FP29-4 Inhibitory effect of IGF1R silencing by small interfering RNA (siRNA) in NCI H295R adrenocortical tumor cell line

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP29-Adrenal Tumors & Pheochromocytoma
Translational
Monday, June 17, 2013: 10:45 AM-11:15 AM
Presentation Start Time: 11:00 AM
Room 134 (Moscone Center)

Poster Board MON-4
Tamaya Castro Ribeiro*1, Alexander Augusto Lima Jorge2, Luciana Ribeiro Montenegro3, Madson Q Almeida4, Bruno Ferraz Souza5, Mirian Yumie Nishi6, Berenice Bilharinho Mendonca7 and Ana Claudia Latronico3
1ospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, 2University of Sao Paulo, Sao Paulo, Brazil, 3Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, Brazil, 4Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, 5Hospital das Clínicas da Faculdade de Medicina da USP, HCFMUSP, São Paulo, 6Disciplina de Endocrinologia e Metabologia, Laboratório de Hormônios e Genética Molecular/ LIM42, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, Brazil, 7Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
Background: The insulin-like growth factor (IGF) signaling pathway has many important roles in normal cell growth and development. This system is implicated in various pathophysiological conditions, and is thought to play a particularly role in tumorigenesis. IGF1R is overexpressed in a variety of human cancers, including adrenocortical tumors. This upregulation can be implicated in the adrenocortical malignancy.

Objective: Investigate if siRNA could downregulate IGF-1R expression in a human adrenocortical cell line and evaluate its effects on cell proliferation and apoptosis.

Methods: The human adrenocortical tumor NCI H295R line was cultured in RPMI 1640 medium. All experiments were carried out in four groups: 1) untreated NCI H295R cells, 2) NCI H295R cells transfected with a siRNA negative control, 3) NCI H295R cells transfected with specific IGF1R siRNA # 1 (exon 18) and 4) NCI H295R cells transfected with specific IGF1R siRNA # 2 (exon 2). IGF-1R gene and protein expression were determined by real-time PCR and Western blot, respectively. The effects of IGF-1R silencing on cancer cell growth was assessed by a colorimetric assay. Apoptosis analysis was based on the caspase-3/7 activity after treatment with siRNA.

Results: The relative values of IGF1R mRNA decreased approximately 50% and Western blot analysis revealed a 30% of reduction in IGF-1R protein. The two specific siRNA for IGF1R had similar efficiency and were able to reduce significantly IGF-1R expression both at the messenger RNA (p < 0.01 for both siRNAs) and protein levels (p = 0.027 for siRNA # 1 and p = 0.009 for siRNA # 2). The untreated cells and the negative control group exhibited equivalent mRNA and protein expressions (p = ns). Downregulation of this gene was accompanied by a reduction in 40% of cell growth in vitro and an increase in 45% in cell apoptosis.

Conclusion: These findings demonstrated that decreasing IGF-1R mRNA and protein expressions with siRNA in NCI H295R cells could inhibit tumoral cell growth in vitro.

Nothing to Disclose: TCR, AALJ, LRM, MQA, BFS, MYN, BBM, ACL

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Financial support:  Fapesp #10/09503-7