GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCt-116 colon cancer cells with 10 M GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OtM) and loss of inner mitochondrial membrane potential (Δψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADp-ribose)polymerase (pARp) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21^Waf1/CipI was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and pI staining assays. In vivo, JMR-132 decreased the volume of Ht-29, HCt-116 and HCt-15 tumors xenografted into athymic mice up to 75% (p < 0.05) and extended tumor doubling time (p < 0.001). Our observations suggest that GHRH antagonist JMR-132 exerts its antiproliferative effect on experimental colon cancer cells through p21 ^Waf1/CipI mediated S-phase arrest along with apoptosis involving the intrinsic pathway.