Androgen sensitive LNCaP cells did not maintain their viability when cul tured in serum free, 0. 25% FBS RPMI, or 0. 5% FBS RPMI media, for more than 36 h. These cells started to detach from tissue selleck chemicals culture plates and cell viability was decreased to less than 40% as determined by the trypan blue dye exclusion method. However, in the presence of 1% FBS, cells remained attached to the tissue culture plate and their growth increased 31% at day 4 and 20% on day 6 as compared with the control values at day 2. Saposin C stimulated proliferation of these cells by 13% at day 2, 35% at day 4, and 33% at day 6 compared to the controls. PC 3 cells appeared to be more sensitive to serum deprivation and the number of live cells decreased 30% by day 4 and 60% by day 6 compared to the control values at day 2.
However, saposin C increased Inhibitors,Modulators,Libraries cell proliferation by 9% at day 2, 19% at day 4, and 88% at day 6, compared to control plates at the same time period. The growth response of DU 145 cells was differ ent from PC 3 or LNCaP cells. In the absence of saposin C, the number of live cells increased 10% at day 4 and 29% at day 6 compared to day 2. These cells also demon strated the highest proliferative response to saposin C at day 4 by 93%. Taken together, these data indicate that saposin C in a dose dependent and cell type specific manner, promotes the survival of the serum deprived prostate cancer cells. Saposin C activates the PI3K/Akt Inhibitors,Modulators,Libraries signaling pathway in prostate cancer cells Several studies have demonstrated that the serine/threo nine kinase Akt is a pivotal survival effector for prostate cancer cells and protects them from apoptotic cell death induction by various types of stresses.
Hence, we next evaluated the effect of saposin C on the Akt signaling pathway Inhibitors,Modulators,Libraries in cells. Direct immunoblotting of serum starved cells for 24 h showed that saposin C upregulates phosphorylative activ ity of Akt at serine 473 in androgen independent PC 3 Inhibitors,Modulators,Libraries and DU 145 cells. This response was biphasic. The response of LNCaP cells was distinct and started at 1. 0 nM that subsequently returned to a basal level at higher Saposin C activates Akt signaling pathway in prostate cancer 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 Inhibitors,Modulators,Libraries 10 min. A representative culture plate was also treated with LY294002 before treating with saposin C. Fifteeng protein per sample was subjected to SDS PAGE under reducing con ditions and immunoblotting was carried out using phospho specific Akt antibodies against serine 473 or threonine 308. B, non radioactive in vitro kinase assay http://www.selleckchem.com/products/Paclitaxel(Taxol).html was performed to determine the effect of saposin C on Akt kinase activity as described in details in Materials and Methods.
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