Des-γ-Carboxyl Prothrombin Stimulates The Growth And Metastasis Of Hepatocellular Carcinoma

BackgroundsProthrombin, which synthesized in liver, is a kind of serum clotting factor and the precursor of thrombin. There are ten glutamic acid residues in the prothrombin precursor, from which y-carboxyglutamic acid residues are synthesized depending on vitamin K-dependent carboxylase. In the absence of vitamin K or in the presence of vitamin K antagonists, lacking these modified glutamic acid residues, des-y-carboxyl prothrombin (DCP) is produced in liver. Because the y-carboxyglutamic acid is a functional area in calcium-binding, it's deficiency leads to loss of the structural basis of calcium binding.DCP has been recognized as a useful tumor marker in HCC. In exploring the structure of DCP, there are two kringle domains similar to those of hepatocyte growth factor (HGF), which was identified as a potent mitogen for mature hepatocytes. The expressions of DCP in serum and tissue are associated with the biological malignant potential of HCC and the development of portal venous invasion of HCC. Previously, we found that DCP could stimulate the proliferation and metastasis of HUVEC cells. In this study, the stimulation of DCP on the growth and metastasis of HCC was observed and the mechanism of the signal pathway was investigated.Methods:In the study of DCP stimulation and mechanism of signal pathway, the human hepatocellular carcinoma cell lines HLE and SK-Hep which are the DCP-negative cells were used. The proliferation of HCC cells was evaluated by the assay of cell counting kit-8 (CCK-8). The apoptosis of HCC cells was measured by Annexin V-PI staining. The Western blotting assay was employed to evaluate the expression levels of phospho-Met, EGFR, phospho-EGFR, MMPs, phospho-ERK1/2, phospho-MEK1/2, phospho-c-Raf and angiogenic factors. Elisa analysis was used to measure the expression of angiogenic factors. Gelatin zymography analysis was employed to assess the effect of DCP on activity of MMPs. Transwell chamber assay was used to measure the promoting effect of DCP on the invasion and migration of HCC cells.In the evaluation of vitamin K2 on inhibition of cancer growth, the human hepatocellular carcinoma cell lines HepG2 and PLC/PRF/5 were used to investigate the effect of vitamin K2 via the decrease of DCP. The 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the HCC cell growth in vitro. The transwell chamber assay was used to investigate the invasion of HCC cells after DCP incubation. Western blot was employed to evaluate the expression of DCP in HCC cells.ResultsDCP significantly stimulated the proliferation of HCC cells. At the concentration of 160ng/ml of DCP for 48h exposure, the growth of HLE and SK-Hep cell was observed and the mitogenic effect for HLE cell plateaued at a 51.2% enhancement. Moreover, the percentage of apoptosis of HLE cells induced by erlotinib was decreased in the presence of DCP. DCP elevated the activity of MMPs in HLE and SK-Hep cells. Therefore, the promotion of HCC cells penetrating the Matrigel was observed. We investigated the signal pathway of DCP stimulation. The high levels of MMPs and EGFR, phosphorylation of Met, EGFR, ERK1/2, MEK1/2 and c-Raf were measured. Furthermore, we blocked the ERK1/2 MAPK signaling pathway with the ERK1/2 inhibitor PD98059 and then examined production of MMP-2 and MMP-9. Inhibition of ERK1/2 activation essentially abolished the DCP-induced MMP-2 and MMP-9 activity. Taken together the observation that DCP rapidly activates the ERK/MAPK signaling pathway and sustains its maximum expression for 120 min, and that PD98059 inhibits DCP induced MMP-2 and MMP-9 activity, our results suggest that the stimulatory effects of DCP on production of MMP-2 and MMP-9 are mediated via activation of the ERK1/2 MAPK signaling pathway.Vitamin K2 might inhibit the proliferation HCC cells via the decrease of DCP. The results showed that vitmin K2 inhibited the expression of DCP in PLC/PRF/5 and HepG2 cells. The proliferation and invasion of HCC cells were inhibited. At the concentration of 40μM of vitamin K2 for 120h incubation, the maximum inhibition of PLC/PRF/5 cells was reached at 48.2%. In the range of 2-40μM of VitK2, the number of PLC/PRF/5 cells migrating through the Matrigel coated membrane were decreased with a plateau of 72.4%. At the same time, the levels of DCP were decreased and the rates of inhibition were well correlated with the inhibition of anti-proliferative effects of vitamin K2, indicating that the inhibition of vitamin K2 was via decreases of DCP.Conclusions:DCP stimulates the growth and metastasis of HCC cells. The mechanism of the stimulation may via the EGFR-Ras-Raf-MEK-ERK cell signal pathway. Vitamin K2 could inhibit the proliferation and invasion of HCC cells via the inhibition of DCP.