The Preclinical Study Of New Molecular Targeted Therapy For Malignant Pheochromocytoma

Purpose: Malignant pheochromocytoma is difficult to diagnostic in early period and little treatment means could be used,so its prognosis is poor.The aim of this study is to find and evaluate new possible targets and strategies for the treatment of m PCC.In the early studies,we found that heat-shock protein 90(HSP90)and cyclooxygenase-2(COX-2)were significantly overexpressed in m PCC,and given that the important roles of these proteins in cells,there is reason to believe that they may be effective therapeutic targets.On this basis,we conducted this preliminary study.Materials and Methods: In the first part of the study,we used the new HSP90 inhibitor NVP-AUY922 to evaluate the HSP90 as a therapeutic target in m PCC treantment.CCK8 and Transwell assays were used to assess the effects of NVP-AUY922 on the proliferation and cell migration of the PCC cell line,PC12.Flow cytometry was used to determine the effects of NVP-AUY922 on apoptosis and cell cycle progression.Activation of PI3K/AKT and MEK/ERK signaling was measured using a western blot analysis.In vivo,a mouse xenograft model was used to test the effects of intraperitoneal injection of NVP-AUY922 on tumor growth.In the second part of the study,we first determined the antiproliferative effect of celecoxib by the CCK8 assay in m PCC cells PC12,and then explored the antitumor effect of celecoxib in m PCC xenograft.We also examined the anticancer effect of celecoxib and m TOR inhibitor everolimus combination through the CCK8 assay and xenograft experiment.To expound the possible mechanisms of enhancement of combination,the phosphorylation status of m TOR and Akt was analyzed by Western blotting.Results: In the first part of the study,NVP-AUY922 inhibited the proliferation of PC12 cells in a time-and concentration-dependent manner,and decreased the rate of migration of PC12 cells.Furthermore,we found that HSP90 inhibition induced cell cycle arrest and apoptosis.In vivo,administration of NVP-AUY922 reduced PCC tumor growth without significant weight loss.Finally,we report the modulation of MEK/ERK and PI3K/AKT signaling in response to NVP-AUY922 exposure.In the second part of the study,the results shown a decreasing of cell viability of celecoxib in a dose and time-dependent manner,and found that celecoxib could only delay the tumor growth but not inhibit.In the other hand,the combination reagents showd additive anti-tumour efficacy compared to treatment with either untreated control or agent alone in vitro and vivo.The level of p-Akt was investigated by Western blotting,and indicated that the levels of p-AKT in celecoxib treated cells were significantly reduced compared with control cells or those treated with everolimus alone.Conclusions: The first part of the stduy results showed that NVP-AUY922 exhibits potent anti-PCC activities in vitro and in vivo,and represents a promising therapeutic small molecule for treating malignant PCC.The results of second part of the study showed that COX 2 inhibition may be a new candidate strategies against m PCC but not as a single target,and the combination of celecoxib and everolimus may has a better antitumor effect.And the additive effect of combination may attribute to the inhibiting of everolimus induced AKT phosphorylation by celecoxib.