Abstract
Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 microm) or the Akt-specific inhibitor-X (Akti-X; 2.5 microm). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr389). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.
Highlights
Proliferation, survival, adhesion, and invasion [1,2,3]
After 2 weeks, the majority of cells exposed to CCL2 were able to survive in contrast to control cells, suggesting that CCL2 confers an advantage upon PC3 cells to survive in adverse conditions, such as limited nutrients and growth factors (Fig. 1D)
CCL2-mediated Cell Survival Is phosphatidylinositol 3-kinase (PI3K)/Akt Pathwaydependent—To determine the mechanism involved in CCL2induced cell survival, we investigated the signaling pathways activated by this chemokine that are critical for cell survival
Summary
Cell Lines—The androgen-independent human prostate cancer cell lines, PC3 and DU 145 were obtained from ATCC (Manassas, VA). Cells were washed three times with Hanks’ balanced salt solution to remove unattached cells, and medium was replaced with 10 ml of RPMI 1640, containing 2 g/ml puromycin. Immunofluorescence—PC3, shS-1, and shS-2 cells were serum-starved in RPMI 1640 medium for 16 h at 37 °C and plated into Lab-Tek II dual chamber culture slides (catalog number 177380; Nunc) and allowed to attach at 37 °C for 6 h. Coverslips were applied with Prolong Gold Antifade reagent (catalog number P-36930; Invitrogen), and slides were transferred to 4 °C for at least 24 h prior to immunofluorescence analysis. Samples were added to washed beads, and the immunoprecipitation was carried out following the recommended procedures of Invitrogen; all washes were performed using cell lysis buffer (Cell Signaling). A probability level of less than 0.05 was considered significant
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