FTY720 induces apoptosis of human hepatoma cell lines through PI3-K-mediated Akt dephosphorylation

Our aim was to study the anticancer effect of the novel immunomodulator FTY720 in vitro and in vivo by investiga-tion of cell cycle entry, cell cycle regulation, cell survival and apoptosis pathways. Three hepatoma cell lines with different p53 statuses (HepG2, Huh-7 and Hep3B) and one non-tumorigenic immortalized liver cell line (MIHA) were used for an in vitro study. The in vivo effects of FTY720 were evaluated in a nude mouse tumor model. Cell cycle distribution and cell cycle regulator proteins p27 Kip1 and cyclin D1, together with the PI3-K/Akt pathway, mitogen-activated protein kinases and cleaved caspase-3 and caspase-9, were evaluated. FTY720 selectively induced cell apoptosis in hepatoma cell lines with overexpression of cleaved caspase-3 and caspase-9, but the same phenomena were not found in MIHA cells. FTY720 induced Akt dephosphorylation at Ser473 mediated by phosphoinositide 3-kinase (PI3-K) inhibition. Dephosphorylation led to down-regulation of p42/p44 and dephosphorylation of Forkhead transcription factor and GSK-3 b and, subsequently, up-regulation of p27 Kip1 and down-regulation of cyclin D1. In our in vivo model FTY720 induced apoptosis of tumor cells by down-regulation of the Akt pathway. FTY720 suppressed tumor growth without notable side-effects in normal liver. In conclusion, FTY720 is a novel anticancer agent that induces apoptosis of hepatoma cell lines both in vitro and in vivo through PI3-K-mediated Akt dephosphorylation in a p53-independent manner.


Introduction
Hepatocellular carcinoma (HCC), which is particularly prevalent in Asia and Africa, is a major malignancy world wide and is one of the most common causes of cancer mortality (1). Although surgical resection is the first treatment of choice for HCC, most cases present at an advanced stage when surgery can no longer cure the disease. Chemotherapy is commonly applied for inoperable HCC, but its results are often discouraging because of chemoresistance conferred by p53 mutation and toxicity to normal cells. Therefore, a novel pharmaceutical therapy needs to be developed. Recently, FTY720, a novel immunomodulator, has been reported to have a strong antitumor effect for breast cancer (2), bladder cancer (3) and leukemia (4). So far, the feasibility of using this drug in HCC treatment has not been studied. The precise cellular mechanisms of FTY720 on cancer cells are not completely understood. Therefore, in this study we aimed to investigate the in vitro and in vivo anticancer potential of FTY720 and ascertain its exact mechanism of inducing apoptosis in HCC cells.
Phosphoinositide 3-kinase (PI3-K), an upstream effector of Ser/Thr kinase Akt (also known as protein kinase B), is the major mediator of survival signals that protect cells from apoptosis (5). PI3-K activity is opposed by the dual specificity phosphatase PTEN (also known as MMAC1 and TEP1), which acts on position 3 of the inositol ring (6). Mutations of PTEN have been demonstrated in many cancers and, in particular, in HCC (7), suggesting that PI3-K and Akt are critical factors in the genesis of cancers (8). Therefore, PI3-K acting in cellular signal transduction pathways plays a central role in cancer cell survival.
The immunomodulator FTY720, a synthetic structural analog of myriocin, a metabolite in ascomycetes, was originally demonstrated to induce apoptosis of infiltrated lymphocytes, especially T cells, and to prolong allograft survival after solid organ transplantation (9,10). Recently, FTY720 was found to be effective at inducing cancer cell apoptosis via caspase-3 activation in the human prostate cancer cell line DU145 (11). FTY720 was also reported to induce apoptosis through mitochondria by inhibition of bcl-2, independent of the Fas pathway (12,13). Furthermore, FTY720 induced G 0 /G 1 arrest in HL60RG and Jurkat cells via dephosphorylation of retinoblastoma protein (14).
The PI3-K and Akt pathway is very important in governing cell survival and prevention of apoptosis in tumors. In this study we have investigated the effect of FTY720 on the Akt cell survival pathway and found that Akt dephosphorylation plays a central role in cell growth arrest and apoptosis upon administration of FTY720 to hepatoma cell lines. Dephosphorylation of Akt Ser473 but not Akt Thr308 might lead to dephosphorylation of Forkhead transcription factor (FKHR), GSK-3b and p42/p44, resulting in p53-independent G 1 arrest, possibly by up-regulation of p27 Kip1 and down-regulation of cyclin D1. It might also activate the apoptotic pathway by up-regulation of caspase-3 and caspase-9, both in vitro and in vivo. hepatoblastoma cell line HepG2 (American Type Culture Collection, HB-8065) were maintained in Dulbecco's modified Eagle's medium (DMEM) with high glucose (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL), 50 U/ml penicillin G and 50 mg/ml streptomycin (Gibco BRL) at 37 C in a humidified atmosphere containing 5% CO 2 . The human non-tumorigenic, immortalized liver cell line MIHA (16) (kindly provided by Dr J.R.Chowdhury, Albert Einstein College of Medicine, New York) was maintained in Chee's medium (Gibco BRL) supplemented with 5% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mmol/l L-glutamine, 50 mmol/l dexamethasone and 20 mU/ml recombinant insulin (Boehringer Mannheim, Indianapolis, IN). FTY720 was kindly provided as a powder by Novartis Pharmaceuticals Ltd (Basel, Switzerland). It was added to the culture medium at different concentrations after dissolution in normal saline. The constitutively active PI3-K plasmid (CD2-p110) was a gift from Dr D.Cantrell (University of Dundee, Dundee, UK).

Cell cycle analysis
After FTY720 treatment, the DNA content and cell cycle distribution of hepatoma cells grown in 6-well plates were determined by flow cytometry. The cells were plated at a low density (5 Â 10 4 cells/well) and were harvested at 0, 4, 8, 16 and 24 h. Another set of controls was employed without FTY720 treatment. They were trypsinized and washed once in phosphate-buffered saline (PBS). They were then fixed in cold 70% ethanol and stored at 4 C. Before testing, the ethanol was removed and the cells were resuspended in PBS. The fixed cells were then washed with PBS and treated with RNase (1 mg/ml) and stained with propidium iodide (50 mg/ml) for 30 min at 37 C. Cell cycle analysis was performed in an EPICS profile analyzer, using ModFit LT2.0 software (Coulter Electronics, Hialeah, FL).
Detection of apoptotic cells by 4,6-diamidino-2-phenylindole (DAPI) staining and TUNEL assay Hep3B cells were plated onto 12 mm coverslips in DMEM medium at~70% confluence for 24 h. The cells were then treated with FTY720 at the IC 10 and IC 50 doses for 0, 4, 8, 16 and 24 h. They were fixed in ice-cold acetone and methanol (1:1), washed with PBS and then stained with DAPI for 5 min. The stained cells were examined under a fluorescence microscope and cells were considered to undergo apoptosis based on the appearance of nuclear fragmentation. A total of 500 cells were counted in 5 fields/sample. The percentage of apoptotic cells was calculated as (number of apoptotic cells Ä total number of cells counted) Â 100. For the TUNEL assay the procedure was the same as for DAPI staining except that the cells were stained in situ with a POD cell death detection kit (Boehringer Manheim, Manheim, Germany) and counterstained with propidium iodide.
Assay for PI3-K activity Cultured Hep3B cells were treated with FTY720 at the IC 10 and IC 50 doses for 4 h and then stimulated with 100 nM insulin for 10 min at 37 C. The cells were then lysed in 1 ml of NP-40 containing lysis buffer with protease inhibitors. Equal amounts of protein lysate (Protein Assay Kit, Bio-Rad, Hercules, CA) were incubated with 5 ml of anti-PI3-kinase antibody (Upstate Biotechnology, Lake Placid, NY) and bound with protein A--agarose beads for 1 h at 4 C. Immunoprecipitates were washed and evaluated for PI3-K activity by competitive ELISA (Echelon Biosciences, Salt Lake City, UT) as described (17). Briefly, PI3-K bound to the beads was incubated for 2 h with 10 mM diacylglycerol phosphatidylinositol 4,5-diphosphate (diC 8 PI(4,5)P 2 ) substrate at room temperature in 50 ml of buffer containing 5 mM HEPES (pH 7), 25 mM ATP and 2.5 mM MgCl 2 . The beads removed by centrifugation and the supernatant, or known concentrations of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], were incubated for 1 h with 50 ml of PI(3,4,5)P 3 -binding reagent and then transferred to a detection plate coated with PI(3,4,5)P 3 . Platebound binding reagent was quantitated using a secondary detection reagent, peroxidase, and peroxidase substrate, with the reaction product measured by absorbance at 450 nm.
In vivo effect of FTY720 in the nude mouse tumor model We studied the in vivo effect of FTY720 on the cell survival and apoptosis pathway using a nude mouse tumor model. The mice were housed in microisolator cages under positive air pressure and maintained at a constant temperature (22 C) and humidity. Surgical operations and drug administration were performed in a laminar flow cabinet. Four-week-old male BALB/c nu/nu mice were used to establish the tumor model by s.c. implantation of 1--2 mm 3 tumor tissue originating from the Hep3B strain. FTY720 was given at 10 mg/kg by i.p. injection when the tumor size reached 32 mm 3 (L Â W 2 /2) at 10 mg/kg for 10 or 20 days. Tumor tissue and liver were sampled for detection of protein expression and morphological examination. Tumor growth was also compared between the rats with and without FTY720 treatment.
Morphological examination TUNEL staining was performed in situ using a POD cell death detection kit (Boehringer Manheim) following the manufacturer's protocol. Apoptotic cells were determined with careful observation of TUNEL-positive nuclei. For hematoxylin and eosin (H&E) staining the samples were stained with H&E. The images were captured by an image analysis system (Eclipse E600; Nikon, Japan).

FTY720 selectively induces significant hepatoma cell death in vitro
To determine the cytotoxic effect of FTY720 on hepatoma cell lines, the MTT assay was performed to determine the IC 10 and IC 50 doses. The MTT assay demonstrated that FTY720 treatment induced dramatic cell death in hepatoma cell lines in a time-and dose-dependent manner ( Figure 1). As shown in Table I, there was no significant difference in drug sensitivity (IC 50 range 12--14 mM) among the three hepatoma cell lines (HepG2, Huh-7 and Hep3B) with different p53 statuses. To examine its toxicity, the effect of FTY720 on MIHA cells was also investigated. The results showed that FTY720 conferred greater chemoresistance to MIHA, with an IC 50 up to 60 mM (Table I).
FTY720 induces p53-independent G 1 arrest in hepatoma cell lines FTY720 was previously reported to induce G 1 arrest in prostate and breast cancers (2,11). To evaluate its possible effects on the cell cycle of cells with different p53 statuses, cell cycle changes were analyzed by flow cytometry in HepG2, Huh-7 and Hep3B cells. The effect of FTY720 on G 1 arrest was examined by excluding cells of small size in the sub-G 1 phase. When FTY720 was administered at the IC 10 and IC 50 doses, all three hepatoma cell lines showed increased G 1 arrest in a dose-and time-dependent manner. The cell cycle distribution of Hep3B cells upon FTY720 treatment is shown in Figure 2. The percentage of sub-G 1 phase cells was analyzed by DAPI staining and TUNEL assay. The DAPI result showed that the number of apoptotic nuclei increased significantly after FTY720 administration compared with the control starting at 8 h at the IC 50 dose and at 24 h at the IC 10 dose (Figure 3). The percentages of apoptotic nuclei as determined by the TUNEL assay were 1. FTY720 induces G 1 arrest and apoptosis through Akt dephosphorylation We then examined the effect of FTY720 on Akt and MAPK. The PI3-K/Akt/MEK/MAPK pathway is essential for cell growth, migration and survival (18). The Akt pathway has been reported, at least in part, to activate the MAPK pathway (19). In the current study we found that the phosphorylation level of Akt Ser473 , but not Akt Thr308 , decreased in a time-and dose-dependent manner upon treatment with FTY720 in Hep3B cells (Figure 4). The Akt protein level remained unaffected ( Figure 4). The phosphorylation level of p42/p44 was also found to be down-regulated in response to FTY720 treatment, while the p42/p44 protein level remained unchanged in the course of FTY720 treatment (Figure 4). Moreover, the phosphorylation level of FKHR and GSK-3b decreased, accompanied by up-regulation of p27 kip1 and down-regulation of cyclin D1 ( Figure 5). Therefore, down-regulation of both phospho-GSK-3b and phospho-FKHR might contribute to the G 1 arrest of hepatoma cells. For FTY720-induced apoptosis, dephosphorylation of Akt Ser473 led to increased cleaved forms of both caspase-9 and caspase-3 ( Figure 5).

FTY720 induces apoptosis through inhibition of PI3-K activity
To examine whether dephosphorylation of Akt Ser473 was mediated by PI3-K, PI3-K activity in Hep3B cells was evaluated by competitive ELISA. FTY720 inhibited the PI3-K activity in a dose-dependent manner 4 h after treatment. There was 39.4 AE 2.9 and 79.2 AE 3.4% PI3-K inhibition at the IC 10 and IC 50 doses, respectively. To further determine that inhibition of PI3-K was critical to the apoptosis-inducing activity of FTY720, a constitutively active PI3-K plasmid (CD2-p110) was transiently transfected into Hep3B cells. Flow cytometry analysis showed that the transfection efficiency of PI3-K cDNA was 59.3% (data not shown). PI3-Ktransfected Hep3B cells showed decreased apoptosis by the TUNEL assay, up-regulation of Akt Ser473 and down-regulation of cleaved caspase-3, caspase-9, p27 kip1 and cyclin D1 protein levels ( Figure 6). However, there was no significant change in Akt Thr308 ( Figure 6) and no obvious change in the percentage of cells in G 1 phase (from 54.84 to 58.98%) compared with parental Hep3B cells (from 54.84 to 67.73%) ( Figure 6).

FTY720 suppresses tumor growth and selectively induces tumor cell apoptosis in vivo
In the nude mouse tumor model tumor growth was suppressed by FTY720 treatment (Figure 7). Consistent with our in vitro results, we also observed down-regulation of Akt Ser473 but not Akt Thr308 in Hep3B-bearing nude mice, together with an increased amount of cleaved caspase-3 and caspase-9 in a time-dependent manner upon FTY720 treatment (Figure 8).

FTY720 induces apoptosis
There was a remarkable difference in the number of apoptotic nuclei in tumor tissues treated with FTY720 on day 10 and patchy necrosis on day 20 ( Figure 9B) when compared with the untreated samples ( Figure 9A) by TUNEL assay and H&E staining. However, the architecture of the liver of the nude mice remained intact after FTY720 treatment ( Figure 10). There was no difference in the number of apoptotic nuclei of the liver between mice with and without FTY720 treatment ( Figure 10).   3. The percentage of sub-G 1 phase cells was analyzed by DAPI staining TUNEL assay. Hep3B cells that showed nuclear fragmentation were considered to be undergoing apoptosis (arrows) (Â200). Increased apoptotic cells were observed in a time-and dose-dependent manner. Results were derived from three independent experiments and the error bars indicate the standard error of the means. Ã P 5 0.05 compared with 0 h (control). Apoptosis was further confirmed by TUNEL assay. Green nuclear staining represents apoptotic nuclei; the cells were counterstained with propidium iodide.
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Discussion
Chemotherapy is the current therapy for inoperable HCC. For single agent therapy doxorubicin is widely adopted clinically. However, HCC is considered to be resistant to chemotherapy (20). The response rates for single agent chemotherapy, usually doxorubicin, are~15--20% (21--23). Combination chemotherapy appears to give a higher response rate (20--30%), although in both cases remissions are usually short and a survival advantage has not been convincingly demonstrated (24). Many reports have linked chemoresistance to p53 mutation and Mdr1 overexpression (25). Therefore, there is an urgent need to develop a novel drug that specifically targets cancer cells but is not harmful to normal liver cells.
Recently, several reports have demonstrated that FTY720 induces apoptosis in vitro in several cancer cell lines, including Jurkat, HL60 and DU145 cells (11,26). In the present study we have demonstrated that FTY720 selectively kills hepatoma cells and inhibits in vivo tumor growth in the nude mouse model without notable side-effects. The above data are promising and make FTY720 a new candidate for HCC therapy.
Although the mechanism of FTY720-induced cell death has been studied, most of the in vitro studies focused only on the pro-apoptotic death signaling pathways, such as the c-Jun, NH 2 -terminal kinase and p38 pathways, and little is known about the anti-apoptotic pathways and the effectiveness of FTY720 in in vivo systems (26). This study aimed to demonstrate for the first time both the in vitro and in vivo effects of FTY720 on HCC by studying the Akt pathway, an important survival pathway. We demonstrated that dephosphorylation of Akt played a central role in FTY720-induced G 1 arrest and apoptosis in hepatoma cells. However, interestingly, FTY720 preferentially dephosphorylated Akt at Ser473 but not significantly at Thr308. This result was not only observed in our Fig. 4. Dephosphorylation of Akt and p42/p44 upon FTY720 treatment. Dephosphorylation of (A) Akt Ser473 was observed in Hep3B cells treated with FTY720 at the IC 10 and IC 50 doses at 0, 4, 8, 16 and 24 h, but was not significant at (B) Akt Thr308 . The total Akt protein level (C) remained unaffected. In addition, the phosphorylation level of p42/p44 (D) decreased in a time-and dose-dependent manner, but no decrease was found in total p42/p44 protein level (E).  in vitro experiment but also in our in vivo nude mouse model. For apoptosis induced by FTY720, dephosphorylation of Akt Ser473 led to dephosphorylation of phospho-caspase-9, resulting in an increase in the cleaved form of caspase-9 and, consequently, caspase-3. Apoptosis induced by Akt dephosphorylation in hepatoma cell lines was found through inhibition of PI3-K activity and was confirmed by transfection of a constitutively active PI3-K enzyme into Hep3B cells. Transfection of PI3-K into Hep3B cells protected them from apoptosis, with increased Akt Ser473 and decreased cleaved caspase-3, cleaved caspase-9, p27 kip1 and cyclinD1, while Akt Thr308 remained unchanged. This result was different from a previous study of FTY720-induced apoptosis in leukemia cell lines, in which both Akt Ser473 and Akt Thr308 were dephosphorylated independent of PI3-K inactivation (4), suggesting  Tumor growth curve for the Hep3B-bearing nude mouse model with and without FTY720. Fig. 8. Dephosphorylation of Akt Ser473 but not Akt Thr308 leads to apoptosis in the nude mouse model. Nude mice bearing Hep3B cells were injected with FTY720 at a dose of 10 mg/kg into the peritoneum and the mice killed on day 10 or 20. Consistent with the in vitro results, the phosphorylation level of Akt Ser473 but not Akt Thr308 decreased in a time-dependent manner. Dephosphorylation of Akt Ser473 led to increases in the cleaved forms of caspase-3 and caspase-9.
T.K.Lee et al.
a differential effect of FTY720 on different tumor cell lines. This discrepancy could be attributed to the fact that sphingosine kinase could be found in different forms in different tissues and showed different tissue distributions (27). Therefore, FTY720 may show different kinetic properties and substrate specificities in different tissues. The level of protein p27 kip1 is regulated directly by Akt phosphorylation and indirectly by FKHR. FKHR is the downstream substrate of Akt. FKHR plays a major role in cell cycle progression by inhibition of p27 kip1 transcription through PI3-K-induced FKHR-L1 phosphorylation (28). In addition to Akt, p42/p44 activation triggers p27 Kip1 degradation independent of CDK2/cyclin E in NIH 3T3 cells (29). There is cross-talk between Akt and p42/ p44. Several reports have demonstrated that expression of p42/ p44 is partly mediated by the Akt pathway (19). Akt can also affect cellular proliferation by inactivating GSK-3b, resulting in stabilization of cyclin D1 (30). p27 kip1 and cyclin D1 have been shown to play an important role in cell cycle progression from G 1 to S phase (31). From our western blot results, upregulation of p27 Kip1 and down-regulation of cyclin D1 were observed in a time-and dose-dependent manner after FTY720 administration and the alterations were consistent with  FTY720 induces apoptosis induction of G 1 arrest. Therefore, up-regulation of p27 Kip1 and down-regulation of cyclin D1 might be correlated with FTY720-induced G 1 arrest. This conclusion was further confirmed by transfection of constitutively active PI3-K enzyme into Hep3B cells. There was no apparent change in p27 kip1 and cyclin D1 protein levels in PI3-K-transfected Hep3B cells, with only an insignificant change in the percentage in G 1 phase from 54.84 to 58.98%, in contrast to parental Hep3B cells, which changed from 54.84 to 67.73%, thus suggesting the role of p27 kip1 and cyclin D1 in FTY720-induced G 1 arrest. Therefore, we propose that FTY720-induced apoptosis is mediated by PI3-K Akt dephosphorylation ( Figure 11). The main hurdle to chemotherapy for HCC is chemoresistance and toxicity to normal liver cells (32). In this study we have found that FTY720 induces hepatoma cell death in a p53-independent manner. This result is an advantage in preventing chemoresistance induced by p53 mutation in HCC (33). FTY720 selectively induces hepatoma cell death but not that of immortalized, non-tumorigenic liver cell lines. Consistent with the in vitro study, we found that FTY720 selectively induces apoptosis in tumors in a time-and dosedependent manner, but not in normal liver in the nude mouse tumor model.
In conclusion, FTY720 is a novel anticancer agent that induces apoptosis of hepatoma cell lines in a p53-independent manner through PI3-K-mediated Akt dephosphorylation. FTY720 may be a promising agent for the treatment of HCC patients.

Supplementary material
Supplementary material is available online at http://www. carcin.oupjournals.org/. Fig. 11. The possible mechanism of FTY720 induced human hepatoma cell line apoptosis is through PI3-K-mediated Akt dephosphorylation.
T.K.Lee et al.