Experimental And Clinical Studies On The Effects Of Rapamycin And Other Immunosuppressive Agents On Hepatocellular Carcinoma Growth, Recurrence And Metastasis

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Asia and Africa. Although the first-line surgical therapy for HCC is liver resection, the concomitant cirrhosis most often leaves liver transplantation (LT) and not liver resection as the only potentially curative option. Live transplantation may also be the best curative treatment for HCC since it removes the tumor with the widest margin as well as the underlying cirrhosis that is responsible for both postoperative hepatic decompensation and tumor recurrence after partial hepatectomy.Because of the scarcity of donor livers, there is low tolerance within the organ allocation system for posttransplantation HCC recurrence and metastasis. Although it is known that the pharmacologic immunosuppression required after liver transplantation for HCC may be accelerated tumor recurrence and metastasis, recent reports suggest that not all immunosuppressive drugs necessarily promote HCC recurrence in transplant recipients. However, the possible influence of different immunosuppressive schedules on HCC recurrence and metastasis had been poorly investigated until recently. In the following study, we will focus the discussion on the pro- and anti-recurrence properties and mechanics of three key immunosuppressive drugs (FK506, rapamycin, and cyclosporine) on HCC. The results may provide evidence for immunosuppressive protocols introduction after OLT for HCC.Section I: Experimental studies of rapamycin and other immunosuppressive agents on growth and metastasis of hepatocellular carcinomaThe effects of three immunosuppressive agents (rapamycin, FK506, and cyclosporine) on tumor growth and metastasis were investigated in LCI-D20 nude mice model with a highly-potentially human hepatocellular carcinoma. Immunohistochemistry and terminal deoxynucleotidyl transferas-mediated dUTP nick end labeling (TUNEL) assay were used to detect the levels of PCNA, microvesseldensity (CD31 stained) and tumor cell apoptosis in vivo, respectively.Three immunosuppressive agents effects on phenotypic and cytoskeleton changes, proliferation, apoptosis, cell cycles, migration, adhesion, and invasiveness on MHCC97H cell line were also studied in vitro by scanning electron microscopy, F-actin polymerization, MTT, flow cytometry, Boyden chamber methods, and gelatin zymograph, respectively. And the expression of molecules (E-cadherin, ICAM-1, and MMP-2) implicated in tumor progression were analyzed by real-time PCR, western blot analysis in vitro, and immunohistochemistry, ELISA in vivo, respectively.The results showed that, in vivo, rapamycin inhibited tumor growth and lung metastasis compared with control in nude mice (0.76±0.38 g vs. 2.09±0.75 g, P=0.001, 3/7 vs. 7/7, P=0.007). The number of metastatic nodules in lung was increased in cyclosporine group as compared with control group (6±2 vs. 4±1, P=0.046). There was no significant difference between FK506 and control in lung metastasis rate (5/7 vs. 7/7, P=0.078). Rapamycin but not FK506 or cyclosporine inhibited tumor cell proliferation, induced apoptosis, and decreased tumor angiogenesis in LCI-D20 model.In vitro, treatment of MHCC97H resulted in striking morphological alterations in cyclosporine group, including numerous pseudopodial protrusions, increased cell motility, and metastasis abilities. Confocal laser scan microscopy of MHCC97H cells stimulated in cyclosporine showed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. Cyclosporine could also evidently increase the secretion of MMP-2 by MHCC97H and tumor cells in LCI-D20 model. Rapamycin blocked proliferation of MHCC97H and induced cell cycle arrest at the G1 checkpoint, but did not induce apoptosis. FK506 and rapamycin inhibited the ability of migration, adhesion, and invasiveness of MHCC97H. They also resulted in a significant decrease in the levels of expression of ICAM-1 and MMP-2.Our findings suggest that cyclosporine can promote hepatocellular carcinoma cell progression by a direct cellular effect, FK506 may not promote hepatocellular carcinoma cell invasiveness, and rapamycin seems to inhibit the growth, motility and invasiveness of hepatocellular carcinoma cell.Section II. Preliminary study on the molecular mechanisms of antiangiogenic effects of rapamycin in hepatocellular carcinomaThe first section demonstrated that rapamycin inhibited tumor growth and recurrence in the LCI-D20 xenograft model in nude mice by suppressing tumor angiogenesis. However, the underlying molecular mechanism was not fully elucidated. In this study, a cDNA microarray analysis followed by Real-time PCR, Western blot, and Immunohistochemistry analysis revealed that VEGF, Hypoxia-inducible factor 1 a (HIF-1a) and Angiopoietin-2 might be inhibited by rapamycin in vivo. Then we demonstrated that rapamycin could also downregulate VEGF expression in protein levels in a time-dependent manner, as well as downregulating HIF-1a expression in MHCC97H cells treated with the hypoxia mimetic agent, CoCl₂, in vitro. While HIF-1a is a well characterized regulator of VEGF expression, we may lead to the initial conclusion that rapamycin could suppress VEGF synthesis and secretion by downregulating HIF-1a expression. Our results also suggest that the anti-angiogenesis treatment of rapamycin may be associated with suppression of VEGF and Angiopoietin-2.In LCI-D20 nude mice model, when comparison was made among control, rapamycin 2mg/kg/day, interferon alpha (IFNα) 1×10⁶ U/kg/day, and combined treatment of rapamycin and IFNa for treated groups; tumor volume was 4.10cm³Â±0.58cm³, 1.82cm³Â±0.90cm³, 0.70cm³Â±0.39cm³, and 0.20cm³Â±31cm³, and incidence of lung metastasis being 100%, 83.3%, 50%, and 0%, respectively. The combined treatment did not increase the inhibition effect on the growth of tumor compared with rapamycin group (P=0.128). However, the combined therapy significantly improved the effect on lung metastasis with single treatment with either rapamycin (P=0.023) or IFNa (P=0.000). Considering the fact that IFNa is used in liver transplant patients with chronic hepatitis C and rapamycin acts as a primary immune suppressant, combination therapy of rapamycin plus IFNa has the potential clinical implication, particularly for the prevention of recurrence and metastasis after liver transplantation for hepatocellular carcinoma.Section HI: Experimental studies on the effects of immunosuppression on tumor growth, recurrence and metastasis in ratsThe pro- and anti- tumor effects of three key immunosuppressive drugs (FK506, rapamycin, and cyclosporine) had been discussed in severe combinedimmunodeficient BALB/c mice in section I and II. Here we investigated the effects of rapamycin, FK506, and cyclosporine on established tumors in rats simultaneously bearing a liver allograft.In one tumor-transplant model, SD rats received subcutaneous syngenic walker-256 carcinosarcoma cells 3 days after orthotopic liver transplantation (Wistar →SD) with character of acute rejection. Rapamycin (1 mg/kg/d), FK506 (1 mg/kg/d) or CsA (20 mg/kg/d) was initiated with transplantation. The controls received a similar volume of saline. The clinical characters, the liver function, the transplantated liver pathologic character and the cytotoxicity of spleen mononuclear from recipient against the Yac-1 cells were observed and measured on tumor postimplantative day 14. The treatment and control groups were evaluated for tumor volume thrice every one week.In a second model system, a transplanted hepatoma model was made in rats by implantation of histologically intact walker-256 tumor fragment into the left lateral lobe of the liver. On the 14th days after implantation, the hepatic tubercle was excised to be examined by histological method to confirm the successful establishment of primary carcinoma of the livers in rats. Then the orthotopic liver transplantation (SD →SD) with character of no acute rejection was performed in different groups. Intrahepatic recurrence and lung metastasis were recorded.In the first model system, results showed that the function of graft was improved, the cytotoxicity against the Yac-1 cells decreased, and the rejection reaction inhibited in the immunosuppressive treatment groups compared with control groups. The rapamycin treatment group showed a significantly decreased tumor volume (1.41±0.87cm3 vs. 3.65±0.87cm3, P=0.047), and CsA or FK506 group increased tumor size versus controls (9.56±2.81cm3 vs. 3.65±0.87cm3, P=0.000; 8.11±1.69cm3 vs. 3.65±0.87cm3, P=0.001) at the end of treatment.In the second model system, when comparison was made among control, FK506, rapamycin, and cyclosporine, incidence of recurrent tumor was 0%, 20%, 16.7% and 0%; incidence of lung metastasis being 42.9%, 60.0%, 33.3% and 0%, respectively. The incidence of lung metastasis was decreased in the rapamycin-treated animals, and tumor recurrence in liver was seen only in the cyclosporine-treated and FK506-treated animals.In conclusion, this study demonstrates that rapamycin simultaneously protects allografts from rejection and attacks tumors in a complex transplant-tumor situation.Notably, CsA or FK506 protects allografts from rejection, but cancer progression is promoted in transplant recipients.Section IV: Retrospective analysis of 134 cases of liver transplantation for hepatocellular carcinoma at the Liver cancer institute of Fudan University, chinaA total of 134 patients received orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) from January 2004 to October 2005 in authors' institute were enrolled in this study to evaluate the clinical value and prognostic factors of OLT for HCC. Their clinicopathological characteristics [including age, gender, the history of hepatitis, Child-Pugh class, α-fetal protein (AFP), blood transfusion, tumor size, number, capsule, micro-vascular invasion, tumor differentiation, pTNM stage, Milan criteria, and immunosuppressive protocol], and follow-up data [including tumor recurrence, overall (OS) and disease-free survival (DFS) of patients] were retrospectively analyzed.The 1- and 2-year OS rates of the 134 patients were 82.75%±3.47% and 68.80%±7.07% and the DFS rates were 69.13%±4.42% and 41.33%±17.20%, respectively. Univariate analysis revealed Child-Pugh class (P=0.000), tumor size (P=0.032), micro-vascular invasion (P=0.019), blood transfusion (P=0.037), Milan criteria (P=0.007) were statistically significant factors affecting OS; and Child-Pugh class (P=0.009) , tumor size (P=0.002), tumor number (P=0.041), micro-vascular invasion (P=0.000), Milan criteria (P=0.000), pTNM stage (P=0.001) were statistically significant factors affecting DFS. Multivariate analysis using Cox proportional hazards regression model demonstrated the Child-Pugh class (P=0.000) and Milan criteria (P=0.001) were the independent and statistically significant factors affecting OS; and the Child-Pugh class (P=0.009), Milan criteria (P=0.004), and micro-vascular invasion (P=0.004) were independent and statistically significant factors affecting DFS.Taking the Milan criteria as a categorized variable, the 134 cases were classified into two subgroups: within the Milan criteria group (n= 61) and beyond group (n=73). In the Milan group, with the Cox regression, only Child-Pugh class (P=0.043) was identified as the independent predictors for OS, only micro-vascular invasion (P=0.014) had independent predictive significance for DFS.In the beyond Milan criteria group, the patients with the sirolimus-based protocol (n= 27) were significantly associated with a better OS (P=0.011) as well as DFS (P=0.037), and later posttransplant recurrence time (263.0± 100.0 天 vs. 140.1± 123.5 天, P=0.035), compared with the FK506-based patients (n=46). Immunosuppressive protocol after OLT was one of the leading independent prognostic factors for both OS (P=0.015) and DFS (P=0.015) in multivariate Cox models analyses. Acute rejection (n=6), thrombocytopenia (n=3), anemia (n=12), and oral aphthous ulcers (n=7) were found in beyond Milan cohort and were easily manageable.We can draw the following theoretical considerations based on our results that The sirolimus based immunosuppression protocol appears to have beneficial effects on tumor recurrence and survival with an acceptable rate of rejection and toxicity in beyond Milan criteria group. The risk of recurrence and metastasis after OLT for HCC may be predicted preoperatively using imaging-based criteria (presence of macroscopic vascular invasion and size/number of tumors, which are surrogates for microscopic invasion), and the predictive model may be refined based on information gained on pathologic study of the explanted livers (e.g. microscopic invasion). And the prognosis of these patients depends not only on the stage of the tumor at the time of diagnosis, but also on the degree of deterioration in liver function.