarified protein samples was subjected to SDS-PAGE under reducing conditions. Western analysis was carried out using monoclonal antibodies against non-cleaved and cleaved caspases-3, -7, and -9 and PARP. For control loading, membranes were probed or reprobed with anti-actin antibody. Each experiment was performed in duplicate, and the assays were repeated three times.

Figure 4

Saposin C reduces growth inhibitory effect of etoposide and acts as an anti-apoptotic factor for prostate cancer cells. A, cells were seeded at 2000 per well in 96-well plates in their complete culture media for 3 (for PC-3 and DU-145) or 4 days (for LNCaP), treated with vehicle (DMSO), saposin C (0.1, 1, or 10 nM), prosaptide TX14A (10 nM), inactive mutant peptide 769M (10 nM), or prosaposin (1 ng/ml) at the indicated concentrations in the presence or absence of etoposide at the indicated concentrations for 3 days. After this period cell number was determined using MTS assay and cell type-specific OD/cell number calibration curve as described in Materials and Methods. B, apoptosis was determined by TUNEL assay. Cells were cultured in multiwell chamber slide up to 40% confluency in their complete culture media, and treated with etoposide in the presence or absence of saposin C (0.1, 1, or 10 nM) for 3 days. Percentage of apoptosis was determined by random selection of 10 microscopic field (at × 200 magnification) and cell count with a hemocytometer. Data expressed at the average of three independent experiments and twelve replicate samples; bars, ± SEM. * indicates P < 0.05, and ** indicates P < 0.01 compared to control (etoposide). Statistical significance of the effect of saposin C on cell growth and apoptosis was evaluated by one-way ANOVA with Bonferroni's corrections. Differences of vehicle (or etoposide)-only treated cells and any other single experimental group of interest (TX14A or prosaposin) was evaluated by Student's t-test and statistical significance was set at P < 0.05.

Figure 5

Effects of saposin C on caspase-3/7 activity (A) and the influence of PI3-kinase inhibitor (B) in etoposide-treated cells. Cell culture, treatment period, growth and caspase activity was described in Figure 4. Caspase-3/7 activity was determined using the Apo-ONE Homoheneous Caspase-3/7 assay kit based on the cleavage of a profluorescent caspase-3/7 substrate (Z-DEVD-R110) and fluorimetric quantitation was performed at an excitation and emission wavelength of 485+20 and 535+25 nm, respectively. After correction of the fluorimetric reading with the blank (vehicle control), final fluorescent intensity was depicted as an arbitrary endpoint relative fluorescent unit, RFLU. PI3-kinase inhibitor (LY294002; LY) was used at final 1.5 μM concentration and saposin C was added at optimal 1 nM (for PC-3 and LNCaP) or 10 nM (for DU-145) concenration. Etoposide (Et) was added at optimal 20 μM (for PC-3 and LNCaP) or 2 μM (for DU-145). Data expressed are the average of three independent experiments and twelve replicate samples; bars, ± SEM. * indicates P < 0.05, and ** indicates P < 0.01. Statistical significance of the effect of saposin C on cell growth and apoptosis was evaluated by one-way ANOVA with Bonferroni's corrections. Differences of vehicle (or etoposide)-only-treated cells and any other single experimental group of interest (TX14A or prosaposin) was evaluated by Student's t-test and statistical significance was set at P < 0.05.

Figure 6

Saposin C activation of MAPK pathway is pertussis toxin-sensitive and PI3K/Akt-dependent. Cells were cultured up to 60% confluency in their maintenance media, washed with PBS, serum-deprived for 20 h, and then fresh basal culture media was added for an additional 4 h in the presence or absence of LY294002 (50 μM, 3 h), Wortmannin (10 μM, 15 min), U0126 (10 μM, 1.5 h), or pertussis toxin (200 ng/ml, 4 h). After pretreatment, saposin C (0.1 nM) was added directly to the cells and incubated for 5 min at 37°C. Cell lysates were prepared and 10 μg protein per sample was subjected to SDS-PAGE and immunoblotting using phospho-specific p42/44 MAPK antibody. For control loading, membranes were also probed or reprobed with p42/44 antibody to detect total p42/44 MAPK. Parallel tissue culture plates, treated in the same manner, were also tested for cell viability by trypan blue dye-exclusion assay. FBS at 10% final concentration was used as a positive control. Each experiment was performed in duplicate, and the assays were repeated three times.