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Sphingosine Suppresses Mesothelioma Cell Proliferation by Inhibiting PKC-δ and Inducing Cell Cycle Arrest at the G₀/G1 Phase

Background/Aims: Sphingosine regulates cellular differentiation, cell growth, and apoptosis. The present study aimed at understanding sphingosine-regulated mesothelioma cell proliferation. Methods: Human malignant mesothelioma cells such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells were cultured. The siRNA to silence the protein kinase C (PKC)-δ-targeted gene was constructed and transfected into cells. MTT assay, cell cycle analysis using a flow cytometry, and cell-free PKC-δ assay were carried out. Results: For all the cell types sphingosine inhibited cell growth in a concentration (1-100 µM)-dependent manner. The sphingosine effect was not prevented by rottlerin, an inhibitor of protein kinase C-δ (PKC-δ); conversely, rottlerin further enhanced the sphingosine effect or rottlerin suppressed mesothelioma cell growth without sphingosine. In the cell-free PKC assay, sphingosine attenuated PKC-δ activity. Knocking-down PKC-δ induced cell cycle arrest at the G0/G1 phase and inhibited cell growth. Conclusion: The results of the present study show that sphingosine suppressed mesothelioma cell proliferation by inhibiting PKC-δ, to induce cell cycle arrest at the G0/G1 phase.


Introduction
Sphingolipids include ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate (S1P). Sphingosine is produced from ceramidase-catalyzed cleavage of fatty acids from ceramide. Evidence has pointed to the role of sphingolipid in the regulation of cellular differentiation, cell growth, and apoptosis. We have earlier found that sphingosine induces apoptosis in hippocampal neurons and astrocytes by activating caspase-3/-9 via a sphingosine-dependent protein kinase (SDK)/14-3-3 protein/Bax/cytochrome c pathway [1]. SDK is produced through proteolytic processing of protein kinase C-δ (PKC-δ) and activated by binding sphingosine [2]. SDK specifically phosphorylates 14-3-3 protein [3,4], thereby dissociating Bax from a complex with 14-3-3 protein, to induce a mitochondriamediated apoptosis. Sphingosine, alternatively, induces apoptosis in rhabdomyosarcoma cells by activating caspase-3/-9 in a Bax-dependent manner [5] or in mouse BALB/c 3T3 clone A31 cells in an SDK-dependent manner [2]. In our recent study, sphingosine induced apoptosis in well differentiated MKN-28 human gastric cancer cells by increasing SDK production from PKC-δ, to phosphorylate 14-3-3 protein, thereby causing disruption of mitochondrial membrane potentials and activating caspase-9 followed by the effector caspase-3. Sphingosine, thus, might be a target for development of anti-tumor drugs.
Malignant mesothelioma is an aggressive tumor arised from previous asbestos exposure. A great deal of challenge has been attempted, yet disappointingly, malignant mesothelioma is considerably resistant to conventional anticancer therapies and no beneficial effect is expected. Then, we were prompted to assess the effect of sphingosine on proliferation of malignant mesothelioma cells. We show here that sphingosine suppresses mesothelioma cell proliferation by inhibiting PKC-δ, to induce cell cycle arrest at the G 0 /G 1 .

Construction and transfection of siRNA
The siRNA to silence PKC-δ-targeted gene (PKC-δ siRNA) and the negative control siRNA (NC siRNA) were obtained from BONAC (Fukuoka, Japan). The PKC-δ siRNA and the NC siRNA were reverse-transfected into cells using a Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA). Cells were used for experiments 48 h after transfection.

Western blotting
Cells were lysed with 1% (w/v) sodium dodecyl sulfate (SDS). Proteins were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) using a TGX gel (BioRad, Hercules, CA, USA) and then transferred to polyvinylidene difluoride membranes. Blotting membranes were blocked with TBS-T [150 mM NaCl, 0.1% (v/v) Tween20 and 20 mM Tris, pH 7.5] containing 5% (w/v) bovine serum albumin and subsequently incubated with an anti-PKC-δ antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or an anti-β-actin antibody (Sigma, St Louis, MO, USA). After washing, membranes were reacted with a horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG antibody. Immunoreactivity was detected with an ECL kit (GE Healthcare, Piscataway, NJ, USA) and visualized using a chemiluminescence detection system (GE Healthcare). Protein concentrations for each sample were determined with a BCA protein assay kit (Pierce, Rockford, IL, USA).

Statistical analysis
Statistical analysis was carried out using Fisher's Protected Least Significant Difference (PLSD) test, unpaired t-test, and Dunnett's test.  without difference in the extent among the cell types ( Fig. 1A, B, C, D). In the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, no significant increase in TUNEL-positive cells was obtained with sphingosine for all the cell types (data not shown). This suggests that sphingosine do not induce mesothelioma cell apoptosis but suppresses mesothelioma cell proliferation. Sphingosine is phosphorylated by sphingosine kinase 1 to produce S1P. Treatment with S1P for 24-48 h had little effect on the number of viable cells at concentrations ranging from 1 to 30 µM for all the cell types ( Fig. 2A, B, C, D). This indicates that sphingosine by itself, but not S1P produced from sphingosine, has the potential to suppress mesothelioma cell proliferation.  were treated with sphingosine (100 µM) for 0-6 h, and Western blotting was carried out using an anti-PKC-δ antibody. Note that no increase in the SDK production (arrows) was found with sphingosine treatment and that similar results were obtained with 4 independent experiments.

Sphingosine suppresses mesothelioma cell proliferation
In our earlier study, sphingosine induced apoptosis in hippocampal neurons and astrocytes via a PKC-δ/SDK pathway [1]. We subsequently examined whether the sphingosine effect is due to PKC-δ/SDK activation. Sphingosine did not increase SDK production in all the cell types (Fig. 3A, B, C, D), ruling out the participation of SDK in the sphingosine effect here. Sphingosine-induced decrease in the number of viable cells was not inhibited by rottlerin (10 µM), an inhibitor of PKC-δ; conversely, further decrease in the number of viable cells was found in the presence of rottlerin (P<0.001 as compared with the effect of sphingosine in the absence of rottlerin for all the cell types, Fisher's PLSD test) (Fig. 4A, B,  C, D). Moreover, treatment with rottlerin alone for 24-48 h decreased the number of viable cells in a concentration (0.1-10 µM)-dependent manner for all the cell types (Fig. 5A, B, C,  D). Taken together, these results suggest that PKC-δ promotes proliferation of mesothelioma cells. Then, we postulated that sphingosine might suppress proliferation of mesothelioma cells by inhibiting PKC-δ.

Mesothelioma cell proliferation is suppressed by inhibiting PKC-δ
To obtain evidence for sphingosine-induced PKC-δ inhibition, we carried out cell-free PKC assay. PKC-δ activity in the absence of sphingosine was 2.26 ± 0.32 pmol/min, but the activity was significantly attenuated in the presence of sphingosine (0.76 ± 0.06 pmol/min) (Fig. 6). This confirms that sphingosine inhibits PKC-δ.

Sphingosine suppresses mesothelioma cell proliferation by inducing cell cycle arrest at the G 0 /G 1 phase
In the cell cycle analysis, knocking-down PKC-δ significantly increased the population of cells at the G 0 /G 1 phase of cell cycling, but it otherwise decreased the population at the G 2 /M phase for all the cell types (Fig. 8). This implies that PKC-δ accelerates cell cycling for mesothelioma cells, i.e., inhibiting PKC-δ causes cell cycle arrest at the G 0 /G 1 phase.

Discussion
The results of the present study demonstrate that sphingosine suppresses mesothelioma cell proliferation. Sphingosine is recognized to bind to and activate SDK, that is produced through proteolytic processing of PKC-δ [2]. Sphingosine induces apoptosis in hippocampal neurons and astrocytes by activating SDK, to phosphorylate 14-3-3 protein, thereby dissociating Bax and disrupting mitochondrial membrane potentials, and then leading to activation of caspase-9 and the effector caspase-3 [1]. In the present study, sphingosine did not increase SDK production, which would exclude the possibility for the implication of SDK in sphingosine-induced suppression of mesothelioma cell proliferation.
Of PKC isozymes cloned PKC-δ as well as the other novel PKCs including PKC-ε, -η, -θ, and -µ, is activated by signaling cascades linked to phospholipase A 2 in a Ca 2+ -independent manner [7,8]. PKC-δ is activated by translocation towards the membrane surface [9]. Sphingosine inhibits PKC activation induced by diacylglycerol, phorbol dibutyrate, calcium, unsaturated fatty acids or other lipids [10,11]. PKC binds to membranes through interactions with diacylglycerol and negatively charged phosphatidylserine. Sphingosine may be localized in regions of acidic lipids, thereby inhibiting PKC binding to membranes and activity [12][13][14][15]. Sphingosine, alternatively, inhibits the enzyme phosphatidic acid phosphohydrolase, that generates diacylglycerol production from phosphatidic acid produced by phospholipase D-catalyzed hydrolysis of phosphatidylcholine [16][17][18]. In the cell-free PKC assay, sphingosine attenuated PKC-δ activity. This indicates that sphingosine inhibits PKC-δ through its direct binding. Notably, rottlerin, an inhibitor of PKC-δ, further enhanced sphingosine-induced suppression of mesothelioma cell growth or rottlerin alone without sphingosine inhibited mesothelioma cell growth. Moreover, knocking-down PKC-δ significantly suppressed mesothelioma cell growth. It is indicated from these results that sphingosine suppresses mesothelioma cell proliferation by inhibiting PKC-δ. In the cell cycle analysis, knocking-down PKC-δ induced cell cycle arrest at the G 0 / 1 phase for all the cell types used here. Overall, these results lead to a conclusion that sphingosine suppresses mesothelioma cell proliferation by inhibiting PKC-δ, to induce cell cycle arrest at the G 0 / 1 phase. Lines of evidence have pointed to the contrasting roles of PKC-δ in cell survival and cell death [19]. The pro-and anti-apoptotic function of PKC-δ not only depends on the cell type but also on the stimulus. PKC-δ promotes survival of a variety of cancer cells such as nonsmall cell lung cancer, breast cancer, pancreatic cancer, liver cancer, and chronic lymphocytic leukemia cells. PKC-δ protects glioma cells from the apoptosis induced by TRAIL, and phosphorylation of PKC-δ on Tyr155 and its cleavage are essential for the anti-apoptotic effect of PKC-δ [20]. Phosphorylation of PKC-δ at Tyr332 is also required for protection against TRAIL-induced apoptosis [21]. Overexpression of PKC-δ increases cell proliferation, anchorage-independent growth, and resistance to apoptotic stimuli by elevating cyclin D1 level and hyperphosphorylating Rb in murine mammary NMuMG cells [22]. Conversely, PKC-δ antisense oligonucleotide and dominant-negative PKC-δ decrease survival of breast cancer MCF-7 and MDA-MB-231 cells [23]. As found with the present study, rottlerin enhances apoptosis in non-small cell lung cancer cells [24]. Rottlerin blocks DNA damageinduced apoptosis, but otherwise it potentiates receptor-induced apoptosis [25].
PKC-δ promotes cell survival via several well-known pro-survival pathways, that include NF-κB, Akt, and ERK. PKC-δ prevents apoptosis in colon cancer cells by inducing inhibitor of apoptosis protein-2 and FLICE-like inhibitory protein via NF-κB [26,27]. PKC-δ suppresses autophagy in breast cancer cells via NF-κB [28]. Tumor necrosis factor (TNF) is recognized to induce translocation of PKC-δ to the nucleus, where it bound to the NF-κB RelA subunit and induced transactivation of p65/RelA [29]. PKC-δ protects MCF-7 breast cancer cells against TNF-related apoptosis-inducing ligand-mediated apoptosis [30]. Phosphoinositide-dependent kinase-1 may also participate in PKC-δ survival signaling in cells containing an activated p21Ras protein [31]. Activation of Akt and ERK, alternatively, is responsible for PKC-δ-mediated increase in anchorage-independent growth and resistance of pancreatic ductal cancer cells to apoptotic stimuli [32]. PKC-δ-induced cell proliferation in murine mammary cells is associated with activation of ERK/MAPK [22]. In contrast, PKCδ-induced suppression of ERK1/2 is associated with the survival of MDA-MB-231 cells [33]. PKC-δ attenuates apoptosis by inducing phosphorylation and proteasomal degradation of the proapoptotic protein Bim via the MEK/MAPK pathway in immortalized and malignant keratinocytes [34]. Syk, a tyrosine kinase, promotes survival of B-cell chronic lymphocytic leukemia cells by stabilizing Mcl-1 in a PKC-δ-dependent manner [35]. PKC-δ activated by Syk phosphorylates and inhibits glycogen synthase kinase-3, causing stabilization of Mcl-1 and inhibition of apoptosis. It is presently unknown what signaling pathways underlie sphingosine/PKC-δ-dependent inhibition of proliferation in mesothelioma cells. To address this question, we are carrying out further experiments.
In conclusion, the results of the present study show that sphingosine inhibits PKC-δ, thereby inducing cell cycle arrest at the G 0 / 1 phase, and then leading to suppression of mesothelioma cell proliferation. This may represent further insight into the sphingosine signaling pathway relevant to cell proliferation and death.