We have previously reported that, in prostate cancer, inhibition of the oncogenic sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway is a key element in chemotherapy-induced apoptosis. Here, we show that selective pharmacologic inhibition of SphK1 triggers apoptosis in LNCaP and PC-3 prostate cancer cells, an effect that is reversed by SphK1 enforced expression. More importantly, we show for the first time that the up-regulation of the SphK1/S1P pathway plays a crucial role in the resistance of prostate cancer cells to chemotherapy. Importantly, pharmacologic SphK1 inhibition with the B-5354c compound sensitizes LNCaP and PC-3 cells to docetaxel and camptothecin, respectively. In vivo, camptothecin and B-5354c alone display a limited effect on tumor growth in PC-3 cells, whereas in combination there is a synergy of effect on tumor size with a significant increase in the ceramide to S1P sphingolipid ratio. To conclude, our study highlights the notion that drugs specifically designed to inhibit SphK1 could provide a means of enhancing the effects of conventional treatment through the prosurvival antiapoptotic SphK1/S1P pathway. [Mol Cancer Ther 2008;7(7):1836–45]

Prostate cancer is the leading cancer in the male population and its management remains a complex problem (1). For men with advanced disease, chemotherapy may be offered after failure of hormonal therapy. Systemic chemotherapy has been evaluated in men with hormone-refractory prostate cancer for many years, with two effective compounds identified.

The microtubules, targeted by camptothecins and docetaxel, seem to be a relevant target in hormone-refractory prostate cancer as shown by the survival improvement observed in docetaxel-based randomized phase III trials (2, 3). The U.S. Food and Drug Administration recently approved docetaxel as standard first-line therapy in patients with metastatic hormone-refractory prostate cancer. However, the benefit is modest—2-month improvement in median survival with no curative potential (2, 3). It has been also recently suggested that camptothecin analogues might be of clinical relevance both in hormone-refractory prostate cancer patients as a first-line chemotherapy (4) and in patients who failed one prior cytotoxic chemotherapy (5).

We recently reported that the efficacy of docetaxel and camptothecin in prostate cancer cell and mouse models correlated with the shift of the ceramide to sphingosine 1-phosphate (S1P) ratio toward proapoptotic ceramide (6). Sphingolipid ceramide and S1P are critical signal transduction molecules regulating cell death and survival. Ceramide mediates a wide array of stress signals such as anticancer treatments leading to apoptosis, whereas S1P exerts prosurvival capabilities by antagonizing ceramide effects. The opposing directions of ceramide- and S1P-mediated signaling gave birth to the concept of a ceramide/S1P rheostat and the assumption that the balance between these two sphingolipids could determine whether a cell survives or dies (7). A key regulator of this balance is sphingosine kinase-1 (SphK1), which phosphorylates sphingosine (the catabolite of ceramide) to generate S1P, because it reduces proapoptotic ceramide levels by driving the sphingolipid metabolism toward antiapoptotic S1P. Accordingly, SphK1 overexpression promotes survival in response to stresses that increase ceramide content (6, 8). SphK1 mRNA content was found elevated in various human (9, 10) and rodent tumor tissues (11). On the contrary, SphK1 down-regulation has been associated with cell death induced by anticancer treatments (6, 8, 12).

We have previously reported that limited efficacy of camptothecin in PC-3 and docetaxel in LNCaP cells was associated with a transient paradoxical increase in SphK1 activity, suggesting that SphK1 stimulation might compromise chemotherapy success (6). In view of these findings, the objectives of the present study were to determine whether direct inhibition of SphK1 could sensitize LNCaP and PC-3 cells to docetaxel and camptothecin, respectively. SphK1 inhibition was achieved using B-5354c, a novel specific inhibitor for SphK1 (13). We report that pharmacologic inhibition strongly sensitized prostate cancer cells to the effects of camptothecin and docetaxel. Moreover, these findings could be confirmed in vivo using an orthotopically transplanted fluorescent PC-3 model in mouse. Collectively, our data suggest that the SphK1/S1P pathway could represent a target for the development of novel chemosensitizers.

Cell Lines

Human prostate cancer PC-3 and LNCaP were obtained from the German Collection of Microorganisms and Cell Cultures and cultured in RPMI 1640 containing 10% fetal bovine serum. SphK1- and green fluorescent protein (GFP)-overexpressing cell models have been described previously (6). All experiments were conducted in the absence of serum at 50% confluence and then treated as indicated in the figure legends.

Reagents

Culture medium, serum, and antibiotics were obtained from Invitrogen. Camptothecin was from Sigma. Docetaxel and camptothecin derivative irinotecan were from Sanofi-Aventis. Escherichia coli diacylglycerol kinase and octyl-d-glucopyranoside were from Calbiochem. Alkaline phosphatase was from Sigma. The SphK1 inhibitor B-5354c was prepared in DMSO and stored at −20°C. [γ-32P]-ATP (3,000 mCi/mmol) was purchased from Perkin-Elmer, and silica gel 60 high-performance TLC plates were from VWR. The Annexin V-FITC kit was purchased from Beckman Coulter; Syto 13 and propidium iodide (PI) were purchased from Molecular Probes. The pan-caspase inhibitor zVAD-fmk was from Bachem and prepared in DMSO.

Cell Viability, Flow Cytometry, and Staining of Apoptotic Nuclei

Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described (14). Annexin V-FITC/PI staining was used for the analysis of apoptotic cells. After trypsinization, cells were labeled with Annexin V-FITC according to the manufacturer's instructions, and PI was added to the samples immediately before flow cytometric measurement as previously described (6). Apoptosis was also visually assessed by double staining cells with Syto 13 (1 μmol/L) and PI (6 μg/mL) for 5 min at 37°C.

SphK1 Assay and Mass Measurement of Ceramide, S1P, and Phospholipids

SphK1 activity was done as described previously (15) and determined in the presence of 50 μmol/L sphingosine, 0.25% Triton X-100, and [γ-32P]ATP (10 μCi, 1 mmol/L) containing 10 mmol/L MgCl2. The labeled S1P was separated by TLC on silica gel 60 with 1-butanol/ethanol/acetic acid/water (80:20:10:10, v/v) and visualized by autoradiography. Activity was expressed as picomoles of S1P formed/min/mg of protein.

Amounts of ceramide were measured by the E. coli enzyme diacylglycerol kinase (16). [32P]ceramide 1-phosphate was extracted, resolved from other reaction products by TLC using chloroform/acetone/methanol/acetic acid/water (50:20:15:10:5, v/v) as developing solvent, and quantitated by liquid scintillation counting. Intracellular S1P content was measured as described by Edsall and Spiegel (17). Briefly, to aqueous phase of Folch extraction buffer C [200 mmol/L Tris-HCl (pH 7.4), 75 mmol/L MgCl2 in 2 mol/L glycine (pH 9.0)] was added (1:6, v/v) and S1P was dephosphorylated by addition of 50 units/sample of alkaline phosphatase for 30 min at 37°C. Reaction was stopped by addition of HCl and organic phase containing sphingosine was separated and evaporated. Evaporated sphingosine was resuspended in sphingosine kinase buffer with 0.25% Triton X-100. Sphingosine was converted to S1P by addition of cytosolic extracts of SphK1-overexpressing PC-3 cells and [γ-32P]ATP (10 μCi, 1 mmol/L) containing 10 mmol/L MgCl2.

Fluorogenic DEVD Cleavage Enzyme Assay

Enzyme reactions were done with 50 μg cytosolic proteins and a final concentration of 20 μmol/L Ac-DEVD-AMC substrate (Bachem) as previously reported (18).

Western Blot Analysis and Antibodies

Cell lysate preparation and Western blotting were carried out as respectively reported (19). Rabbit anti–poly(ADP-ribose) polymerase (Cell Signaling Technology), rabbit anti-caspase-3 (gift from Dr. Nicholson, Merck-Frosst, Pointe Claire-Dorval, Quebec, Canada), rabbit anti-caspase-7 (Cell Signaling Technology), and mouse anti-FLAG (clone M2; Sigma) were used as primary antibodies. Proteins were visualized by enhanced chemiluminescence detection system using anti-rabbit or anti-mouse horseradish peroxidase–conjugated IgG (Bio-Rad). Equal loading was confirmed by probing the blots with the mouse anti-actin antibody (clone AC-15; Sigma).

Animals

NMRI/Nu (nu/nu) 6-wk-old male mice were obtained from Elevage Janvier. The mice were housed in a barrier facility on High Efficiency Particulate Air–filtered racks. At 7 to 8 wk of age, the animals were used in accordance with the principles and procedures outlined in Council Directive 86/609/EEC about the protection of animals used for experimental and other scientific purposes. The local Animal Care and Use Committee of Institut National de la Sante et de la Recherche Medicale approved all animal studies.

Surgical Orthotopic Implantation of PC-3 Prostate Cancer Cells

Intraprostatic human prostate cancer xenografts were established in nude mice by surgical orthotopic implantation as originally described (20). Briefly, mice were anesthetized by isoflurane inhalation and placed in the supine position. A lower midline abdominal incision was made and 20 μL tumor cell suspension (1 × 106 cells) was injected into the dorsal lobe of the prostate using a 30-gauge needle and glass syringe (Hamilton). After implantation, the surgical wound was closed in two layers with 4-0 Dexon interrupted sutures. All procedures were done with a dissecting microscope (KAPS-SoM82). Three weeks after surgical orthotopic implantation, the mice were randomized into different groups for three treatments with irinotecan and/or B-5354c (each week for 2 wk). One group served as the negative control and received a sham injection instead of an active treatment.

Autopsy, Histology, and In vivo Fluorescence Imaging

Two days after the final treatment, all mice were anesthetized and percutaneous GFP imaging analysis was done, and the mice were then euthanized with carbon dioxide asphyxiation for direct internal imaging. A long midline incision was made to access the abdominal and the thoracic cavities. The fluorescent primary tumor was removed en bloc with the seminal vesicles, and a meticulous fluorescence-assisted exploration (see description below) was conducted to establish the presence of periaortic nodal extensions, as well as adrenal, liver, pancreas, and lung metastases. The primary tumors were then removed, saved, and routinely processed for H&E histology to confirm the nature of the disease, or processed for sphingolipid analyses as described above.

GFP fluorescence in the tumors was detected with a Leica MZFL III fluorescence stereomicroscope (Leica). High-resolution 16-bit images of 1,392 × 1,040 pixels were captured on a personal computer by using a thermoelectrically cooled charge-coupled device camera (model CoolSNAP HQ; Roper Scientific). Magnifications (×8 or ×35) were used to visualize the whole tumor and lymph nodes, or micrometastases, respectively. Selective excitation was produced with a Mercury Arc Lamp (HBO, Osram) and a GFP filter (Leica). Color images were obtained using a Micro*Color tunable RGB filter (CRI). The images were processed for contrast and brightness, and the GFP fluorescence was analyzed with MetaVue 6.2 software (Princeton Instruments).

Isobolographic Analysis

An isobologram analysis was used to establish whether combination of conventional chemotherapies with the B-5354c compound was associated with synergism, additive effects, or antagonism (21).

Data Representation and Statistical Analysis

The statistical significance of differences between the means of two groups was evaluated by unpaired Student's t test. Differences in the number of metastases per mouse were examined using a nonparametric Wilcoxon rank sum test. All statistical tests were two sided. Calculations were done using InStat (GraphPad Software). Representative images are presented for the experiments carried out with fluorescence.

The SphK1 Inhibitor B-5354c Induces a Caspase-Dependent Apoptosis in Prostate Cancer Cells by Shifting the Sphingolipid Rheostat toward Ceramide

B-5354c induced extensive cell death in a dose- and time-dependent manner in both PC-3 and LNCaP cells (Fig. 1A). Staining with Syto 13/PI revealed characteristic condensed nuclei indicative of apoptosis (data not shown). Apoptosis triggered by B-5354c was caspase dependent as pretreatment with the pan-caspase inhibitor zVAD-fmk could markedly reverse its effect (Fig. 1B). We next used the fluorogenic substrate Ac-DEVD-AMC, which corresponds to the motif cleaved in the proteins targeted by executioner caspases, to measure the activity of these caspases. A significant increase in DEVDase activity (Fig. 1C) was observed in both PC-3 and LNCaP cells treated by B-5354c, which was confirmed by Western blot against caspase-3 and caspase-7 (Supplementary Fig. S1).6

6

Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Accordingly, the DNA enzyme repair poly(ADP-ribose) polymerase, a substrate of these caspases, was cleaved after treatment with B-5354c (Supplementary Fig. S1).6 The effect of B-5354c on the status of the sphingolipid rheostat was examined. Exposure of PC-3 and LNCaP cells to B-5354c resulted in a profound time-dependent SphK1 inhibition that culminated at 24 h (Fig. 1D). This was further illustrated by the decrease in S1P content (Fig. 1D), which was concurrently associated with increase in ceramide levels (Fig. 1D). Consequently, the ceramide to S1P ratio was amplified overtime and, after 24 h of treatment, a 4- to 7-fold increase was observed in LNCaP and PC-3 cells, respectively (calculated from the relative amounts of S1P and ceramide levels shown in Fig. 1D).

Figure 1.

SphK1 inhibitor B-5354c induces apoptosis in a caspase-dependent manner by tilting the ceramide/S1P rheostat toward ceramide. A, PC-3 and LNCaP cells were treated with 1, 5, or 10 μmol/L of B-5354c for the indicated times and loss of cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Points, mean of five independent experiments; bars, SD. B, PC-3 and LNCaP cells were preincubated for 2 h with 20 μmol/L zVAD-fmk or not and then treated for 24 h with 10 μmol/L B-5354c and apoptosis was assessed by staining with Annexin V-PI. Columns, mean of three independent experiments; bars, SD. The two-tailed P values between the means are as follows: ***, P < 0.001. C, DEVDase activity was measured with fluorogenic Ac-DEVD-AMC substrate in cell lysates from PC-3 and LNCaP cells treated for 24 h with B-5354c (10 μmol/L). Similar results were obtained in three independent experiments. D, cells were incubated with 10 μmol/L B-5354c for the indicated times and then tested for SphK1 activity and S1P and ceramide levels. Columns, mean of five independent experiments done in duplicate; bars, SD.

Figure 1.

SphK1 inhibitor B-5354c induces apoptosis in a caspase-dependent manner by tilting the ceramide/S1P rheostat toward ceramide. A, PC-3 and LNCaP cells were treated with 1, 5, or 10 μmol/L of B-5354c for the indicated times and loss of cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Points, mean of five independent experiments; bars, SD. B, PC-3 and LNCaP cells were preincubated for 2 h with 20 μmol/L zVAD-fmk or not and then treated for 24 h with 10 μmol/L B-5354c and apoptosis was assessed by staining with Annexin V-PI. Columns, mean of three independent experiments; bars, SD. The two-tailed P values between the means are as follows: ***, P < 0.001. C, DEVDase activity was measured with fluorogenic Ac-DEVD-AMC substrate in cell lysates from PC-3 and LNCaP cells treated for 24 h with B-5354c (10 μmol/L). Similar results were obtained in three independent experiments. D, cells were incubated with 10 μmol/L B-5354c for the indicated times and then tested for SphK1 activity and S1P and ceramide levels. Columns, mean of five independent experiments done in duplicate; bars, SD.

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SphK1 Overexpression Inhibits B-5354c–Induced Apoptosis in PC-3 and LNCaP Cells by Shifting the Sphingolipid Rheostat toward S1P

To establish proof of concept that SphK1 inhibition was critical for induction of apoptosis by B-5354c, transfection of PC-3 and LNCaP cells with SphK1 was done to examine whether these cells will be rendered resistant to B-5354c treatment. Transfection efficiency was verified by Western blotting (Fig. 2A) and SphK1 activity of LNCaP/SphK1 and PC-3/SphK1 cells was increased to over 1,000 pmol/mg protein/min (Fig. 2B). Enforced expression of SphK1 rendered both PC-3 and LNCaP cells highly resistant to treatment by B-5354c as shown in Fig. 2C, which was coupled with a minimal increase in ceramide content (Fig. 2D) and lower decrease in S1P levels (Fig. 2E). Consequently, the ceramide to S1P ratio was almost 3-fold lower in PC-3–overexpressing SphK1 cells treated with B-5354c for 24 h when compared with neo cells (1.49:0.53 versus 1.62:0.24, respectively) similar to what is observed in LNCaP cells (1.15:0.41 versus 1.28:0.86, respectively).

Figure 2.

SphK1 enforced expression in PC-3 and LNCaP cells shifts the lipid biostat toward prosurvival S1P and protects against B-5354c–induced apoptosis. A, SphK1 expression was analyzed by Western blotting using an anti-FLAG antibody. B, basal SphK1 activity was measured in PC-3 and LNCaP cells overexpressing or not SphK1. C, apoptosis was quantified by Annexin V-PI assay in PC-3 and LNCaP cells treated in the presence of 10 μmol/L B-5354c for 24 h. D, cells were incubated with 10 μmol/L B-5354c for 24 h and then tested for ceramide and S1P levels. Columns, mean of three independent experiments done in triplicate for sphingolipid dosage and five independent experiments for apoptosis assay; bars, SD. The two-tailed P values between the means are as follow: ***, P < 0.001.

Figure 2.

SphK1 enforced expression in PC-3 and LNCaP cells shifts the lipid biostat toward prosurvival S1P and protects against B-5354c–induced apoptosis. A, SphK1 expression was analyzed by Western blotting using an anti-FLAG antibody. B, basal SphK1 activity was measured in PC-3 and LNCaP cells overexpressing or not SphK1. C, apoptosis was quantified by Annexin V-PI assay in PC-3 and LNCaP cells treated in the presence of 10 μmol/L B-5354c for 24 h. D, cells were incubated with 10 μmol/L B-5354c for 24 h and then tested for ceramide and S1P levels. Columns, mean of three independent experiments done in triplicate for sphingolipid dosage and five independent experiments for apoptosis assay; bars, SD. The two-tailed P values between the means are as follow: ***, P < 0.001.

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SphK1 Inhibition by B-5354c Sensitizes Prostate Cancer Cells to Chemotherapy-Induced Apoptosis

We recently reported that PC-3 and LNCaP were poorly responsive, respectively, to camptothecin and docetaxel because of a low elevation of the ceramide to S1P ratio shift toward proapoptotic ceramide (6). Interestingly, we noted that treatment with camptothecin in PC-3 and docetaxel in LNCaP induced a transient SphK1 activity increase (up to 24 h) followed by an inhibition (6). Therefore, we asked whether inhibiting directly SphK1 could sensitize PC-3 cells to camptothecin and LNCaP to docetaxel. As already reported (6, 22, 23), PC-3 cells exhibited a low sensitivity to camptothecin (0.5 μmol/L) with an apoptosis level of ∼10% at 48 h culminating at around 20% to 25% after 72 h of incubation (Fig. 3A, left) and was further confirmed by Syto 13/PI staining (data not shown). Higher dose of camptothecin (up to 1 μmol/L) did not enhance apoptosis (data not shown). On the contrary, a time-dependent apoptosis was observed in cells treated with 20 nmol/L docetaxel (Fig. 3A, left; ref. 6). When coincubated with camptothecin and SphK1 inhibitor B-5354c, PC-3 cells did dramatically undergo apoptosis (Fig. 3A, right). Of note, using the isobologram method, there was a clear synergistic effect between camptothecin and B-5354c (Supplementary Fig. S2).6 The superiority of this combined treatment was confirmed by Syto 13/PI staining (data not shown). Conversely, combining B-5354c with docetaxel did not improve the sensitivity to docetaxel (Fig. 3A, right). This chemosensitizing effect of B-5354c when added to camptothecin was seen at the executioner caspase level with a remarkable DEVDase activity increase that was not observed in cells coincubated with B-5354c and docetaxel (Fig. 3C).

Figure 3.

B-5354c sensitizes PC-3 and LNCaP cells to apoptosis. Apoptosis was quantified by Annexin V-PI assay in PC-3 (A) and LNCaP (B) cells treated with or without 0.5 μmol/L camptothecin (CPT), 20 nmol/L docetaxel, or 5 μmol/L B-5354c for the indicated times (left) or for 48 h (right). DEVDase activity in protein extracts from cells incubated for 24 h, in the presence or absence of 0.5 μmol/L camptothecin or 20 nmol/L docetaxel with or without 5 μmol/L B-5354c, was measured with the fluorogenic Ac-DEVD-AMC substrate in PC-3 (C) and LNCaP (D) cells. Striped columns, combined treatments.

Figure 3.

B-5354c sensitizes PC-3 and LNCaP cells to apoptosis. Apoptosis was quantified by Annexin V-PI assay in PC-3 (A) and LNCaP (B) cells treated with or without 0.5 μmol/L camptothecin (CPT), 20 nmol/L docetaxel, or 5 μmol/L B-5354c for the indicated times (left) or for 48 h (right). DEVDase activity in protein extracts from cells incubated for 24 h, in the presence or absence of 0.5 μmol/L camptothecin or 20 nmol/L docetaxel with or without 5 μmol/L B-5354c, was measured with the fluorogenic Ac-DEVD-AMC substrate in PC-3 (C) and LNCaP (D) cells. Striped columns, combined treatments.

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In line with previous reports, camptothecin treatment induced a time-dependent apoptosis in the LNCaP cell model (2426), whereas docetaxel was much less efficient consistent with observations that docetaxel is a weak inducer of apoptosis in this cell line (Fig. 3B, left; refs. 6, 24). However, when combined with SphK1 inhibitor B-5354c (5 μmol/L), a strong apoptosis was observed in LNCaP cells treated with docetaxel (Fig. 3B, right). Of note, combined camptothecin and B-5354c treatment did not enhance the sensitivity of LNCaP to camptothecin (Fig. 3B, right). Similar to the observations made with camptothecin and B-5354c in PC-3 cells, a synergistic effect between docetaxel and B-5354c in LNCaP cells was found after isobolographic analysis (Supplementary Fig. S2).6 The increased efficacy detected in LNCaP treated with both docetaxel and B-5354c was also reflected at the executioner caspase level with a strong increase in DEVDase activity that was not observed in cells treated with B-5354c and camptothecin (Fig. 3D).

As shown in Fig. 4A and C, B-5354c (5 μmol/L) could markedly lower SphK1 activity in both PC-3 and LNCaP cells. On the contrary, camptothecin in PC-3 and docetaxel in LNCaP remarkably increased SphK1 activity within 24 h of treatment (Fig. 4A and C). When coincubated with camptothecin, B-5354c could totally block the stimulation of SphK1 induced by camptothecin in PC-3 (Fig. 4A). Mirroring the effect on SphK1, we found that camptothecin could strongly up-regulate the content of S1P in PC-3 cells (Fig. 4B, bottom) while having smaller effect on ceramide levels (Fig. 4B, top). B-5354c could hinder the S1P increase induced by camptothecin (Fig. 4B) and it could potentiate the elevation of proapoptotic ceramide (Fig. 4B).

Figure 4.

B-5354c chemosensitizes PC-3 and LNCaP cells by counteracting the stimulation of SphK1 induced by camptothecin or docetaxel. PC-3 (A and B) and LNCaP (C and D) cells were incubated for 24 h with the indicated concentrations of B-5354c, camptothecin, or docetaxel, and then SphK1 activity (A and C) and ceramide and S1P (B and D) levels were determined. Columns, mean of five independent experiments; bars, SD. The two-tailed P values between the means are as follow: ***, P < 0.001; **, P < 0.01; *, P < 0.1. ns, not significant. Striped columns, combined treatments.

Figure 4.

B-5354c chemosensitizes PC-3 and LNCaP cells by counteracting the stimulation of SphK1 induced by camptothecin or docetaxel. PC-3 (A and B) and LNCaP (C and D) cells were incubated for 24 h with the indicated concentrations of B-5354c, camptothecin, or docetaxel, and then SphK1 activity (A and C) and ceramide and S1P (B and D) levels were determined. Columns, mean of five independent experiments; bars, SD. The two-tailed P values between the means are as follow: ***, P < 0.001; **, P < 0.01; *, P < 0.1. ns, not significant. Striped columns, combined treatments.

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In LNCaP cells, B-5354c could markedly inhibit the stimulation of SphK1 (Fig. 4C) and the production of S1P (Fig. 4D, bottom) induced by docetaxel, whereas it could amplify the increase in ceramide (Fig. 4D, top). In contrast, no distinct alteration of the SphK1 activity or sphingolipid levels was found in PC-3 cells and LNCaP when combined B-5354c + docetaxel and B-5354c + camptothecin treatments were respectively used (Fig. 4).

B-5354c Increases Efficacy of Irinotecan in an Orthotopic PC-3/GFP Model Established in Nude Mice

The chemosensitizing role of B-5354c was examined in vivo using a surgical orthotopic implantation of human PC-3 cells overexpressing GFP. The histology of the tumor was consistent with poorly differentiated prostate cancer (data not shown). As shown by high-magnification microscopy, smaller tumors were seen with 70 mg/kg irinotecan (Fig. 5A). Although B-5354c treatment alone exhibited a limited effect on tumor growth, the association between irinotecan and B-5354c remarkably diminished tumors size indicative of a synergistic effect (Fig. 5A). The tumor mass measurement showed that combination of irinotecan and B-5354c was much more effective than irinotecan (Fig. 5B). We next evaluated the SphK1 activity of primary tumor samples obtained from treated mice. We observed a lower SphK1 activity in mice cotreated with B-5354c and irinotecan (more than 50% less) compared with sham-treated animals (Fig. 5C). A dramatic reduction in the S1P content (Fig. 5D) was observed in B-5354c–treated and irinotecan-treated mice compared with untreated or irinotecan-treated animals. Conversely, the ceramide content was dramatically increased in B-5354c–treated and irinotecan-treated mice compared with untreated or irinotecan-treated animals (Fig. 5D). These in vivo data thus support the idea suggested in vitro (Fig. 3A) that SphK1 inhibition can potentiate camptothecin effects on PC-3 cells.

Figure 5.

Effects of camptothecin and B-5354c on growth of established GFP-expressing human PC-3 cells in nude mice. Three weeks after surgical orthotopic implantation, mice were randomized into four groups (seven to eight animals per arm). These animals were then subjected to 3 weekly 70 mg/kg irinotecan treatment, 20 mg/kg B-5354c treatment, combination of 70 mg/kg irinotecan plus 20 mg/kg B-5354c treatment, or a sham treatment by i.p. injections. A, representative fluorescent primary prostate tumors from sham-treated, irinotecan-treated, B-5354c–treated, and combination of irinotecan and B-5354c–treated groups at the time of autopsy. Red arrows, primary orthotopic GFP tumor; yellow, bladder. B, tumor mass of excised primary GFP-labeled tumor. Columns, mean of seven to eight animals; bars, SE. SphK1 activity (C) and ceramide and S1P levels (D) were measured in tissue extracts obtained from sham-treated, irinotecan-treated, B-5354c–treated, and combination of irinotecan and B-5354c–treated animals. Columns, mean of seven to eight animals; bars, SE. The two-tailed P values between the means are as follow: ***, P < 0.001; **, P < 0.01; *, P < 0.1.

Figure 5.

Effects of camptothecin and B-5354c on growth of established GFP-expressing human PC-3 cells in nude mice. Three weeks after surgical orthotopic implantation, mice were randomized into four groups (seven to eight animals per arm). These animals were then subjected to 3 weekly 70 mg/kg irinotecan treatment, 20 mg/kg B-5354c treatment, combination of 70 mg/kg irinotecan plus 20 mg/kg B-5354c treatment, or a sham treatment by i.p. injections. A, representative fluorescent primary prostate tumors from sham-treated, irinotecan-treated, B-5354c–treated, and combination of irinotecan and B-5354c–treated groups at the time of autopsy. Red arrows, primary orthotopic GFP tumor; yellow, bladder. B, tumor mass of excised primary GFP-labeled tumor. Columns, mean of seven to eight animals; bars, SE. SphK1 activity (C) and ceramide and S1P levels (D) were measured in tissue extracts obtained from sham-treated, irinotecan-treated, B-5354c–treated, and combination of irinotecan and B-5354c–treated animals. Columns, mean of seven to eight animals; bars, SE. The two-tailed P values between the means are as follow: ***, P < 0.001; **, P < 0.01; *, P < 0.1.

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Our fluorescent model also allowed us to monitor metastatic dissemination (Table 1). Although no treatment was able to prevent periaortic lymph nodes, there were significant differences with regard to the other metastasis sites. Irinotecan alone reduced the number of metastases (average per animal: 1.00) in comparison with sham-treated animals (average per animal: 3.13). Of interest, although the SphK1 inhibitor by itself did not have a significant effect on metastases dissemination (average per animal: 1.71), cotreatment with B-5354c did significantly improve the effect of irinotecan (average per animal: 0.57).

Table 1.

Efficacy of irinotecan with or without B-5354c against metastases

Treatments
Sham
Irinotecan
B-5354c
Irinotecan + B-5354c
No. mice with metastases/total no. mice8/8 (100%)7/7 (100%)7/7 (100%)7/7 (100%)
Metastasis sites     
    Periaortic lymph nodes 2, 2, 2, 2, 2, 2, 2, 2 1, 2, 2, 2, 1, 2, 2 2, 2, 2, 2, 2, 2, 2 2, 2, 2, 2, 2, 2, 2 
    Periadrenal 2, 2, 2, 2, 2, 2, 2, 2 0, 2, 0, 1, 0, 0, 2 2, 0, 2, 1, 2, 1, 2 0, 2, 2, 0, 0, 0, 0 
    Liver 1, 0, 0, 0, 1, 0, 1, 1 0, 1, 0, 0, 0, 0, 0 0, 0, 1, 0, 1, 0, 0 0, 0, 0, 0, 0, 0, 0 
    Mesenteric 1, 0, 0, 0, 0, 0, 1, 1 0, 0, 0, 0, 0, 0, 1 0, 0, 0, 0, 0, 0, 0 0, 0, 0, 0, 0, 0, 0 
    Lung 1, 0, 0, 0, 0, 1, 0, 0 0, 0, 0, 0, 0, 0, 0 0, 0, 0, 0, 0, 0, 0 0, 0, 0, 0, 0, 0, 0 
Total no. metastases 41 19* 26 18 
Treatments
Sham
Irinotecan
B-5354c
Irinotecan + B-5354c
No. mice with metastases/total no. mice8/8 (100%)7/7 (100%)7/7 (100%)7/7 (100%)
Metastasis sites     
    Periaortic lymph nodes 2, 2, 2, 2, 2, 2, 2, 2 1, 2, 2, 2, 1, 2, 2 2, 2, 2, 2, 2, 2, 2 2, 2, 2, 2, 2, 2, 2 
    Periadrenal 2, 2, 2, 2, 2, 2, 2, 2 0, 2, 0, 1, 0, 0, 2 2, 0, 2, 1, 2, 1, 2 0, 2, 2, 0, 0, 0, 0 
    Liver 1, 0, 0, 0, 1, 0, 1, 1 0, 1, 0, 0, 0, 0, 0 0, 0, 1, 0, 1, 0, 0 0, 0, 0, 0, 0, 0, 0 
    Mesenteric 1, 0, 0, 0, 0, 0, 1, 1 0, 0, 0, 0, 0, 0, 1 0, 0, 0, 0, 0, 0, 0 0, 0, 0, 0, 0, 0, 0 
    Lung 1, 0, 0, 0, 0, 1, 0, 0 0, 0, 0, 0, 0, 0, 0 0, 0, 0, 0, 0, 0, 0 0, 0, 0, 0, 0, 0, 0 
Total no. metastases 41 19* 26 18 
*

P = 0.0313, between irinotecan- and sham-treated animals.

P = 0.0625, between B-5354c–treated and sham-treated animals; P = 0.325, between B-5354c–treated and irinotecan-treated animals.

P = 0.0313, between irinotecan + B-5354c–treated and sham-treated animals; P = 0.1563, between irinotecan + B-5354c–treated and irinotecan-treated animals; P > 0.9999, between irinotecan + B-5354c–treated and B-5354c–treated animals.

We have previously reported that the oncogenic SphK1 could act as a sensitizer to chemotherapy in prostate adenocarcinoma cell and mouse models (6). PC-3 cells, for example, are very sensitive to taxanes but not to camptothecins in contrast to LNCaP cells (6). The differential effects of camptothecins and taxanes on PC-3 and LNCaP cells have been reflected by the status of the ceramide/S1P rheostat governed by SphK1. A long-lasting and robust SphK1 inhibition was followed by a strong antitumoral effect both in vitro and in vivo, a situation that was not seen with drugs that are known to show limited efficacy in vitro (6). SphK1 activity is crucial for survival of prostate cancer cells because its inhibition with B-5354c (13) induced a caspase-dependent apoptosis by tipping the ceramide/S1P balance toward ceramide. This result confirms our previous work with the compound II, another pharmacologic SphK1 inhibitor (27), which triggered apoptosis in prostate cancer cells regardless of their sensitivity/resistance status to a given drug (6). Importantly, the apoptotic effect of B-5354c is blocked by SphK1 overexpression.

The pioneering work of Nava et al. (28) established that in prostate cancer cells SphK1 inhibition correlates with sensitivity to γ-irradiation. Since then, similar effects were shown in chemotherapy-induced apoptosis in prostate (6) and leukemic cells (8, 29). The critical role of SphK1 in regulating anticancer therapy-induced apoptosis is supported by its overexpression that can markedly inhibit apoptosis (6, 8, 30). Previously, we have shown that resistance to camptothecin and docetaxel in PC-3 and LNCaP cells, respectively, is associated with stimulation of SphK1 activity (6). We have noted a similar pattern in pancreatic and lung cancer cells resistant to gemcitabine where a stimulation of SphK1 activity was observed after gemcitabine treatment.7

7

O. Cuvillier, personal data.

In that respect, stimulation of SphK1 activity might represent an important antiapoptotic pathway counteracting the effects of anticancer therapies.

Because camptothecin and docetaxel treatments stimulate the SphK1/S1P survival pathway in PC-3 and LNCaP cells, respectively, it is thought that SphK1 inhibition may be the primary event that leads to a sensitization of the effects of chemotherapy. Here, we have clearly established that SphK1 pharmacologic inhibitor B-5354c could potentiate the effect of camptothecin in PC-3 and docetaxel in LNCaP cells. By overriding the SphK1 stimulation induced by these chemotherapeutic agents, B-5354c could prevent cancer cells from generating prosurvival S1P, thus allowing these agents to exert their full effect on microtubules. Importantly, SphK1 inhibition does not sensitize PC-3 cells to docetaxel nor LNCaP cells to camptothecin, which have no stimulatory effect on SphK1 activity (6).

Collectively, our data show that some chemotherapy agents can up-regulate the prosurvival SphK1/S1P pathway and that pharmacologic inhibition of SphK1 can be useful for sensitizing prostate cancer cells to chemotherapy. It has been formerly reported that sphingosine derivatives such as dimethylsphingosine or dihydrosphingosine (safingol), which act as competitive inhibitors for SphK1 (31), could sensitize to irradiation in LNCaP prostate cancer cells (28) or to fenretinide in various cancer models (32). However, the rationale for sensitization obtained by these sphingosine derivatives was not elucidated in these studies. Our investigation is the first to report in vivo sensitization to anticancer regimen with B-5354c, a SphK1 inhibitor (13, 33). The effect of chemotherapy-induced SphK1 activation is transient. In our previous study (6), we have found that beyond 72 h of treatment with camptothecin or docetaxel SphK1 activity dropped below the control levels in PC-3 and LNCaP cells, respectively. This could explain the inhibition of SphK1 in PC-3 tumors after 14 days of irinotecan treatment. Although such treatment achieves a moderate SphK1 inhibition, it fails to induce a significant tumor regression, clearly showing that it is the early-time SphK1 inhibition that is required for the effective tumor clearance.

In contrast with the SphK1 inhibitors used before this current publication, B-5354c seems to have more attractive features, particularly a greater specificity (13, 33). Lineweaver-Burk plot analysis showed that B-5354c is a noncompetitive-type inhibitor with respect to sphingosine (13); thus, B-5354c may interact with domains distinct from the sphingosine-binding site to regulate SphK1 activity. Importantly, B-5354c does not inhibit other lipid kinases, such as phosphatidylinositol 3-kinase, nor protein kinase C even at doses above 500 μmol/L (33) in contrast to sphingosine derivatives such as dl-threo-sphingosine or dimethylsphingosine (3436). Moreover, although sphingosine derivatives have been used in vivo, these compounds exhibited undesirable cytotoxic side effects, including hemoglobinuria, inflammatory response, or loss of body weight (37). A toxicity profile of i.p. injection of B-5354c inhibitor was run in our experiments and no weight loss nor gross abnormalities were observed in the animals, no i.p. inflammation, no adhesions that could have impaired B-5354c absorption, and intraabdominal organs (kidney, liver, and gastrointestinal tract) were healthy looking.

Recently, the effects of a novel nonlipid SphK1 inhibitor, the compound V, were reported (27). Although it displayed a significant antitumor activity toward a murine adenocarcinoma model in animals, no chemosensitizing experiments were conducted. Moreover, in a similar fashion to sphingosine derivatives, this compound also lacked specificity because it was found equipotent for inhibiting SphK1 and phosphatidylinositol 3-kinase (27).

To conclude, our study supports the hypothesis that drugs specifically designed to inhibit SphK1 could provide a promising means of enhancing the effects of conventional chemotherapy due to the activation of the prosurvival antiapoptotic SphK1/S1P pathway that compromises their efficacy.

No potential conflicts of interest were disclosed.

Grant support: Institut National de la Santé et de la Recherche Médicale Interface Program, Centre National de la Recherche Scientifique, Institut National du Cancer, Association pour la Recherche sur le Cancer, and Fondation pour la Recherche Médicale (O. Cuvillier). D. Pchejetski is a recipient of Association Etudes et Recherches Urologiques.

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.

We thank Dr. B. Wattenberg (Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia) for generous gift of reagents.

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Supplementary data