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Cancer Research 68, 1204, February 15, 2008. doi: 10.1158/0008-5472.CAN-07-2621
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

FTY720 Induces Apoptosis in Hepatocellular Carcinoma Cells through Activation of Protein Kinase C {delta} Signaling

Jui-Hsiang Hung1, Yen-Shen Lu3, Yu-Chieh Wang1, Yi-Hui Ma1, Da-Sheng Wang1, Samuel K. Kulp1, Natarajan Muthusamy2, John C. Byrd2, Ann-Lii Cheng3 and Ching-Shih Chen1

1 Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy and 2 Division of Hematology and Oncology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio and 3 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan

Requests for reprints: Ching-Shih Chen, Division of Medicinal Chemistry, College of Pharmacy, Parks Halls, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210. Phone: 614-688-4008; Fax: 614-688-8556; E-mail: chen.844{at}osu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was aimed at elucidating the mechanism by which FTY720, a synthetic sphingosine immunosuppressant, mediated antitumor effects in hepatocellular carcinoma (HCC) cells. The three HCC cell lines examined, Hep3B, Huh7, and PLC5, exhibited differential susceptibility to FTY720-mediated suppression of cell viability, with IC50 values of 4.5, 6.3, and 11 µmol/L, respectively. Although FTY720 altered the phosphorylation state of protein kinase B and p38, our data refuted the role of these two signaling kinases in FTY720-mediated apoptosis. Evidence indicates that the antitumor effect of FTY720 was attributable to its ability to stimulate reactive oxygen species (ROS) production, which culminated in protein kinase C (PKC){delta} activation and subsequent caspase-3–dependent apoptosis. We showed that FTY720 activated PKC{delta} through two distinct mechanisms: phosphorylation and caspase-3–dependent cleavage. Cotreatment with the caspase-3 inhibitor Z-VAD-FMK abrogated the effect of FTY720 on facilitating PKC{delta} proteolysis. Equally important, pharmacologic inhibition or shRNA-mediated knockdown of PKC{delta} protected FTY720-treated Huh7 cells from caspase-3 activation. Moreover, FTY720 induced ROS production to different extents among the three cell lines, in the order of Hep3B > Huh7 >> PLC5, which inversely correlated with the respective glutathione S-transferase {pi} expression levels. The low level of ROS generation might underlie the resistant phenotype of PLC5 cells to the apoptotic effects of FTY720. Blockade of ROS production by an NADPH oxidase inhibitor protected Huh7 cells from FTY720-induced PKC{delta} activation and caspase-3–dependent apoptosis. Together, this study provides a rationale to use FTY720 as a scaffold to develop potent PKC{delta}-activating agents for HCC therapy. [Cancer Res 2008;68(4):1204–12]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FTY720 is a synthetic sphingosine immunosuppressant, which is currently undergoing clinical trials for the prevention of kidney graft rejection (1) and the treatment of relapsing multiple sclerosis (2). Previous studies indicate that the effect of FTY720 on prolonging the survival of allografts is attributable to the ability of its phosphorylated metabolite to inhibit T-lymphocyte infiltration by targeting several of the sphingosine-1-phosphate (S1P) receptors (3, 4). In addition to immunosuppression, FTY720 has also been shown to induce apoptosis in several human cancer cell lines, including Jurkat T cells (5), multiple myeloma cells (6), and those of liver (7, 8), prostate (9–12), breast (13), kidney (14), and bladder (15). This antitumor effect is noteworthy because of the involvement of S1P receptor–independent mechanisms (16). To date, a number of signaling pathways have been proposed to account for the ability of FTY720 to facilitate apoptosis in different cancer cell lines, including those mediated by protein kinase B (Akt) (5, 6, 8), mitogen-activated protein (MAP) kinases (6, 10), focal adhesion kinase (10), Rho-GTPase (12), signal transducers and activators of transcription 3, I{kappa}B{alpha}, nuclear factor-{kappa}B, and Bcl-xL (6). Mechanistically, the targeting of this wide spectrum of signaling elements underscores the effectiveness of FTY720 in suppressing cell growth in a broad range of cancer cells that exhibit distinct mechanisms in governing cell cycle progression and apoptosis.

Hepatocellular cancer occurs both sporadically and is also related to chronic viral infection, environmental exposure, and alternative causes of hepatic cirrhosis. The US Surveillance, Epidemiology, and End Results database estimates that 19,160 men and women will be diagnosed with and 16,780 men and women will die of cancer of the liver and intrahepatic bile duct in 2007 within the United States (17). The incidence of hepatocellular carcinoma is even higher in the Asian cultures due to the higher frequency of chronic active viral hepatitis. Until only recently, effective treatment of hepatocellular cancer has been essentially absent (18). Recently, the RAF inhibitor sornafinib has been shown to be beneficial for the treatment of metastatic hepatocellular carcinoma and was approved for marketing by the Food and Drug Administration in this indication (19). However, this therapy only works in a subset of patients and is not curative. This emphasizes both the potential for multitargeted kinase inhibitors in hepatocellular carcinoma and the need to identify new therapies.

In this study, we describe a novel mechanism by which FTY720 induces apoptosis in hepatocellular carcinoma cells (HCC). We obtain evidence that FTY720 facilitates reactive oxygen species (ROS)-dependent activation of protein kinase C {delta} (PKC{delta}), resulting in caspase-3–mediated apoptotic death in HCC cells. PKC{delta}, a member of the novel PKC subfamily, has been shown to play a pivotal role in mediating apoptosis induced by oxidative stress (20, 21), Fas ligands (22), and various genotoxic agents including DNA damaging agents (23) and paclitaxel (24), thereby representing an important target for cancer therapy. Consequently, this mechanistic finding provides a molecular basis to pharmacologically exploit FTY720 to develop potent PKC{delta}-targeted antitumor agents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. FTY720 was synthesized according to a published procedure (25). The identity and purity were verified by nuclear magnetic resonance, high-solution mass spectrometry, and elemental analysis. The following pharmacologic agents were purchased from the respective vendors: okadaic acid, PD98059, SB203580, Ro6976, rottlerin, and U3122 (Calbiochem); diphenyleneiodonium chloride (DPI; Cayman Chemical); 2,7-dichlorofluorescein diacetate (DCFDA; Invitrogen); (Ac-DMQD)2-Rh110 (Anaspec); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; TCI America). Cell death detection ELISA kit and the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit were obtained from Roche Diagnostic and Pierce, respectively. Antibodies against various proteins were obtained from the following sources: PKC{alpha}, PKC{varepsilon}, PKC{zeta}, PKC{delta}, p-Tyr155-PKC{delta}, p-Thr507-PKC{delta}, and p-Thr308-Akt (Santa Cruz Biotechnology); p-Tyr311-PKC{delta}, p-Ser664-PKC{delta} (Abcam); Akt and p-Ser473-Akt (Cell Signaling); glutathione S-transferase (GST)-{pi} (DAKO). The pKD-PKC{delta}-v2 plasmid encoding shRNA against PKC{delta}, goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugates, and rabbit anti-mouse IgG-HRP conjugates was purchased from Upstate.

Cell culture. Huh7, PLC5, and Hep3B human HCC cell lines were purchased from the American Type Culture Collection. Cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 10 µg/mL gentamicin at 37°C in a humidified incubator containing 5% CO2. Culture medium was replaced every 2 days.

MTT assay. Cell viability was assessed by using the MTT assay in six replicates. HCC cells were seeded at 3 x 105 per well in 24-well flat-bottomed plates and incubated in 10% FBS–supplemented DMEM for 24 h. Cells were treated with FTY720 at various concentrations in the same medium. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. After 24 h, the drug-containing medium was replaced with 200 µL of 10% FBS–supplemented DMEM containing 0.5 mg/mL MTT, and cells were incubated in the CO2 incubator at 37°C for 4 h. Medium was removed, the reduced MTT was solubilized in 500 µL per well of DMSO, and 100 µL aliquots from each well was transfer to 96-well plates to measure absorbance at 570 nm.

Annexin V/propidium iodide assay. For assessment of apoptosis, both floating and adherent cells were collected and analyzed. Briefly, ~7 x 105 cells per dish were plated onto 10-cm dishes and incubated at 37°C for 16 h. The cells were treated with DMSO or 10 µmol/L FTY720 for the time indicated. The cells were washed twice with PBS and collected by trypsinization. After centrifugation at 400 x g for 5 min at room temperature, the cells were stained with Annexin V and propidium iodide (1 µg/mL). The cell apoptosis distributions were determined on a FACScort flow cytometer and analyzed by ModFitLT V3.0 software program.

Western blotting. Cells, seeded in 10-cm dishes (7 x 105 cells per dish), were incubated for 16 h, subjected to different drug treatments, and harvested by scrapping. Cell lysates were prepared by exposing cells to 2 x SDS lysis buffer [0.1 mol/L Tris, 0.4% SDS, and 20% glycerol (pH 6.8)]. Protein concentrations of cell lysates were measured using a Micro BCA protein assay reagent kit (Pierce). To the cell lysate, the same volume of SDS-PAGE sample loading buffer [100 mmol/L Tris-HCl, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, and 0.1% bromphenol blue (pH 6.8)] was added, and the cells were boiled for 10 min. Equal amounts of proteins were resolved in SDS-polyacrylamide gels. After electrophoresis, gel was transferred to nitrocellulose membranes using a semidry transfer cell. The transblotted membrane was washed twice with TBS containing 0.1% Tween 20 (TBST). After blocking with TBST containing 5% nonfat milk for 1 h, the membrane was incubated with the appropriate primary antibody in 1% TBST nonfat milk at 4°C overnight. The membrane was washed thrice with TBST for a total of 15 min. The secondary anti-mouse IgG-HRP conjugates or anti-rabbit IgG-HRP conjugates (1:2,000 dilution) was subsequently incubated with the membrane for 1 h at room temperature and was washed extensively for 50 min with TBST. The blots were visualized with the enhanced chemiluminescence (Amersham) Western blot detection system according to the manufacturer's instructions.

Cell death detection ELISA assay. Induction of apoptosis was assessed with a Cell Death Detection ELISA kit, which is based on the quantitative determination of cytoplasmic histone–associated DNA fragments in the form of mononucleosomes and oligonucleosomes after induced apoptotic death. In brief, Huh7 cells (5 x 104) were seeded and incubated in 96-well plates containing DMEM supplemented with 10% FBS for 16 h and were then subjected to various drug treatments for 24 h. After incubation, the plates were centrifuged at 200 x g for 10 min and incubated with 200 µL of lysis buffer at room temperature for 30 min. Twenty microliters of supernatant from each sample were used in the ELISA by following the manufacturer's instruction.

Detection of ROS. DCFDA is an indicator for ROS that is nonfluorescent until its acetate groups are removed by intracellular esterases and oxidation occurs in the cell. The ROS production in Huh7 cells after FTY720 treatment was detected using 5 µmol/L DCFDA. Huh7 cells (7 x 105) in 10 cm-dishes were treated with FTY720 for 1 h, washed with PBS twice, and then exposed to 5 µmol/L DCFDA for 30 min at 37°C. Cells were collected and analyzed by flow cytometry.

Detection of hydrogen peroxide. Quantitative determination of hydrogen peroxide in the culture medium was performed by using a colorimetric hydrogen peroxide kit (Assay Designs, Inc.) according to the vendor's instruction. In brief, Huh7 cells (7 x 105) were seeded and incubated in 10-cm plates containing 10% FBS–supplemented DMEM for 16 h. Because the culture medium would interfere with the detection of hydrogen peroxide, it was replaced with an equal volume of Hanks' balanced Salt Solution (HBSS) containing 10 µmol/L FTY720. After 3-h of exposure, 50 µL of the cultured HBSS solution was incubated with 100 µL of the colorimetric solution provided in the kit in 96-well plates, and incubated at room temperature for 30 min. Absorbance at 570 nm was measured.

ShRNA-mediated knockdown of PKC{alpha}, PKC{varepsilon}, and PKC{delta}. Huh7 cells (1 x 106) were cotransfected with 1.8 µg of individual shRNA plasmids (pKD-PKC{alpha}-v4, pKD-PKC{delta}-v2, and pKD-PKC{varepsilon}-v5) and 0.2 µg of pCDNA 3.1 (+) plasmids using Invitrogen Lipofectamine 2000 reagent according to the manufacturer's protocol. Cells were then selected by G418 for 2 weeks. The stable clones were established and confirmed by Western blotting using individual antibodies against PKC{alpha}, PKC{varepsilon}, and PKC{delta}.

Ectopic expression of constitutively active Akt. The pcDNA 3.1 (+)/CA-Akt-HA plasmid that encodes AktT308D/S473D, a constitutively active form of Akt, was provided by Dr. Matthew D. Ringel (The Ohio State University, Columbus, OH). Huh7 cells were transfected via nucleofection by using program T-022 of the AMAXA Nucleofector system according to the manufacturer's instructions. Expression of constitutively active Akt was confirmed by Western blotting using antibodies against Akt and hemagglutinin tag.

Subcellular fractionation. Subcellular fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit according to the manufacturer's instruction. In brief, Huh7 (7 x 105) cells in 10-cm dishes were exposed to 10 µmol/L FTY720 in 10% FBS–containing DMEM for different time intervals. After treatment, the cells were washed with cold PBS, scraped, and harvested by centrifugation. The cell pellets were suspended in 200 µL of Cytoplasmic Extraction Reagent I solution and incubated on ice for 10 min, followed by adding 11 µL of Cytoplasmic Extraction Reagent II solution and incubation on ice for 1 min. The cell suspensions were centrifuged at 16,000 x g for 5 min to collect supernatant as the cytoplasmic fraction. The pellets were resuspended with 100 µL of Nuclear Extraction Reagent on ice for 40 min. The cell suspension was centrifuged at 16,000 x g for 10 min at 4°C to collect supernatant as the nuclear fraction.

Analysis of caspase-3 activity. Caspase-3 activity was determined using (Ac-DMQD)2-Rh110 as the fluorogenic substrate for active caspase-3. Briefly, Huh7 (7 x 105) cells in 10-cm dishes were subjected to different drug treatments for 24 h and were resuspended in 100 µL of PBS containing 10 µmol/L 2-Rh110 for 15 min at room temperature. Each sample was then added with 400 µL PBS, and fluorescence signals generated by caspase-3–cleaved substrate were analyzed by flow cytometry.

Cytochrome c release. Drug-treated Huh7 cells were collected and triturated with 100 µL of chilled hypotonic lysis solution [220 mmol/L mannitol, 68 mmol/L sucrose, 50 mmol/L KCl, 5 mmol/L EDTA, 2 mmol/L MgCl2, and 1 mmol/L DTT in 50 mmol/L PIPES-KOH (pH 7.4)] for 45 min. The solution was centrifuged at 600 x g for 10 min to collect the supernatant. The supernatant was further centrifuged at 14,000 rpm for 30 min, and equal amounts of proteins (50 µg) from the supernatant were resolved in 15% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes and analyzed by immunoblotting with anti-cytochrome c antibodies.

Statistical analysis. The JMP5.0.1 software package was used to perform all analyses. Data were analyzed by the Student's t test. Differences were considered significant at a P value of <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential susceptibility of HCC cell lines to FTY720-induced apoptotic death. The in vitro antitumor efficacy of FTY720 was evaluated in three human HCC cell lines, Huh7, Hep3B, and PLC5, which are resistant to cytotoxic drugs due to loss of p53 function and/or overexpression of Bcl-xL (26). As these three cell lines harbor different cellular and genetic abnormalities, they showed differential susceptibility to the antiproliferative effect of FTY720. The IC50 values for Hep3B, Huh7, and PLC5 cells were 4.5, 6.3, and 11 µmol/L, respectively (Fig. 1A ). The antiproliferative activity of FTY720 was, at least in part, attributable to apoptosis, as evidenced by poly(ADP)ribose polymerase (PARP) cleavage in each of these three cell lines and Annexin V/propodium iodide staining in Huh7 cells in a dose- and/or time-dependent manner (Fig. 1B and C).


Figure 1
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  		Figure 1. Huh7, Hep3B, and PLC5 cells exhibit differential susceptibility to FTY720-induced apoptotic death. A, dose-dependent effect of FTY720 on suppressing the cell viability of the three cell lines. Cells were exposed to FTY720 at the indicated doses in 10% FBS–supplemented DMEM for 24 h, and the cell viability was assessed by MTT assays. Points, mean; bars, SD (n = 6). B, dose- and time-dependent effect of FTY720 on PARP cleavage in the three cell lines by Western blot analysis. C, flow cytometric analysis of apoptotic death in Huh7 cells with DMSO vehicle or 10 µmol/L FTY720 in 10% FBS–containing DMEM for 12 or 24 h. Huh7 cells were cultured with FTY720 for the time indicated. The cells were analyzed by flow cytometry after staining with fluorescein-conjugated Annexin V and propidium iodide (PI). The percentages in the graphs represent the percent of cell numbers in the respective quadrants. Columns, mean; bars, SD.

 
Involvement of PKC{delta} in FTY720-mediated apoptotic death. To date, various signaling pathways have been reported to account for FTY720-induced apoptosis in different cancer cell systems, which might be reconciled by differences in the molecular abnormalities associated with the oncogenesis of individual cancer types. Of these proposed mechanisms, the ability of FTY720 to affect the activation status of p38 MAP kinase and Akt in prostate cancer cells (10) and HCC cells (8), respectively, was especially noteworthy.

To discern the role of Akt and p38 in FTY720-mediated apoptosis in HCC cells, we investigated the effect of FTY720 on the phosphorylation of these two signaling kinases in Huh7, Hep3B, and PLC5 cells. As these HCC cell lines contained functional PTEN, they exhibited low levels of Akt phosphorylation at Ser473 compared with the PTEN-defective PC-3 cancer cells (Fig. 2A ). In contrast, this PTEN functional status had no direct correlation with the Thr308 phosphorylation, as all these three HCC cell lines exhibited high levels of p-Thr308-Akt relative to PC-3 (Fig. 2B). This site-specific phenomenon is consistent with the finding that levels of PTEN modulate Akt phosphorylation on Ser473, but not on Thr308, in rhabdomyosarcomas cells (27).


Figure 2
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  		Figure 2. Role of Akt inhibition and PKC{delta} activation in FTY720-mediated apoptosis. A, endogenous expression levels of PTEN, p-Ser473-Akt, and p-Thr308-Akt, in Huh7, PLC5, and Hep3B cells. PC-3 prostate cancer cells, which are PTEN null, were used as a negative control. B, dose-dependent effect of FTY720 on the phosphorylation states of Thr308-Akt and p38 in Huh7, Hep3B, and PLC5 cells by Western blotting. C, ectopic expression of AktT308D/S473D, a constitutively active form of Akt, does not protect Huh7 cells from FTY720-induced cell death. Left, expression of AktT308D/S473D (CA-Akt-HA) in Huh7 transient transfection. Western blot analysis used antibodies against hemagglutinin (HA) tag, Akt, p-GSK3β, and GSK3β. Right, viability of Huh7 cells overexpressing AktT308D/S473D vis-à-vis cells transfected with empty pCMV vector in the presence of DMSO vehicle or 10 µmol/L FTY720 in 10% FBS–supplemented DMEM for 24 h. Columns, mean; bars, ±SD (n = 3). D, effect of the pharmacologic inhibitors of various signaling enzymes on FTY720-mediated cell death in Huh7 cells (left). Huh7 cells were exposed to 5 µmol/L FTY720 in the presence of one of the following inhibitors, okadaic acid (100 nmol/L), PD98059 (10 µmol/L), SB203580 (10 µmol/L), Ro6976 (10 µmol/L), rottlerin (10 µmol/L), and U3122 (10 µmol/L), for 24 h in 10% FBS–supplemented DMEM for 24 h. Cell viability was assessed by MTT assays. Right, dose-dependent effect of rottlerin on protecting Huh7 cells against FTY720-induced cell death. Huh7 cells were exposed to DMSO vehicle or the indicated dose of rottlerin in the absence or presence of 10 µmol/L FTY720 for 24 h, and cell viability was analyzed by MTT assay. Columns, mean; bars, ±SD (n = 6). E, rottlerin (10 µmol/L) inhibits the effect of FTY720 (10 µmol/L) on cytochrome c (left) and PARP cleavage (right) in Huh7 cells. Huh7 cells were exposed to 10 µmol/L FTY720 in the presence of 10 µmol/L rottlerin or DMSO vehicle in 10% FBS–containing DMEM for the indicated time intervals. Mitochondria-free lysates and total lysates were prepared as described in Materials and Methods for the Western blot analysis of cytochrome c release and PARP cleavage, respectively.

 
As shown in Fig. 2B, exposure to FTY720 for 24 h led to a dose-dependent decrease in p-Thr308-Akt levels in these three HCC cell lines (Fig. 2B), which is consistent with that previously reported (8). Moreover, the relative potency in Akt dephosphorylation correlated with the respective susceptibility to FTY720-induced cell death. In contrast, the effect of FTY720 on modulating p38 phosphorylation was cell line specific among the three cell lines examined, i.e., only PLC5 cells exhibited a dose-dependent increase in p-p38 levels after drug treatment.

To further examine whether Akt inhibition represented a major underlying antitumor mechanism for FTY720, we assessed the effect of the ectopic expression of a constitutively active form of Akt (AktT308D/S473D) on FTY720-induced cell death by transiently transfecting Huh7 cells with HA-CA-Akt plasmids (Fig. 2C). The constitutively activated status of Akt was manifested by the multifold increase in the phosphorylation level of GSK3β. However, this ectopic AktT308D/S473D expression did not provide a significant protection against the suppression of cell viability by 10 µmol/L FTY720. Together, these findings argued against the involvement of Akt and p38 in FTY720-induced apoptosis at least in these two sensitive cell lines.

To delineate the underlying mechanism, we further assessed the effect of a panel of pharmacologic inhibitors, including those of protein phosphatase 2A (PP2A; okadaic acid), MAP kinase kinase (PD98059), p38 kinase (SB203580), PKC{alpha}/β (Ro6970), PKC{delta} (rottlerin), and phospholipase C (U3122) on rescuing Huh7 cell from FTY720-induced cell death. Some of these signaling enzymes might potentially be targeted by FTY720 to trigger apoptosis signaling. For example, a recent report suggests the involvement of PP2A activation in FTY720-mediated Akt dephosphorylation in a T-cell–derived leukemia cell line (5). However, of these inhibitors, only the PKC{delta} inhibitor rottlerin exhibited the ability to counteract the cell killing activity of 5 µmol/L FTY720, whereas others either exacerbated or had no appreciable effects on FTY720-mediated suppression of cell viability (Fig. 2D, left). In addition, rottlerin, even at 1 µmol/L, was effective in protecting Huh7 cells from the antitumor activity of FTY720. As PKC{delta} mediates mitochondria-dependent apoptosis, cotreatment of Huh7 cells with rottlerin could block the effect of FTY720 on cytochrome c release into the cytoplasm and PARP cleavage (Fig. 2E). Consequently, these findings suggested that PKC{delta} activation played a pivotal role in FTY720-mediated apoptotic death. It is noteworthy that PKC{delta} was differentially expressed among the three HCC cell lines (Fig. 3A ). Relative to Huh 7 and Hep3B cells, PLC5 cells exhibited low levels of PKC{delta} expression, which might, in part, attribute to their relative insensitivity to FTY720-induced apoptosis.


Figure 3
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  		Figure 3. Knockdown of PKC{delta}, but not PKC{alpha} and PKC{varepsilon}, protects Huh7 cells against FTY720-induced apoptosis. A, differential expression of PKC{delta} in Huh7, Hep3B, and PLC5 cells by Western blotting. B, shRNA-mediated knockdown of PKC{delta} protects Huh7 cells from FTY720-induced apoptosis. Left, Western blot analysis of the expression levels of PKC{alpha}, PKC{varepsilon}, PKC{delta}, and PKC{zeta} in Huh7 cells subjected to different treatments, as indicated. Right, effect of PKC{delta} knockdown on suppressing FTY720-facilitated nucleosomal fragmentation. Cells were treated with 10 µmol/L FTY720 in 10% FBS–containing DMEM for 24 h. DNA fragmentation was quantitatively measured by a cell death detection ELISA kit. Columns, mean; bars, ±SD (n = 3). C and D, knockdown of PKC{alpha} and PKC{varepsilon}, respectively, cannot protect Huh7 cells from FTY720-induced cell death. Huh7 cells were transfected with control or plasmids encoding shRNA against PKC{alpha} or PKC{varepsilon}, and those stable clones were treated with FTY720 as indicated. Cell viability was measured by the MTT assay. Columns, mean; bars, ±SD (n = 6).

 
Role of PKC{delta} in FTY720-induced apoptosis. To validate this premise, we examined the effect of shRNA-mediated knockdown of PKC{delta} vis-à-vis two other PKC isoforms (PKC{alpha} and PKC{varepsilon}, representing conventional and novel PKC isoforms, respectively) on rescuing FTY720-mediated apoptotic death in Huh7 cells. Huh7 cells were transfected with plasmids encoding shRNA against individual PKC isoforms, followed by clonal selection. This selection led to three stable clones with substantially reduced PKC{delta} expression, and one stable clone each for PKC{alpha} and PKC{varepsilon} (Fig. 3B–D). This shRNA knockdown was highly specific because no cross silencing of other PKC isoforms examined ({alpha}, {varepsilon}, and {zeta}) was noted. As shown, only the knockdown of PKC{delta} rendered Huh7 cells resistant to FTY720-mediated apoptotic death (Fig. 3B), whereas that against PKC{alpha} or PKC{varepsilon} exhibited no appreciable effect (Fig. 3C and D).

Effect of FTY720 on PKC{delta} activation. We showed that treatment of FTY720 led to PKC{delta} activation in Huh7 cells via two distinct mechanisms, i.e., phosphorylation and proteolytic cleavage. Of the four potential phosphorylation sites examined, FTY720 caused a rapid increase in the phosphorylation at Try311, Thr507, and Ser664, without affecting that of Tyr155 (Fig. 4A, left ). Moreover, this FTY720-facilitated phosphorylation at Thr507 and Ser664 could be blocked by 1 µmol/L rottlerin (right), suggesting that PKC{delta} mediated autophosphorylation on these two sites.


Figure 4
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  		Figure 4. FTY720 facilitates phosphorylation and caspase-3–dependent cleavage of PKC{delta} in Huh7 cells. A, time-dependent effect of FTY720 (10 µmol/L) on the phosphorylation of PKC{delta} at different sites (left). Right, effect of 1 µmol/L rottlerin on FTY720-facilitated PKC{delta} phosphorylation at Thr507 and Ser664. B, time-dependent effect of FTY720 (10 µmol/L) on the proteolytic cleavage of PKC{delta} in whole cell lysates (left) and in the cytosolic versus nuclear fractions (right). C, flow cytometric analysis of dose-dependent effect of FTY720 on increasing caspase-3 activity in Huh7 cells. Left, increased caspase-3 activity was observed in a dose-dependent manner after 24-h exposure to FTY720. Right, relative caspase-3 activities, normalized to DMSO control, at the indicated concentrations of FTY720. Columns, means; bars, ±SD (n = 3). D, the caspase inhibitor Z-VAD-FMK blocks the effect of FTY720 (10 µmol/L) on proteolytic cleavage of PKC{delta}. Cells were treated with DMSO vehicle or 10 µmol/L FTY720 in the presence of increasing doses of Z-VAD-FMK for 24 h, and cell lysates were subjected to immunoblotting with antibodies against anti-PKC{delta} and β-actin.

 
Evidence suggests that phosphorylation at Tyr311 and that at Tyr155 connote different physiologic roles in regulating the function of PKC{delta} (see Discussion). Moreover, proteolytic activation of PKC{delta}, which generated an active kinase domain, was also noted after 12 h of exposure to FTY720 (Fig. 4B, left). In addition to the cytoplasm, PKC{delta} and its processed form were also detected, to a lesser extent, in the nucleus (right), suggesting the potential involvement of a nucleus-dependent pathway in the antitumor effects of FTY720.

As there exists a mechanistic link between caspase-3 activation and the proteolytic cleavage of PKC{delta} (28), we examined the effect of FTY720 on modulating the activity of caspase-3 in Huh7 cells by using flow cytometric analysis. As shown, exposure to FTY720 led to a dose-dependent stimulation of caspase-3 activity (Fig. 4C). For example, FTY720 caused 3.5-fold and 4.8-fold increases in caspase activity at 5 and 10 µmol/L, respectively. Equally important, cotreatment with the caspase inhibitor Z-VAD-FMK abrogated the effect of FTY720 on facilitating the proteolytic cleavage of PKC{delta} (Fig. 4D), confirming the involvement of caspase-3 in PKC{delta} proteolysis. Overall, this suggests that proteolytic cleavage of PKC{delta} is a secondary event that occurs after activation of caspase family members.

FTY720 activates PKC{delta} through a ROS-dependent mechanism. We next sought to determine the upstream activator of caspases in hepatocellular cancer cell lines that promoted PKC{delta} activation and cleavage. A variety of reported caspase enzyme–activating stimuli have been shown to trigger PKC{delta} activation in different cell systems, including ROS (29), ceramide (30), tumor necrosis factor {alpha} (31), Fas ligand (31), and radiation (32). The present study shows that the effect of FTY720 on PKC{delta} activation was attributable to its ability to stimulate ROS generation in HCC cells.

Flow cytometric analysis using the ROS-sensitive probe DCFDA indicates that FTY720 at 10 µmol/L stimulated an immediate robust increase in ROS production in Huh7 cells, and that the level of intracellular ROS levels remained high even at 6 h after the treatment (Fig. 5A ). The time course of this ROS production paralleled that of PKC{delta} phosphorylation. Moreover, FTY720 induced different levels of ROS production among the three HCC cell lines, in the order of Hep3B > Huh7 >> PLC5. It is noteworthy that this FTY720-stimulated ROS generation inversely correlated with the expression of GST-{pi} (Fig. 5C), a phase II detoxification enzyme, which has been implicated in protection against apoptosis and drug resistance in cancer cells (33). However, the major ROS produced in response to FTY720 remained unclear because no significant increase in H2O2 generation was detected in FTY720-treated Huh7 cells (data not shown).


Figure 5
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  		Figure 5. FTY720 stimulates ROS production in HCC cells. A, time-dependent effect of FTY720 (10 µmol/L) on ROS production. Left, Huh7 cells were exposed to 10 µmol/L FTY720 in 10% FBS–containing DMEM for the indicated time intervals, stained with DCFDA, and subjected to flow cytometric analysis as described in Materials and Methods. Right, relative ROS production in Huh7 cells treated with 10 µmol/L FTY720 for the indicated time intervals. Columns, means; bars, ±SD (n = 3). B, dose-dependent effect of FTY720 on stimulating different extents of ROS production in Hep3B, Huh7, and PLC5 cells after 1 h of treatment. Columns, means; bars, ±SD (n = 3). C, differential expression of GST-{pi} protein in the three HCC cell lines.

 
The effect of FTY720 on increasing intracellular ROS levels was independent of PKC{delta} activity. As shown in Fig. 6A , although the NADPH oxidase inhibitor DPI could significantly inhibit FTY720-stimulated production of ROS, rottlerin exhibited no appreciable effect on ROS levels. Moreover, DPI was able to suppress the effect of FTY720 on stimulating the phosphorylation and proteolytic cleavage of PKC{delta} (Fig. 6B), indicating the causative role of ROS in PKC{delta} activation. As a consequence, blockade of ROS generation by DPI protected Huh7 cells from FTY720-induced caspase-3 activation and apoptotic death, with potency similar to that of rottlerin (Fig. 6C). In contrast, DPI lacked appreciable effect on rescuing FTY720-facilitated Akt dephosphorylation, suggesting that Akt deactivation was not mechanistically linked to ROS-PKC{delta} signaling and did not play a crucial role in FTY720-mediated cell death (Fig. 6D). Overall, this suggests that FTY720 production of ROS and secondary activation of PKC{delta} contributes to the cytotoxicity observed in hepatocellular carcinoma cell lines treated with this agent.


Figure 6
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  		Figure 6. Mechanistic link between ROS generation and PKC{delta} activation. A, effect of the ROS inhibitor DPI and the PKC{delta} inhibitor rottlerin on FTY720-mediated ROS production. Huh7 cells were exposed to DMSO vehicle, rottlerin (10 µmol/L), or DPI (10 µmol/L) for 30 min, followed by cotreatment with DMSO vehicle or 10 µmol/L FTY720 for 1 h, stained with DCFDA, and subjected to flow cytometric analysis for ROS. B, DPI inhibits the effect of FTY720 (10 µmol/L) on PKC{delta} phosphorylation (left) and proteolytic cleavage (right). For the assessment of PKC{delta} cleavage, Huh7 cells were exposed to DPI (10 µmol/L) for 30 min, followed by cotreatment with FTY720 (10 µmol/L) in 10% FBS–containing medium for 24 h. C, DPI (10 µmol/L) and rottlerin (10 µmol/L) protect Huh7 cells from the effect of FTY720 (10 µmol/L) on stimulating caspase-3 activation (left) and nucleosomal fragmentation (right). Cells were treated with individual agents as aforementioned. Columns, means; bars, ±SD (n = 3). D, DPI lacks effect on inhibiting FTY720-mediated Akt phosphorylation. Cells were treated with individual agents as aforementioned.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In light of the therapeutic potential of FTY720, the mechanism underlying its antitumor effect warrants investigations. In this study, we obtained evidence that FTY720 suppressed the proliferation of HCC cells, at least in part, by stimulating ROS production, leading to PKC{delta} activation and subsequent caspase-3–dependent apoptosis. From a mechanistic perspective, ROS production represents an early hallmark event in the apoptotic effect of many therapeutic agents, including N-(4-hydroxyphenyl)-retinamide (34, 35), arsenic trioxide (36), parthenolide (37), and cisplatin (38). Relative to these agents, FTY720 represents a structurally distinct type of small-molecule agent with unique mechanistic features, which could be exploited to foster novel therapeutic strategies for HCC treatment.

It is well-documented that drug-induced ROS stress triggers a cascade of redox-dependent signaling events at difference cellular levels, culminating in mitochondria-dependent apoptosis (39). This study obtains evidence in HCC cells that PKC{delta} represents a major downstream effector of FTY720-generated ROS to facilitate caspase-3–dependent apoptosis, and that blockade of ROS production by the NADPH oxidase inhibitor DPI would protect the cells from the effect of FTY720 on PKC{delta} activation and consequent apoptosis. From a mechanistic perspective, this signaling pathway provides a molecular basis to account for the differential susceptibility of different HCC cell lines to FTY720-mediated apoptosis. As shown, the expression levels of PKC{delta} and GST-{pi} represent two key determinants for the cellular capability to cope with FTY720-incurred oxidative stress, which underlie the resistant phenotype of PLC5 cells to the apoptotic effects of FTY720. Consequently, these two biomarkers might be used as predictors to select patients to receive FTY720 therapy. Moreover, GST-{pi} overexpression has been reported in a number of human malignancies, and there exists a strong correlation between high tumor GST-{pi} levels and failure of patients to respond to chemotherapy and low patient survival rates (40). For a mechanistic perspective, therapeutic interference of GST-{pi} expression/activity by antisense or small-molecule agents provides a viable strategy to overcome this resistance (41, 42).

PKC{delta} has been shown to play an intriguing role in regulating apoptosis, either proapoptotic or antiapoptotic, in different cell systems (43, 44). This dichotomous behavior might be, in part, controlled by phosphorylation at different tyrosine residues of PKC{delta} by different kinases. For example, stress signals such as etoposide and H2O2 promoted the phosphorylation of tyrosine residues at 64 and 187 (45), and especially 311 (29), respectively, which presumably committed PKC{delta} to activating caspases through phosphorylation. On the other hand, phosphorylation on Tyr155 was reported to promote the antiapoptotic effect of PKC{delta} in Sindbis virus–infected glioma cells (46). Accordingly, the selective phosphorylation of PKC{delta} at Try311, but not Tyr155, in FTY720-treated Huh7 cells underscores the proapoptotic nature of this signaling pathway.

An earlier report indicates that FTY720 induced apoptosis in HCC cells through phosphoinositide 3-kinase–mediated Akt dephosphorylation (8). This premise, however, was disputed by the finding that overexpression of constitutively active Akt could not rescue Huh7 cells from FTY720-mediated cell death. However, it is noteworthy that the mode of antitumor action of FTY720 might vary in different types of tumor cells. For example, in hematologic malignant cells such as those of chronic myelogenous leukemia and chronic lymphocytic leukemia, FTY720 induced caspase-independent apoptosis by activating PP2A signaling (47, 48). This PP2A activation, however, was not noted in HCC or prostate cancer cells.4 This cell line specificity underlines the pleiotropic nature of FTY720 in killing cancer cells. In light of the heterogeneity in molecular and cellular abnormalities associated with oncogenesis and tumor progression, the ability of FTY720 to target different clinically relevant signaling mechanisms in different cancer types might have therapeutic relevance in cancer therapy. Consequently, this rationale constitutes the impetus of defining the causative mechanisms for the antitumor effects of FTY720 in different types of cancer cells.

In conclusion, this study shows the involvement of ROS-PKC{delta} signaling in FTY720-mediated apoptosis in HCC cells, of which the therapeutic relevance is multifold. First, as PKC{delta} is mostly activated by genotoxic signals such as ionizing radiation (32) and etoposide (49), FTY720 represents a novel PKC{delta} activator without known genotoxic effects. Second, dissociation of the antitumor effect of FTY720 from its S1P activity provides a molecular basis to use it as a scaffold to develop potent antitumor agents devoid of the immunosuppressive activity. Third, FTY720 and its derivatives will be inherently suitable to treat cancers exhibiting increased oxidative stress with high levels of cellular ROS and a low antioxidant capability. Human HCC is prevalent in somatic GST-{pi} gene silencing due to CpG island DNA hypermethylation (50), which provides a mechanistic rationale to include this type of ROS-generating agents in HCC therapy.


   Acknowledgments
 
Grant support: Public Health Service Grant CA112250 from National Cancer Institute, a Leukemia and Lymphoma Society grant, and the Lucius A. Wing Endowed Chair Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
4 Y.S. Lu and C.S. Chen, unpublished data. Back

Received 7/11/07. Revised 11/ 9/07. Accepted 12/11/07.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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