The senescence-associated secretory phenotype (SASP) from mesenchymal stromal cells impairs growth of immortalized prostate cells but has no effect on metastatic prostatic cancer cells

Abstract

Senescent cells secrete inflammatory cytokines, proteases, and other factors, which are indicated as senescence-associated secretory phenotype (SASP). There are contrasting studies on the role of the SASP in cancer. Studies suggested that cancer cells may misuse the senescent secretome for their growth. Other investigations evidenced that the SASP may induce cancer growth arrest, senescence, or apoptosis. These conflicting data can be reconciled considering that cancer cells can coax senescent cells to secrete factors for their survival, thus abrogating the SASP's anti-cancer effect. Cancer stage may also have an impact on the capacity of the SASP to block tumor proliferation and promote senescence. Indeed, senescence is associated with a permanent cell cycle arrest, which needs functional cell cycle checkpoints. We evaluated the SASP effect on the in vitro biological properties of PNT2 and PC3 cells, which are immortalized prostate cells and metastatic prostatic cancer cells, respectively. We evidenced that SASPs, coming either from mesenchymal stromal cells treated with H₂0₂ or with low X-ray doses, induced senescence of immortalized cells but not of cancer cells. Hence, the SASP released by acute senescent cells should be considered as an effective weapon against pre-tumorigenesis events rather than an anti-cancer mechanism acting on malignant cells.

Keywords: mesenchymal stem cells; secretome; senescence.

Figure 1

Responsiveness of immortalized and metastatic cancer cells to A-SASP from senescent MSCs. (A) PNT2 and PC3 cell proliferation was determined by Cell Counting Kit-8 (CCK-8) colorimetric assay (Dojindo, Germany). On the left, PNT2 proliferation in control medium (CTRL) and in media containing either an A-SASP or C-SASP. On the right, PC3 proliferation (data are expressed with standard deviation SD, n = 3, *p < 0.05; **p < 0.01). (B) Representative cell cycle FACS analysis of PNT2 and PC3 cultures treated with SASPs. (n=3 ± SD). (C) Representative micrographs of BrdU immunostaining (red) on PNT2 and PC3 cultures treated with SASPs. Cell cytoplasms were stained with actin (green). The graph shows the percentage of cycling (BrdU-positive) PNT2 and PC3 cells in the presence of different SASPs (n = 3 ± SD, *p < 0.05). (D) Representative micrographs of Ki67 immunostaining (red) on PNT2 and PC3 cultures treated with SASPs. Cell nuclei were stained with DAPI (blue). The graph shows the percentage of cycling (Ki-67-positive) PNT2 and PC3 cells in the presence of different SASPs (n = 3 ± SD). (E) Representative apoptosis FACS analysis. The experiments were carried out after treatment with SASPs. The assay allows the identification of early (Annexin V + and 7ADD −) and late apoptosis (Annexin V + and 7ADD +). The histogram shows the global percentage of Annexin V-positive cells. Data are expressed with standard deviation (n = 3 ± SD). (F) Representative micrographs of colony in suspension from PNT2 and PC3 cultures treated with SASPs. Colonies were identified by crystal violet staining. The table shows the 590 nm absorbance of crystal violet released by colonies after de-staining samples in 100% methanol (n = 3 ± SD) [26]. (G) MUG quantitative senescence assay in PNT2 and PC3 cultures. The graph shows mean percentage value of senescence. 4-MUG is a beta-galactosidase substrate that does not emit fluorescence until cleaved by the enzyme to generate the fluorophore 4-methylumbelliferone. In the different experimental conditions, weperformed an assay on cell lysates to monitor the fluorophore production; the results are shown in the graph and are expressed as arbitrary units (n = 3 ± SD **p< 0.01).

Figure 2

Western blot analysis of an SASP effects the SASP on immortalized and metastatic cancer cells. The picture shows the expression levels of proteins involved in regulation of cell cycle, senescence, and apoptosis. GAPDH was used as a loading control. The graph shows mean expression levels, n = 3 ±SD, *p < 0.05, **p < 0.01).

Figure 3

Comparison of SASP contents. Venn diagram showing common and specific proteins among secretomes obtained from X-ray-irradiated or H₂0₂-treated or senescent-replicative MSCs.