The potential role of early-phase graft injury in induction of late-phase chemoresistance after liver transplantation

Abstract

INTRODUCTION: Although liver transplantation dramatically increased 1-year and 3-year survival rate of the patients with advanced HCC, the postoperative tumor recurrence remains to be an obstacle without an efficient treatment. Chemotherapy, as a regular treatment for recurrent tumor, its efficiency was largely limited by the high incidence of chemoresistance. Our previous study reported that tumor recurrence rate was higher after liver transplantation using small-for-size (50%) graft compared with whole graft. However, the relationship between early-phase liver graft injury and the generation of chemoresistance after liver transplantation is still unknown. We aim to investigate the potential role of acute-phase liver graft injury on induction of late-phase chemoresistance after liver transplantation and to explore the underlying mechanism. MATERIALS AND METHODS: A rat orthotopic liver transplantation model using cirrhotic recipients was established with applying whole or small-for-size (50%) graft. The recipients were injected with a rat hepatoma cell line (MH7777) via portal vein after reperfusion to mimic the generation of recurrent tumor. Proline tyrosine kinase 2 (Pyk2) expression level was compared between small-for-size and whole grafts, as well as the tumor developed from small-for-size and whole grafts. Pyk2 overexpressed HCC cell stable clones were established. The role of Pyk2 in cisplatin resistance was investigated by in vitro studies on HCC cell proliferation and apoptosis under cisplatin environment, and further confirmed by in vivo xenograft tumorigenesis and orthotopic nude mice models. Afterwards, the gene expression profile of Pyk2 overexpressed HCC cells was screened by cDNA microarray to explore the underlying mechanism. RESULTS: Intragraft gene expression of Pyk2 increased gradually after liver transplantation (4 h, 2 d, 4 d and 7 d) in both small-for-size and whole grafts. mRNA levels of Pyk2 were significantly higher in small-for-size grafts at day 4 (55.71 vs 24.25 folds of normal liver, p=0.005) and day 7 (90.51 vs 48.5 folds, p=0.005) after liver transplantation. Pyk2 expression level was also found to be significantly higher in recurrent liver tumor from small-for-size graft at day 14 (1.75 folds of whole graft, p=0.01) and day 21 (2.41 folds of whole graft, p=0.01) after liver transplantation. In the in vitro functional studies, MTT assay and colony formation assay illustrated that overexpression of Pyk2 can promote cell proliferation under cisplatin treatment. Moreover, Annexin-V-FLOUS assay also demonstrated that overexpression of Pyk2 can improve the survival rate of HCC cells against cisplatin treatment. cDNA microarray screening showed that MDR1, GAGE, STAT1, MAP7 and CASP9, which related to cell proliferation, drug resistance, or apoptosis were significantly up-regulated or down-regulated in Pyk2 overexpressed HCC cells. In the in vivo studies, xenograft tumorigenesis and orthotopic nude mice models demonstrated that suppression of Pyk2 could sensitize liver tumor to cisplatin treatment by induction of significant necrosis and apoptosis via down-regulation of Akt signaling. CONCLUSION: Pyk2 was significantly up-regulated in both of small-for-size grafts and the tumor developed from small graft after transplantation. Pyk2 overexpression in HCC cells remarkably induced cisplatin resistance. Therefore, early-phase liver graft injury may induce late-phase chemoresistance after liver transplantation by up-regulating Pyk2.