Tumor recurrence after liver transplantation still remains a significant problem in patients with hepatocellular carcinoma. The small GTPase Rho/Rho-associated kinase (ROCK) pathway is involved in the motility and invasiveness of cancer cells. We investigated whether tacrolimus activated the Rho/ROCK signal pathway to promote the invasiveness of rat hepatocellular carcinoma cells. We also investigated whether the ROCK inhibitor Y-27632 suppressed tumor recurrence after experimental liver transplantation in a rat hepatocellular carcinoma model. Orthotopic liver transplantation was performed in hepatocellular carcinoma cell line McA-RH7777-bearing rats. Tacrolimus was administered to liver transplant rats and these rats were divided into two groups: the Y-27632-treated (10 mg/kg, for 28 days) group and the Y-27632-untreated group. Tacrolimus enhanced the cancer cell migration and stimulated phosphorylation of the myosin light chain (MLC), a downstream effector of Rho/ROCK signaling. Y-27632 suppressed the cancer cell migration and tacrolimus-induced MLC phosphorylation. Suppression of tumor recurrence after liver transplantation and significant prolongation of survival were observed in the Y-27632-treated rats in comparison with theY-27632-untreated rats. Tacrolimus stimulates the Rho/ROCK signal pathway to enhance the invasiveness of hepatocellular carcinoma, and the ROCK inhibitor Y-27632 can be used as a new antimetastatic agent for the prevention of tumor recurrence after liver transplantation.
Hepatocellular carcinoma (HCC) is frequently observed in patients with chronic virus-associated liver disease. These patients often have a poor hepatic functional reserve that does not allow liver resection and ablation (1). Orthotopic liver transplantation (OLT) can offer a cure for both chronic liver disease and HCC (2). Poor long-term results were obtained in early studies due to the high tumor recurrence rate, from 30% to 70%. These led to reconsideration of the indication of liver transplantation in the presence of HCC (3,4). In 1996, Mazzaferro and colleagues demonstrated that strict selection of patients with HCC on the basis of tumor staging prior to transplantation enabled satisfactory tumor-free survival (5). The criteria outlined by Mazzaferro (the so-called Milano criteria) are now followed by a large number of transplant units worldwide. Unfortunately, only a small number of patients with HCC are potential transplantation candidates, and many patients with HCC who do not meet the criteria have no chance of cure. Although various strategies have been tested to improve outcomes in these patients, tumor recurrence still remains a significant problem when long-term results are analyze (6–9).
The high incidence of neoplasm and its aggressive progression, which are associated with immunosuppressive therapy, are thought to be due to the resulting impairment of the organ recipient's immunosurveillance system (10). HCC recurrence after liver transplantation has been shown to be associated with the trough blood level of the immunosuppressive agent cyclosporine (CsA) (11,12). Development of cancer with aggressive phenotypes has been correlated with CsA immunosuppression (13). Recently, Hojo et al. reported a mechanism for the increased occurrence of malignancy, which is independent of host immunity, in which CsA induces phenotypic changes (including invasiveness of nontransformed cells) by a cell-autonomous mechanism (14). However, the molecular mechanisms are still unknown. Although there is evidence of neoplastic potential of tacrolimus (15) that has had a major impact on improving patient outcome following liver transplantation, a direct cause-and-effect relationship between tacrolimus use and cancer promotion still remains a subject of debate.
The Rho protein is a well-known member of the p21 Ras superfamily of small GTPases, which exhibit both GDP/GTP binding and GTPase activities (16,17). Rho regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology (18), motility (19), cytoskeletal dynamics (20) and tumor progression (21,22), and it also functions as a molecular switch in the cells (16,17). Rho activates Rho-associated kinase (ROCK), and this then increases myosin light chain (MLC) phosphorylation (23). Phosphorylated MLC induces myosin contraction and subsequently the assembly of actin stress fibers and focal adhesions (24). Activation of Rho/ROCK signaling is also known to stimulate the assembly of actin stress fibers and enhance the motility and invasion of rat hepatoma cells (25) and human ovarian cancer cells (26). Studies using clinical specimens showed a relationship between the expression level of RhoC, an isoform of Rho, and tumor aggressiveness in breast cancer (27) and ovarian cancer (28). Furthermore, RhoC also enhances the metastasis of melanoma cells (29).
Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, a recently described specific inhibitor of ROCK, specifically binds amino acids 1011–1020 and 1167–1176, respectively of p160ROCK, a Rho-associated coiled-coil forming kinase, and inhibits the kinase activity in a dose-dependent manner (30). The compound has been reported to inhibit Rho-mediated cell migration and reduce smooth muscle cell contraction selectively by inhibiting the Ca2+-sensitization mechanism (30–33). This ROCK inhibitor has been shown to inhibit the dissemination of cancer cells implanted into the peritoneal cavity (25). However, the mechanism underlying the contribution of tacrolimus to the invasiveness of HCC is not understood. Furthermore, no data is available on the effect of ROCK inhibitors on the recurrence of HCC after liver transplantation.
This study was designed to test the hypothesis that tacrolimus induces phenotypic changes including invasiveness and increased cell motility via the Rho/ROCK pathway and that a ROCK inhibitor can reduce the recurrence of HCC after liver transplantation in HCC-bearing rats.
Materials and Methods
Y-27632 was supplied by Mitsubishi Pharma Corporation (Osaka, Japan). Lysophosphatidic acid (LPA), tacrolimus, bovine serum albumin (BSA), 25% glutaraldehyde, mouse monoclonal anti β- actin antibodies, and mouse monoclonal anti MLC antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit polyclonal anti phospho-myosin light chain 2 (Thr18/Ser19) antibodies, anti-p44/42 mitogen-activated protein kinase (MAPK) antibodies, and anti-phospho-p44/42 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
The rat hepatocellular carcinoma cell line, McA-RH7777 was obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37°C.
Inbred male DA (RT1a) and Buffalo (RT1b) rats weighing 200–250 g were obtained from SLC (Atsugi, Japan) and Clea Japan Inc. (Tokyo, Japan), respectively. All animals were fed rat food as desired with free access to water. They were housed in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science.
Intrahepatict implantation model
The technique for intrahepatic tumor implantation (IHTI) is basically the same as that described by Yang and colleagues, with minor modifications (34). Cells of the rat HCC cell line, McA-RH7777 were inoculated into the portal vein of a Buffalo rat under the anesthesia of isoflurane anesthesia. The rat HCC tissue, recovered from the passaged animals 3 months after tumor cell injection, was cut into small cubes of size approximately 2 × 1 × 1 mm3. The Buffalo rats were anesthetized with isoflurane. A small subxiphoid midline incision was made, and the left lateral lobe of the liver was exposed. A small superficial incision was made into the liver, and one cube (size, 2 × 1 × 1 mm3) of McA-RH7777 tumor was implanted. The incision of the liver was closed with 7-0 suture to avoid any possible early peritoneal seeding of rat HCC.
Orthotopic liver transplantation and in vivo treatment with Y-27632
Orthotopic Liver Transplantation (OLT) was performed in the strong-rejector combination of DA rat to Buffalo rat. OLT was performed by the cuff technique (35) with some modifications in hepatic artery reconstruction as previously described (36). The animals were anesthetized with isoflurane. The donor livers were completely skeletonized. After intravenous injection of 100 IU of heparin, 2 mL of cold Ringer's lactate solution was perfused through the portal vein, and the liver was removed. The livers were then transplanted orthotopically into recipient rats. The suprahepatic vena cava of the graft was anastomosed to the recipient's vena cava with a running suture. The graft portal vein and infrahepatic vena cava were connected to those of the recipient by the cuff technique. The bile ducts of the graft and recipient were connected using a splint tube. Portal vein clamping time was approximately 15 min. Rats that underwent IHTI of rat HCC tissue were randomized into the OLT group and the control group: the OLT rats were then further subdivided into the only tacrolimus-treated group and the tacrolimus plus Y-27632-treated group. Allogeneic OLTs were performed 30–35 days after IHTI. Tacrolimus (0.1 mg/kg per day) was administered intramuscularly for 14 days, and cefazolin (40 mg/kg, intramuscularly) was administered after the operation. After liver transplantation, the rats were randomized into tacrolimus plus Y-27632-treated group and only tacrolimus-treated group. The same volume of either Y-27632 (10 mg/kg per day, 10 mg lysed in 2 mL saline) or saline was administered orally by gavages for 28 days. All rats were killed 1 year later after IHTI, and autopsies were performed immediately. After macroscopic examination, the liver and lung were removed. The samples were fixed in 10% formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin-eosin for histological examination.
Chemotactic migration of cells in response to a gradient of LPA or tacrolimus was measured in a Transwell cell culture chamber. In brief, a polycarbonate membrane filter with 8-μm pores (Coaster, Cambridge, MA), which was coated with bovine fibronectin (Telios, San Diego, CA, USA), was placed in a 24-well chamber (Coaster, Cambridge, MA, USA) containing 1 nM to 10 μM of LPA or 5–100 ng/mL of tacrolimus, and cells (2.0 × 104 cells in 200 μL/well) were loaded into the upper chamber. Ligand solutions and the cell suspension were prepared in DMEM containing 0.5% BSA. Some cells were pretreated with 3, 30 or 100 μM Y-27632 for 1 h. After incubation at 37°C in 5% CO2 for 24 h, the filter was disassembled. The cells on the filter were fixed with 5% glutaraldehyde (Sigma) in phosphate-buffered saline (PBS) and stained with propidium iodide (PI) solution (Dojindo, Kumamoto, Japan). The number of cells that migrated to the lower side of the filter was counted in the five fields by using a phase-contrast microscope.
To determine the level of phosphorylation of MLC20 and MAPK, cells were grown to confluency in minimal essential medium (MEM) containing 10% FBS. In some experiments, the cells were incubated with tacrolimus for 12 h and with Y-27632 for 1 h as pre-incubation step. Cells were lysed immediately after incubation, collected by centrifugation at 150 g for 5 min, lysed in Laemmli's sodium dodecyl sulfate (SDS) sample buffer, boiled at 95°C for 10 min, and the cell lysates were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, separated proteins were transferred to a nitrocellulose membrane and immunoblotted with antibodies. The blot membrane was scanned with a flat scanner and analyzed using NIH Image software.
Cell growth assay
Cells were placed at a density of 1 × 104 cells/well in flat-bottomed 96-well plates and grow overnight in 100 μL of culture medium supplemented with 10% FBS. Subsequently, the cells were incubated in serum-free medium containing 0.1% BSA with various concentrations of LPA or tacrolimus. Incubations were continued for 24 h prior to the addition of 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT, non-radioactive proliferation assay, Promega Corp, Madison, WI, USA) for 4 h. Cellular MTT was solubilized with acidic isopropranol, and the optical density was measured at 570 nm using a 96-well plate reader and the survival fraction was then quantified.
Statistical differences in the means were examined by one-way ANOVA combined with Bonferroni's test. Animal survival was evaluated using the Kaplan–Meier method and compared using the log rank test. Differences were considered statistically significant if p < 0.05.
Tacrolimus activates Rho/ROCK signal pathway to enhance cell migration of rat hepatocellular carcinoma cells and the ROCK inhibitor reduced tacrolimus-induced migration of rat hepatocellular carcinoma cells
McA-RH7777 cells exhibited some spontaneous migration without LPA stimulation, and the addition of LPA to the lower chamber markedly induced rat HCC cell migration. The effect was significant at concentrations as low as 10 nM, and maximum stimulation was observed at 1 μM. A further increase in the concentration of LPA induced less migration, producing a typical bell-shaped curve for chemotactic movement (Figure 1A). The addition of tacrolimus to the lower chamber also significantly induced cell migration at a concentration of 5 ng/mL, and maximum stimulation was observed at 20 ng/mL. A further increase in the concentration of tacrolimus produced less migration (Figure 2A).
The enhanced migration of rat HCC cells induced by LPA or tacrolimus was significantly reduced by the addition of the ROCK inhibitor Y-27632 (Figures 1B and 2B). Furthermore, The ROCK inhibitor significantly reduced migration of rat HCC cells compared with the control (Figure 2B).
To determine whether the Rho/ROCK pathway was also involved in tacrolimus-induced cell migration, we next assessed the phosphorylation state of MLC, a downstream effector of Rho/ROCK signaling by western blotting with polyclonal antibodies specific diphophorylated MLC on Thr18/Ser19. We tested the effect of tacrolimus on Thr18/Ser19-diphosphorylation of MLC in rat HCC cells. Quantitative analysis using a scanning densitometer confirmed that diphosphorylated MLC activity was significantly increased above the control level at 12 h after tacrolimus exposure (20 ng/mL; Figure 3). We then tested the effect of Y-27632 on Thr18/Ser19-diphosphorylation of MLC in rat HCC cells. The activity of phosphorylated MLC was significantly suppressed by the addition of Y-27632 (3 or 30 μM; Figure 3).
Tacrolimus does not increase the proliferation of hepatocellular carcinoma cells
MTT assays were performed to determine whether LPA or tacrolimus affects rat HCC cell growth. After incubation of rat HCC cells with LPA for 24 h, rat HCC cell growth was significantly increased (Figure 4A). On the other hand, after incubation of rat HCC cells with tacrolimus for 24 h, the basal cell growth were not significantly altered (Figure 4B). Furthermore, Y-27632 did not influence the growth of rat HCC cells (Figure 4C). To determine whether tacrolimus promotes MAPK phosphorylation, rat HCC cells were incubated for 12 h with tacrolimus and then analyzed by western blotting assays. Treatment of rat HCC cells with tacrolimus (20 ng/mL) had no effect on the phosphorylation level of MAPK (Figure 5). Treatment of rat HCC cells with Y-27632 (3 μM) did not inhibit MAPK phosphorylation (Figure 5).
The ROCK inhibitor suppresses recurrence of hepatocellular carcinoma after rat liver transplantation
All animals that did not undergo liver transplantation died of pulmonary metastasis with a mean survival period of 116.7 days (Figure 6). Since the DA-liver grafted rats died of rejection with a mean survival of 14days (37), tacrolimus (0.1 mg/kg) was administered subcutaneously into transplanted rats daily for 2 weeks after liver transplantation to prevent rejection. The mean survival of rats that underwent liver transplantation was 175.1 days (Figure 6). Due to tumor recurrence, DA-liver grafting did not significantly prolong the survival of Buffalo rats in the rat HCC model. We next investigated the effect of Y-27632 on the recurrence of HCC after liver transplantation in the rat HCC model. The average size of HCC on the day of liver transplantation did not differ significantly between the Y-27632-treated and Y-27632-untreated rats: the mean diameters of HCC were 2.3 ± 0.4 cm and 2.4 ± 0.5 cm, respectively (Figure 7). Y-27632 (10 mg/kg) was administered orally every day for 28 days after liver transplantation. Y-27632 significantly prolonged the survival of DA-liver grafted rats, and the mean survival period was 303.1 days (Figure 6). Six of 7 DA-liver grafted rats that had not been treated with the ROCK inhibitor showed multiple metastatic nodules in the lungs, whereas no metastatic pulmonary nodules were found in 7 DA-liver grafted rats treated with the ROCK inhibitor (Table 1). The metastatic nodules in the lungs retrieved at autopsy and hepatomas removed at the time of liver transplantation showed moderately to poorly differentiated HCC (Figures 7 and 8).
Table 1. Rock inhibitor decrease pulmonary metastases in DA-liver graft rats
Number of rats with pulmonary metastases
Rate in each group underwent an intrahepatic tumor implantation (IHTI). Allogeneic orthotopic liver transplantations were performed in transplanted groups 30–35 days later after IHTI. After all rats were died or killed at 1 year later, tumor formation were estimated macroscopically.
Transplanted rats without ROCK inhibitor
Transplanted rats with ROCK inhibitor
This is the first study showing that the immunosuppressant tacrolimus enhanced the motility of HCC cells via the activation of the Rho/ROCK signal pathway. This is also the first study that showed that a ROCK inhibitor suppressed tumor recurrence after liver transplantation in HCC-bearing rats.
Our findings indicate that tacrolimus at a concentration of 20 ng/mL, which corresponds to clinical serum levels, promotes the transmigration of tumor cells via the Rho/ROCK pathway. This conclusion is supported by the following observations: (1) tacrolimus markedly induced rat HCC cell motility in a cell migration assay and the ROCK inhibitor suppressed migration of rat HCC cells and (2) MLC phosphorylation was augmented by the addition of tacrolimus, and the level of phosphorylated MLC was decreased by addition of the ROCK inhibitor. The Rho signaling pathway and actomyosin system have been shown to be involved in the motility and invasion of various cancer lines, including hepatoma cells (25) and human ovarian cancer cells (26). Studies using clinical specimens have shown a relationship between the expression level of Rho C, an isoform of Rho, and tumor aggressiveness in breast cancer (27). Considered collectively in the light of new data that shows an association between Rho/ROCK signaling and migration and invasion, the present results suggest that the Rho/ROCK signaling plays a central role in migration and invasion, and the effect is enhanced by tacrolimus. Two other Rho family small GTPases, Rac and Cdc42, are thought to be responsible for generating distinct actin-containing structures. For example, Rac and Cdc42 mediate growth factor-induced lamellipodia and filopodia formation, respectively, and activation of Cdc42 triggers the subsequent activation of Rac and Rho, suggesting that Rho family GTPases are coordinated to control cell motility (16,17). In our experiments, the effects of tacrolimus on Rac and Cdc42 remain unknown.
Next, we studied the effect of tacrolimus on the proliferation of rat HCC cells. Results from MTT assays performed in this study demonstrated that tacrolimus did not promote the proliferation of rat HCC cells. Tacrolimus (20 ng/mL) also did not increase the level of phosphorylation of MAPK in rat HCC cells. In fact, Schumacher et al. have shown that tacrolimus enhanced the proliferation of cells of the human HCC cell line SK-Hep 1 but not that of the human HCC cell line Hep 3B (38). Cao et al. have shown that tacrolimus had a concentration-dependent antiproliferative effect on cells of the liver cancer cell line SMMC-7221 (39). Tacrolimus-induced cell proliferation may depend on the cell type. In our study, tacrolimus, at least at the clinical dose, did not activate the MAPK to enhance the proliferation of rat HCC cells. We also studied the effect of Y-27632 on the proliferation of rat HCC cells. In the current study, Y-27632 did not inhibit the MAPK in rat HCC cells and did not suppress the proliferation of rat HCC cells. However, it has been reported that Y-27632 inhibited thrombin-stimulated DNA synthesis in rat aortic smooth muscle cell (40). As Imamura and colleagues reported that Y-27632 had no effect on the phosphorylation of MAPK in rat ascites hepatoma (MM1) cells (41), our results also showed Y-27632 did not inhibit the MAPK in rat HCC cells. The inhibitory effect of Y-27632 on cell proliferation is still controversial.
Our laboratory has been particularly interested in studying the effect of liver transplantation on survival and tumor recurrence in a rat HCC model. Previously, we described a syngeneic non-immunosuppressed transplantation model of rat HCC (42). Survival was prolonged when the animals underwent transplantation at an early stage of liver cancer. Transplantation after the development of multiple liver cancers resulted in poor survival (43,44). This time course mimics the clinical picture of liver transplantation for HCC. Furthermore, a calcineurin inhibitor such as CsA promoted tumor recurrence after liver transplantation in rat HCC (45), as shown in liver transplant patients (11,12).
Recently, cell motility mediated by the Rho/ROCK signaling pathway has been shown to play a critical role in intrahepatic metastasis of human HCC (46). Moreover, treatment of tumor-bearing animals with a ROCK inhibitor has been shown to decrease peritoneal dissemination of Rat MM1 hepatoma cells (25) and intrahepatic metastasis of human Li7 HCC cells (47). However, since no data is available on the antimetastatic effects of a ROCK inhibitor in a rat liver transplantation model, we investigated the effect of a ROCK inhibitor on the recurrence of HCC after liver transplantation in HCC-bearing rats. In this study, compared with survival of Buffalo rats that did not undergo liver transplantation, treatment with tacrolimus did not prolong the survival of Buffalo rats carrying DA-liver grafting in a rat HCC model that was not treated with the ROCK inhibitor: this was due to tumor recurrence. This result suggests that immunosuppressive therapy using tacrolimus enhances cancer invasiveness and metastasis after liver transplantation because tacrolimus itself not only causes the impairment of the organ recipient's immunosurveillance system but also promotes cancer cell motility by a cell-autonomous mechanism, as shown in our results. However, the ischemia reperfusion component in the liver transplant may stimulate tumor metastasis rather than tacrolimus. To precisely distinguish between the effects of tacrolimus and the effect of ischemia reperfusion on HCC tumor cell growth and metastasis, we need further examinations using syngeneic transplants under the same ischemia and preservation conditions. Our results also showed that the ROCK inhibitor Y-27632 significantly prolonged the survival of DA-liver grafted rats. Furthermore, DA-liver grafted rats that were not treated with the ROCK inhibitor and that died from recurrence of HCC showed the multiple metastatic nodules in the lungs, whereas the ROCK inhibitor significantly suppressed the tumor recurrence in the lung. These findings suggest that since the ROCK inhibitor has no effect on the host's immunosurveillance system, it suppresses tumor recurrence mainly by inhibiting the tacrolimus-enhanced cancer cell invasiveness. It is conceivable that the ROCK inhibitor inhibits the adherence and extravasation of circulating cancer cells, which were seeded during surgery, to capillary beds of various organs (particularly lungs) by suppressing the cancer cell invasiveness. These data suggest that this ROCK inhibitor could be a new potential agent for the prevention of tumor recurrence after liver transplantation in patients with HCC.
In summary, we have shown that tacrolimus activates the Rho/ROCK signal pathway to stimulate cell motility of rat HCC cells and enhances the invasiveness of rat HCC cells. Further, we have also shown that the ROCK inhibitor suppressed tumor recurrence after liver transplantation in a rat HCC model. ROCK inhibitors may be novel antimetastatic agents that could be used for the prevention of tumor recurrence after liver transplantation in patients with HCC.
The authors thank Y. Ishida for excellent technical assistance. This work was supported partly by Grant-in-Aid for Scientific Research (KAKENHI 16591321 (to H.T.), and 16922196, 17591471 (to Y.M.)) from the Ministry of Education, Science, Sports, and Culture of Japan and a grant (to H.T.) from the Tsuchiya Memorial Medical Foundation (Hiroshima, Japan).