Modulation of cell cycle progression by combined rapamycin-octreotide treatment in pituitary tumor cells

We have shown that combined rapamycin-octreotide treatment has a superior antiproliferative effect on pituitary tumor cells compared to each drug alone. In order to elucidate the mechanisms behind their action, pituitary tumor AtT-20 cells were treated with 1nM octreotide, 1nM rapamycin and their combination for 24 and 48 hours. The focus was on cyclins, CDKs and their inhibitors that take place in the G1 to S transition, since rapamycin was shown to act on this phase. Western blot analysis revealed that rapamycin alone or combined with octreotide decreased cyclin D1 and D3 and CDK4 and 6 levels, while octreotide had no effect. The oncogene c-myc is involved in the control of cell cycle progression and cell differentiation. mTOR/p70-S6K pathway upregulates c-myc protein levels. Rapamycin decreased c-myc and addition of octreotide did not potentiate this action, despite the fact that octreotide alone also decreased c-myc levels. On the other hand, checking for changes in the levels of the cyclin kinase inhibitor p27 revealed that single treatment with octreotide or rapamycin increased its protein levels, confirming previous studies. Interestingly, the combined octreotide-rapamycin treatment led to a dramatic increase in p27 protein levels, providing with a potential mechanism for the better antiproliferative effect of the combined compared to the single treatment. It is important to note that p27/kip1 is a tumour suppressor gene that plays a significant role in pituitary tumorigenesis, since p27 knock-out mice present with pituitary tumours and p27 protein is dysregulated in many human pituitary adenoma types. Therefore, it is possible that combined rapamycin-octreotide treatment exerts its antiproliferative action in pituitary tumor cells by upregulating the tumor suppressor p27.