Concomitant inhibition of class I HDAC and COX-2 exerts a antitumor effect in a human pancreatic cancer model

- Introduction :
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in developed countries. Early-stage pancreatic cancer is usually clinically silent, and disease only becomes apparent after the tumor invades surrounding tissues or metastatises to distant organs. Moreover, the current chemotherapeutic treatments have no or few effects on this type of cancer, increasing only slightly the median survival of the patients. The survival rate at 5-years is only 3%. There is a need to develop new effective therapies for PDAC patients together with a robust and fast in vivo model allowing drug screening. In this study, We tested whether the combined inhibition of cyclooxygenase-2 (COX-2) and class I histone deacetylase (HDAC) may result in a better control of PDAC. We improved the formation of pancreatic tumor on Chorioallantoic membrane (CAM), an alternative to murine model.
- Methods :
The impact of the concomitant HDAC and COX-2 inhibition on cell growth, apoptosis and cell cycle was assessed in vitro on human pancreas BxPC-3 cells treated with chemical inhibitors (SAHA, MS-275 and celecoxib) or HDAC1/3/7 siRNA. To test the potential antitumoral activity of this combination in vivo, we improved, characterized and used model of pancreas tumors growing on chick chorioallantoic membrane.
- Results :
The inhibition of HDAC1/3 by SiRNA or MS-275 treatment reduced significantly the growth of BxPC-3 cells in vitro. Furthermore, we showed by QPCR and immunoblotting that both HDAC1 and HDAC3 inhibition induced the expression of COX-2 at least via the NF-kB pathway. Based on this observation, we decided to test the effect of MS-275 combined with celecoxib a COX-2 inhibitor. This combination was more effective then either drug used alone to reduce the growth of BxPC-3 cells. By FACS analysis we showed that MS-275/celecoxib combination decreased significantly the proportion of cells in S phase and increased significantly and drastically the proportion in G0/G1 at 24, 48 and 72h. By immunobloting this GO/G1 arrest was confirmed by accumulation of cell cycle repressors (P21, P27) and disappearance of hyper phosphorylated form of RB protein.
Following a procedure development, we produced on CAM 60 mm3 functionally vascularized tumors mimicking human pancreatic tumors on CAM model. The clinical relevance of this model is supported by the CK7+/CK19+/CK20-/CEA+/Ki67+/CD56- immunolabeling. Recently we have discovered several novel biomarkers of human PDAC: MYOF, TGFBI, LTBP2. These antigens were expressed in tumors grown on CAM, reaffirming its clinical relevance.
The concept of the co-treatment by MS-275 and celecoxib was validated using this model. We showed that celecoxib alone did not significantly reduce tumor growth. MS-275 alone decreased tumor growth by 50% and combination of celecoxib and MS-275 stalled entirely the tumor growth.
- Conclusions :
Our data demonstrate a significant synergic anti-tumoral action of HDAC and COX-2 inhibitors, which set a basis for the development of potentially effective new combinatory therapies for PDAC patients.