Role of complex Cdk4/Cyclin D1 in somatostatin subtype 2 receptor-mediated inhibition of cell proliferation of a medullary thyroid carcinoma cell line in vitro

Somatostatin (SRIH) inhibits cell proliferation by interacting with five distinct SRIH receptor subtypes (SSTR) by several pathways in many tissues. We previously demonstrated that SRIH, by activating SHP-1, inhibits cell proliferation of the human Medullary Thyroid Carcinoma (MTC) cell line, TT, which expresses all SSTR. However, the effects of SRIH on cell cycle proteins have not been investigated, so far. We therefore investigated the effects of SRIH and of a selective SSTR2 agonist on cell cycle protein expression, mainly focusing on Cyclin D1 and its associated kinases. TT cells were serum starved for 48 h and then treated with SRIF or with a selective SSTR2 agonist, BIM-23120 for up to 60 h. Cell proliferation was verified by a colorimentric assay and by [3H]thymidine incorporation. Moreover, cell cycle progression was studied by cytofluorimetry. Cell cycle protein expression at different time points was investigated by Western blot. Cyclin D1 expression was also investigated by quantitative RT-PCR. Our data show that SRIH and the selective SSTR2 agonist, BIM-23120, reduce cell proliferation and DNA synthesis, as well as induce a delay of the cell cycle in G2/M phase. Moreover, treatment with SRIH or with BIM-23120 decreases Cyclin D1 and cdk4 protein levels, with a parallel reduction in Rb phosphorylation levels at Ser-780. These data indicate that the subtype 2 receptor-mediated antiproliferative effect of SRIH on TT cell proliferation may be exerted through a decrease in Cyclin D1 levels, and further underline the importance of SSTR2 in mediating the antiproliferative effects of SRIH, indicating a direct and strong effect on Cyclin D1-cdk4 comple