Title

 

Authors

M. Dovizio

Doctorate School in Clinical and Experimental Medical Science
Dept. of Medicine and Science on Aging, University “G. d’Annunzio”, Chieti, Italy

 

Abstract

Extensive experimental evidence shows that platelets support tumour metastasis by contributing to tumour cell survival and spreading(1). Cyclooxygenase(COX)-2,  is overexpressed in tumor cells and, through the generation of prostaglandin(PG)E₂ plays a central role in tumorigenesis by the inhibition of apoptosis, induction of proliferation and angiogenesis(2). It has been shown that platelets may regulate COX-2 expression in inflammatory cells by a posttranscriptional mechanism involving the induction and cytoplasmatic translocation of mRNA-stabilizing protein HuR(3). Thus, I aimed to address the hypothesis that platelets participate in COX-2 overexpression in human adenocarcinoma cells(HT29) in vitro and identify the molecular mechanisms. First, I compared the time-course of the induction of COX-2 mRNA and protein  in HT29 cells cultured alone or with platelets up to 20h, by using qT-PCR and Western blot, respectively. Under the same experimental conditions, I assessed the release of platelet-derived mediators [thromboxane(TX)B₂  and platelet-derived growth factor(PDGF)-BB] by immunoassays. COX-2 mRNA decay in HT29 cells was assessed performing experiments using actinomycin-D(1.25 μ g/ml), a transcriptional inhibitor. Platelet-dependent translocation of HuR from the nucleus to the cytosol was evaluated by confocal microscopy. The contribution of  platelet COX-1-derived TXB₂ to COX-2 expression in HT29 cells was assessed by using aspirin-treated platelets. In other experiments, I explored the involvement of direct contact between platelets and HT29 cells in COX-2 expression by using a transwell insert(pore size of 0.4 μm). Finally, the role of platelet-derived soluble substances in COX-2 expression was studied by treating HT29 cells with platelet releasate and with PDGF-BB. The contribution of  Na⁺/H⁺ exchanger activity [an important  event in cellular transformation by PDGF (4)] in COX-2 expression was assessed by using the inhibitor dimethyl-amyloride.
In HT29 cells, platelets caused a 2-fold increase of COX-2 mRNA levels at 2h that remained stable up to 20h(p<0.05, n=5-8). Differently, COX-2 protein expression was significantly increased by 3-fold (p<0.01, n=11) only after 20h in association with COX-2 mRNA stabilization and HuR translocation to the cytoplasmic compartment. HT29 cells caused a time-dependent platelet activation and adhesion. Thus, the platelet product TXB₂ was released in a time-dependent manner [(ng/ml), MEAN±SEM:9.1±1.6, 18.7±3.9, 39.2±6.7 at 2, 4 and 20h, respectively(n=5)].  In contrast, PDGF-BB(localized in platelet a-granules) levels increased only after 20h of incubation.
COX-2 protein induction in HT29 cells by platelets was not affected by pretreatment of platelet with aspirin.  Interestingly, I found a 2-fold increase of HT29 cell COX-2 expression in the presence of transwell insert(p<0.05, n=7) or by platelet releasate (p<0.05, n=5), thus suggesting the participation of  a soluble factor released by platelets on cancer cell COX-2 overexpression. In fact, exogenous PDGF-BB caused a concentration-dependent COX-2 induction in HT29 cells. Interestingly, the selective inhibitor of Na⁺/H⁺ exchanger, dimethyl-amyloride(10μM) interfered with platelet-dependent induction of COX-2 in HT29 by 40%(p<0.05, n=4).
In conclusion, my results show an important role of platelets in overexpression  of COX-2 in colon cancer cells through a post-transcriptional mechanism involving COX-2 mRNA stabilization. This effect required both a direct contact between platelets and tumor cells and the activity of soluble factors released from platelets. I discovered  that Na⁺/H⁺ exchanger  activation plays a key role in  the overexpression of colon cancer cell COX-2 triggered by platelets. These results open the way to novel strategy for the treatment of colorectal cancer.

1. Gay et al, Nat Rev Cancer.2011;11:123-34.
2. Cao et al, J Cell Physiol.2002;190:279-86.
3. Dixon et al, JCI.2006;116:2727-38.
4. Ma et al.. JBC.1994; 269:30734-9.