Endogenous Humanin has antiapoptotic effect in a somatolactotrope tumor cell line.

GOTTARDO MF; PIDRE MATIAS; IMSEN M; MORENO AYALA M A; ZUCCATO C; JAITA G; CANDOLFI M; ROMANOWSKI VICTOR; SEILICOVICH A

Orlando

Congreso; ENDO Annual Meeting; 2017

Humanin (HN) is a 24-amino acid peptide originally isolated from a cDNA library of surviving neurons of Alzheimer´s disease. HN has a cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Rattin (HNr), an homologous peptide of HN in rat, has also a neuroprotective action. Previously, we have shown that the expression of HNr in GH3 cells (somatolactotrope tumor cell line) is higher than anterior pituitary normal cells, moreover, exogenous HN inhibited the apoptotic effect of TNF-α in these cells. In the present study, we evaluated the action of HN inhibition on apoptosis and tumor progression in GH3 cells. We designed a shRNA (short hairpin RNA) that inhibits HNr expression in GH3 cells. We determined the effect of HN inhibition on apoptosis at 16, 24 and 48 hs. The percentage of apoptotic cells was determined by TUNEL assay. shRNA increased apoptosis at 24 h (C 24h: 0.6 %; shRNA 24 h: 2.0 %) and 48 hs (C 48h: 3.8 %; shRNA 48h: 9,4 %, p< 0.05). GH3 cells were incubated with shRNA in presence of TNF-α (50 ng/ml), shRNA increased the basal and induced apoptosis by TNF-α (C: 23.7 %; shRNA: 47.1 %; TNF-α: 38.3 %; shRNA-TNF-α: 56.3 %, p< 0.05, χ2). These results not only suggest that the endogenous HNr would play a cytoprotective role but also has a proapoptotic action against a stimulus.In order to evaluate the role of inhibition of mRNA HNr in animal models of pituitary adenomas, we generate a recombinant baculovirus that expressed shRNA (BVshRNA). The baculovirus (BV) are enveloped viruses formed by a molecule of dsDNA to infect insects in larval instar. To carry out in vivo tests in an animal model of pituitary tumors, described previously in the vector pBacPAK9 transfer cassette, which has regions of homology to the genome of the baculovirus AcMNPV, allowing the generation of recombinant baculoviruses by double recombination cloned homologous. We observed that BVshRNA infected GH3 cells and increased apoptosis on GH3 cells (C: 1.2; BVshRNA: 5.8) indicating that endogenous HN again has an antiapoptotic effect. For in vivo experiments, female nude mice were injected s.c with 3 x 106 GH3 cells. When tumor volume reached 200 mm3, mouse were injected intratumorally (2 injections 40 µl per tumor) with BVcontrol or BVshRNA with a MOI of 1x109. We measured tumor progression at 15 days post-inoculation. We note that the infected BVshRNA mice had smaller tumor size, indicating that endogenous HNr increases tumor growth and we observed that mice with BVshRNA have major survival than control.Our results suggest that endogenous HN plays an antiapoptotic role in GH3 cells. Alterations in HN expression may play a role in the development of anterior pituitary tumors. Generating a baculovirus with a RNA interference for HN allows the possibility of developing therapies using Humanin as a potential target for the treatment of tumors.