doi:10.1016/j.bbrc.2006.02.070 
Copyright © 2006 Elsevier Inc. All rights reserved.
High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation
Yukihiro Akaoa, 
, 
, Yoshiko Bannob, Yoshihito Nakagawaa, Nobuko Hasegawaa, Tack-Joong Kimc, Takashi Murated, Yasuyuki Igarashic and Yoshinori Nozawaa
aGifu International Institute of Biotechnology, Kakamigahara 504-0838, Japan
bDepartment of Cell Signaling, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
cDepartment of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
dNagoya University Graduate School of Medicine, Nagoya University School of Health Sciences, Nagoya 461-8673, Japan
Received 8 February 2006.
Available online 21 February 2006.
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Abstract
Although most of pharmacological therapies for cancer utilize the apoptotic machinery of the cells, the available anti-cancer drugs are limited due to the ability of prostate cancer cells to escape from the anti-cancer drug-induced apoptosis. A human prostate cancer cell line PC3 is resistant to camptothecin (CPT). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of S1P receptors, S1P1 and S1P3, as compared with those of LNCaP cells. The knockdown of SPHK1 by small interfering RNA and inhibition of S1P receptor signaling by pertussis toxin in PC3 cells induced significant inhibition of cell growth, suggesting implication of SPHK1 and S1P receptors in cell proliferation in PC3 cells. Furthermore, the treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1P1/S1P3 receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT.
Keywords: Sphingosine kinase 1; S1P receptors; Chemotherapy-resistance; Camptothecin; Prostate cancer
Fig. 1. Comparison of SPHK activity, protein, and mRNA levels of SPHK1, and S1P receptor expression in human prostate cancer cell lines PC3 and LNCaP. (A) SPHK activity was measured in PC3 and LNCaP cell lysates (100 μg proteins) as described under Materials and methods. The produced [32P]S1P was detected by autoradiography (upper panel) and the corresponding bands were quantified by densitometry. The results are expressed as means ± SD for three independent experiments (lower panel). (B) Cell lysates of LNCaP, PC3, and DU145 were subjected to Western blot analysis using the antibody against human SPHK1 as described under Materials and methods. The data were representative of three independent experiments. (C) SPHK1 mRNA levels of PC3 and LNCaP cells were examined by RT-PCR as described under Materials and methods. (D) S1P receptors, S1P1, S1P2, and S1P3 levels of PC3 and LNCaP cells were examined by RT-PCR as described under Materials and methods. β-Actin was used as loading control.
Fig. 2. Effects of SPHK1 inhibitor, N,N-dimethylsphingosine (DMS), and knockdown of SPHK1 by siRNA on cell growth in PC3 cells. (A) PC3 cells were treated with or without 10 μM DMS for 24 h and assayed for SPHK activity as described under Materials and methods. (B) The cells (1.0 × 105/ml) were treated with or without 5 and 10 μM DMS in the medium containing 0.5% FBS for 24 h, and the viable cell numbers were measured by staining with trypan blue. (C) Small interfering RNAs directed to SPHK1 were transfected into PC3 cells and 48 h after the transfection the cell lysates were subjected to Western blot analysis with SPHK1 antibody as described under Materials and methods. The detailed procedure is described under Materials and methods. β-Actin was examined as an internal standard. The result is representative of three experiments. (D) The viable cell number was examined at 48 h after the transfection. The results are expressed as means ± SD for three different experiments.
Fig. 3. Effects of S1P on DMS-induced cell growth suppression and pertussis toxin (PTX) on cell growth in PC3 cells. (A) The cells (1.0 × 105/ml) were treated with or without 10 μM DMS for 30 min in the medium containing 0.5% FCS prior addition of the indicated concentrations of S1P. At 24 h after the S1P treatment the cell number was measured as described under Materials and methods. The difference between DMS 10 μM treatment and DMS 10 μM plus S1P 100 nM was significant. p < 0.001. (
), The other open stars indicated significancy (p < 0.01). (B) The cells (2.0 × 105/ml) were treated with or without the indicated concentrations of PTX and growth suppression was evaluated by the viable cell number at 24 h after the treatment. The results are expressed as means ± SD for three different experiments.
Fig. 4. Increases in SPHK activity in PC3 cells treated with CPT. SPHK activity was measured after treatment with 0.5, 1.0, and 3.0 μM CPT for the indicated intervals as described under Materials and methods. The results are expressed as means ± SD for two experiments, each performed in duplicate.
Fig. 5. Changes in protein and mRNA levels of SPHK1 in PC3 cells treated with CPT. PC3 cells were treated with the indicated concentrations of CPT for 24 and 48 h. (A) The cell lysates were subjected to Western blotting using with the antibody against human SPHK1 as described under Materials and methods. (B) After the CPT treatment the levels of SPHK1 mRNA were measured by semi-quantitative PT-PCR as described under Materials and methods. The data of Western blotting (A) and RT-PCR analysis (B) shown in upper panels are representative of three independent experiments. Lower panels showing densitometric data obtained by Western blotting (A) and RT-PCR (B) were expressed as means ± SD for the three experiments.
Fig. 6. Changes in the expression levels of S1P1, S1P2, and S1P3 mRNAs after the treatment with CPT in PC3 cells. PC3 cells were treated with the indicated concentrations of CPT for 24 h and the expression levels of S1P1, S1P2, and S1P3 mRNAs were determined by semi-quantitative PT-PCR as described under Materials and methods. β-Actin was examined as an internal standard. The data shown in upper panel are representative of three independent experiments. Lower panel shows densitometric data expressed as means ± SD for the three experiments.
Abstract
Purchase PDF (290 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (14)
Purchase PDF (409 K)
