Figure 10

Ghrelin inhibits basal apoptosis of canine mammary carcinoma cells. The effect of n-octanoylated ghrelin peptide (Peptides International) and GHS-R-specific peptide antagonist [D-Lys³]-GHRP6 (Peptides International) treatment on apoptosis of CMT-W1 (A), CMT-W1M (B), CMT-W2 (C) and CMT-W2M (D) cell lines, assessed by the Annexin V/PI test (BD Bioscience, USA). The number of apoptotic cells represented as a percentages of Annexin-V-positive cells (±SD) obtained with FACS Aria II (Becton Dickinson) is significantly decreased after the 1nM ghrelin treatment in CMT-W1, and CMT-W2 cell lines and after 1 nM and 10 nM ghrelin treatment in CMT-W1M, and CMT-W2M cell lines. The administration of ghrelin receptor inhibitor ([D-Lys3]-GHRP6) at concentration of 200 μM significantly increased the number of apoptotic cells in the CMT-W1M cell line. The experiment has been conducted in three replicates. p < 0.05 was marked as *, p < 0.01 was marked as ** and p < 0.001 was marked as ***. One-way ANOVA followed by Tukey HSD post-hoc test were applied. (E) The representative histograms and cytograms of CMT-W1M cell line double stainined with AnnexinV-FITC and propidium iodide (PI). Cells located on the right side of the histograms represented apoptotic cells. On the cytograms are showed normal, early apoptotic, late apoptotic and necrotic cells. Left bottom quadrant shows normal cells, top left quadrant shows necrotic cells (stained with PI only; damaged cell membrane but no phosphatydilserine exposure), right bottom quadrant shows the early apoptotic cells (stained with Annexin V only; intact cell membrane) and top right quadrant shows cells in late stage of apoptosis (stained with Annexin V and PI - phosphatydilserine exposure and damaged cell membrane).