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 muM 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 (Delta Psi m). 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/Cip1) 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/Cip1) mediated S-phase arrest along with apoptosis involving the intrinsic pathway.
Hohla
Florian
F
Department of Internal Medicine, General Hospital Oberndorf, Oberndorf, Austria.
Buchholz
Stefan
S
Schally
Andrew V
AV
Seitz
Stefan
S
Rick
Ferenc G
FG
Szalontay
Luca
L
Varga
Jozsef L
JL
Zarandi
Marta
M
Halmos
Gabor
G
Vidaurre
Irving
I
Krishan
Awtar
A
Kurtoglu
Metin
M
Chandna
Sudhir
S
Aigner
Elmar
E
Datz
Christian
C
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects; D013007:Growth Hormone-Releasing Hormone / Q000037:antagonists & inhibitors / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D051379:Mice; D008819:Mice, Nude; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016196:S Phase; D017337:Sermorelin / Q000031:analogs & derivatives* / Q000494:pharmacology; D014183:Transplantation, Heterologous; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; C534070:GHRH(1-29)NH2, PhAcTyr(1)-Arg(2)-P(H)e(4-CL)(6)-Ala(8)-Tyr(Me)(10)-His(11)-Abu(15)-His(20)-Nle(27)-Arg(28)-HLCr(29)-; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D017337:Sermorelin; D013007:Growth Hormone-Releasing Hormone; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053453:Caspase 9