Pituitary tumors are the most common primary intracranial neoplasms. Humanin (HN) and Rattin (HNr), a rat homolog of HN, are short peptides with a cytoprotective action. In the present study, we aimed to evaluate whether endogenous HNr plays an antiapoptotic role in pituitary tumor cells. Thus, we used RNA interference based on short-hairpin RNA (shRNA) targeted to HNr (shHNr). A plasmid including the coding sequences for shHNr and dTomato fluorescent reporter gene was developed (pUC-shHNr). Transfection of somatolactotrope GH3 cells with pUC-shHNr increased apoptosis, suggesting that endogenous HNr plays a cytoprotective role in pituitary tumor cells. In order to evaluate the effect of blockade of endogenous HNr expression in vivo, we constructed a recombinant baculovirus (BV) encoding shHNr (BV-shHNr). In vitro, BV-shRNA was capable of transducing more than 80% of GH3 cells and decreased HNr mRNA. Also, BV-shHNr increased apoptosis in transduced GH3 cells. Intratumor injection of BV-shHNr to nude mice bearing s.c. GH3 tumors increased the number of apoptotic cells, delayed tumor growth and enhanced survival rate, suggesting that endogenous HNr may be involved in pituitary tumor progression. These preclinical data suggests that the silencing of HN expression could have a therapeutic impact on the treatment of pituitary tumors.
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Melmed S (2009) Acromegaly pathogenesis and treatment. J Clin Invest 119:3189–202
Melmed S (2003) Mechanisms for pituitary tumorigenesis: the plastic pituitary. J Clin Invest 112:1603–1618
Gillam MP, Molitch ME, Lombardi G, Colao A (2006) Advances in the treatment of prolactinomas. Endocr Rev 27:485–534
Benveniste RJ, King WA, Walsh J, Lee JS, Delman BN, Post KD (2005) Repeated transsphenoidal surgery to treat recurrent or residual pituitary adenoma. J Neurosurg 102:1004–1012
Osamura RY, Kajiya H, Takei M et al (2008) Pathology of the human pituitary adenomas. Histochem Cell Biol 130:495–507
Gottardo MF, Jaita G, Magri ML et al (2014) Antiapoptotic factor humanin is expressed in normal and tumoral pituitary cells and protects them from TNF-alpha-induced apoptosis. PLoS ONE 9:e111548
Gottardo MF, Moreno Ayala M, Ferraris J et al (2017) Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-kappaB activation. J Cell Commun Signal 11:329–340
Hashimoto Y, Niikura T, Ito Y et al (2001) Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer’s disease-relevant insults. J Neurosci 21:9235–9245
Hashimoto Y, Niikura T, Tajima H et al (2001) A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer’s disease genes and Abeta. Proc Natl Acad Sci USA 98:6336–6341
Niikura T, Tajima H, Kita Y (2006) Neuronal cell death in Alzheimer’s disease and a neuroprotective factor, humanin. Curr Neuropharmacol 4:139–147
Zapala B, Kaczynski L, Kiec-Wilk B et al (2010) Humanins, the neuroprotective and cytoprotective peptides with antiapoptotic and anti-inflammatory properties. Pharmacol Rep 62: 767–777
Niikura T, Hashimoto Y, Tajima H et al (2003) A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer’s disease-relevant insults. Eur J Neurosci 17:1150–1158
Jia Y, Ohanyan A, Lue YH et al (2015) The effects of humanin and its analogues on male germ cell apoptosis induced by chemotherapeutic drugs. Apoptosis 20:551–561
Lue Y, Swerdloff R, Liu Q et al (2010) Opposing roles of insulin-like growth factor binding protein 3 and humanin in the regulation of testicular germ cell apoptosis. Endocrinology 151:350–357
Gong Z, Tas E, Muzumdar R (2014) Humanin and age-related diseases: a new link? Front Endocrinol 5:210
Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958
Lambeth LS, Smith CA (2013) Short hairpin RNA-mediated gene silencing. Methods Mol Biol 942:205–232
Snove O Jr, Rossi JJ (2006) Expressing short hairpin RNAs in vivo. Nat Methods 3:689–695
Pidre ML, Ferrelli ML, Haase S, Romanowski V (2013) Baculovirus display: a novel tool for vaccination. In: Romanowski V (ed) Biochemistry genetics and molecular biology. Intech, Rijeka, pp 137–164
Hu YC (2008) Baculoviral vectors for gene delivery: a review. Curr Gene Ther 8:54–65
Kwang TW, Zeng X, Wang S (2016) Manufacturing of AcMNPV baculovirus vectors to enable gene therapy trials. Mol Ther Methods Clin Dev 3:15050
Je YH, Chang JH, Choi JY et al (2001) A defective viral genome maintained in Escherichia coli for the generation of baculovirus expression vectors. Biotech Lett 23:575–582
Haase S, McCarthy CB, Ferrelli ML, Pidre ML, Sciocco-Cap A, Romanowski V (2015) Development of a recombination system for the generation of occlusion positive genetically modified Anticarsia gemmatalis multiple nucleopolyhedrovirus. Viruses 7:1599–1612
Ferraris J, Boutillon F, Bernadet M, Seilicovich A, Goffin V, Pisera D (2012) Prolactin antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression. J Physiol Endocrinol Metab 302:E356–E364
Ferraris J, Zárate S, Jaita G, Boutillon F, Bernadet M, Auffret J, Seilicovich A, Binart N, Goffin V, Pisera D (2014) Prolactin induces apoptosis of lactotropes in female rodents. PLoS ONE 9:e97383
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408
Candolfi M, Jaita G, Zaldivar V, Zárate S, Ferrari L, Pisera D, Castro MG, Seilicovich A (2005) Progesterone antagonizes the permissive action of estradiol on tumor necrosis factor-alpha-induced apoptosis of anterior pituitary cells. Endocrinology 146(2):736–743
Candolfi M, Jaita G, Pisera D, Ferrari L, Barcia C, Lui JC, Castro MG, Seilicovich A (2006). Adenoviral vectors encoding tumor necrosis factor-alpha and FasL induce apoptosis of normal and tumoral anterior pituitary cells. J Endocrinol 189(3):681–690
Mottaghi-Dastjerdi N, Soltany-Rezaee-Rad M, Sepehrizadeh Z, Roshandel G, Ebrahimifard F, Setayesh N (2014) Genome expression analysis by suppression subtractive hybridization identified overexpression of Humanin, a target gene in gastric cancer chemoresistance. Daru 22:14
Matsuoka M, Hashimoto Y (2010) Humanin and the receptors for humanin. Mol Neurobiol 41:22–28
Yen K, Lee C, Mehta H, Cohen P (2013) The emerging role of the mitochondrial-derived peptide humanin in stress resistance. J Mol Endocrinol 50:R11–R19
Niikura T, Chiba T, Aiso S, Matsuoka M, Nishimoto I (2004) Humanin: after the discovery. Mol Neurobiol 30:327–340
Eriksson E, Wickstrom M, Perup LS et al (2014) Protective role of humanin on bortezomib-induced bone growth impairment in anticancer treatment. J Natl Cancer Inst https://doi.org/10.1093/jnci/djt459
Surampudi P, Chang I, Lue Y et al (2015) Humanin protects against chemotherapy-induced stage-specific male germ cell apoptosis in rats. Andrology 3:582–589
Cohen P (2014) New role for the mitochondrial peptide humanin: protective agent against chemotherapy-induced side effects. J Natl Cancer Inst 106:dju006
Felberbaum RS (2015) The baculovirus expression vector system: a commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnol J 10:702–714
Makkonen KE, Airenne K, Yla-Herttulala S (2015) Baculovirus-mediated gene delivery and RNAi applications. Viruses 7:2099–2125
Luo WY, Shih YS, Lo WH et al (2011) Baculovirus vectors for antiangiogenesis-based cancer gene therapy. Cancer Gene Ther 18:637–645
This work was supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas, National Research Council (PIP 11420110100353 to M.C.; PIP 11220120100261 to A.S, doctoral fellowships to MFG, MP, ASA), Agencia Nacional de Promoción Científica y Tecnológica (PICT 2013-0310, PICT-2015-3309 to M.C.; PICT 2014-0334 to A.S.; PICT 2014-1827 to V.R., doctoral fellowship to CZ), the University of Buenos Aires (20020130100020 to A.S.) and the National University of La Plata (X703 to V.R.).
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the Ethics Committee of the School of Medicine, University of Buenos Aires.
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Generation of recombinant baculovirus for delivery of shRNAs in mammalian cells. Representative images of Sf9 insect cells co-transfected with bAcGoza bacmid and the plasmid pBacPAK9-shHNr; observation made at 96 h post-transfection. Left panel: DAPI; medium panel: cells with recombinant baculovirus expressing dTomato protein; right panel: cells infected with recombinant baculovirus BV-shHNr observed under phase contrast microscopy (TIF 124 KB)
Transfection efficiency of the plasmid with HNr interfering RNA. GH3 cells were transfected with 1 μg of plasmid DNA pUC-shHNr, incubated for 16, 24 or 48 h and processed for the detection of HNr by immunofluorescence. (a) Representative microphotographs of transfected cells. Arrows indicate transfected cells showing dTomato and HNr expression. Nuclei were counterstained with DAPI. Left panels: DAPI; medium panels: HNr; right panels: dTomato. (b) Percentage of transfected (dTomato-positive) cells. Each column represents the percentage ± CL of dTomato-positive GH3 cells (n ≥ 1000 cells/group) (TIF 143 KB)
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Gottardo, M.F., Pidre, M.L., Zuccato, C. et al. Baculovirus-based gene silencing of Humanin for the treatment of pituitary tumors. Apoptosis 23, 143–151 (2018). https://doi.org/10.1007/s10495-018-1444-0
- Pituitary tumor