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Role of leukotriene B4 in celecoxib-mediated anticancer effect
Biochemical and Biophysical Research Communications 402 (2010) 308–311
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Role of leukotriene B4 in celecoxib-mediated anticancer effect Peng Gao, Lei Guan, Jie Zheng ⇑ Department of Pathology and Pathophysiology, School of Medical Science, Southeast University, Nanjing 210009, Jiangsu, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 26 September 2010 Available online 19 October 2010 Keywords: Celecoxib Leukotriene B4 Colon cancer Prostate cancer
a b s t r a c t Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has anticancer effect on many cancers associated with chronic inflammation by both COX-2-dependent and COX-2-independent mechanisms. The non-COX-2 targets of celecoxib, however, are still a matter of research. Leukotriene B4 (LTB4) has been implicated in prostate and colon carcinogenesis, but little is known about the potential role of LTB4 in celecoxib-mediated anticancer effect. In this study, we evaluated whether LTB4 was involved in celecoxib-mediated inhibitory effect on human colon cancer HT-29 cells and human prostate cancer PC-3 cells. Our data showed that survival of both cell lines was obviously suppressed after celecoxib treatment for 72 h in a concentration-dependent manner. However, only in HT-29 cells, this inhibitory effect could be reversed by LTB4, which promoted survival of HT-29 cells rather than PC-3 cells. Consistent with these results, lioxygenase (LOX) potent inhibitor nordihydroguaiaretic acid (NDGA) had a higher inhibitory effect on HT-29 cells than PC-3 cells. Additionally, ELISA results showed that celecoxib could suppress expression of LTB4 in both cell lines, whereas, inhibition of PGE2 was only detected in HT-29 cells. These results indicate that the anticancer effect of celecoxib is COX-2-independent in HT-29 and PC-3 cells and in HT-29 cells primarily via down-regulating LTB4 production. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Epidemiological studies provide evidence that a high-fat diet can be associated with an increased risk for cancer, in particular colorectal, prostate and pancreatic cancer [1,2]. Arachidonic acid is one major ingredient of the membranes and via two major metabolic pathways, the cyclooxygenase (COX) pathway and the lipoxygenase (LOX) pathway, metabolized to prostaglandins and leukotrienes, which are represented by prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), respectively [2]. These biologically active lipids are involved in chronic inflammation and cancer [1–4]. There are two known COX isoforms, COX-1 and COX-2, with distinct expression patterns and biological activities [2]. COX-1 is constitutively expressed in most tissues, whereas, COX-2 is absent from most tissues but is highly induced by pro-inflammatory stimuli and is often actively expressed in various epithelial cancers [3,5]. COX-2-derived prostaglandins, particularly PGE2, have been associated with antiapoptotic as well as proliferation-promoting effects [2,3]. Since celecoxib is able to inhibit both cell proliferation in vitro and tumor growth in vivo, at first glance it appears that the pronounced anticancer effects of celecoxib are primarily due to its ability to inhibit COX-2. Other studies, however, have demon⇑ Corresponding author. Fax: +86 25 83324887. E-mail addresses: [email protected] (P. Gao), [email protected] (L. Guan), [email protected] (J. Zheng).
strated that non-COX-2 targets, such as 3-phosphoinositidedependent protein kinase-1 (PDK-1) [6] and p53 [7], are involved in celecoxib-mediated antiproliferative effects in prostate [8] and colon [9–12] cancer cells in vitro. Until now most of the effort has focused on the role of cyclooxygenase products in carcinogenesis, very little is known about the roles of lipoxygenase products, particularly LTB4, in this process. However, emerging data suggest that LTB4 has important roles in the prostate and colon carcinogenesis [4,13,14]. Based on these observations, we evaluated the role of LTB4 in celecoxib-mediated anticancer effect on colon cancer HT-29 cells and prostate cancer PC-3 cells in vitro. 2. Materials and methods 2.1. Materials RPMI 1640 and F12 media were purchased from Invitrogen (Green Island, NY). Fetal bovine serum (FBS) was from Sijiqing (Hangzhou, Zhejiang, China). PGE2 and LTB4 enzyme-linked immunosorbent assay (ELISA) kits were from R&D (Abingdon, UK). Celecoxib was from Pfizer (New York, NY). Nordihydroguaiaretic acid (NDGA) was purchased from Sigma Chemicals (St. Louis, MO). LTB4 was purchased from Cayman Chemicals (Ann Arbor, MI). For studies, these agents were dissolved in dimethyl sulfoxide (DMSO) and were then added to the cells in medium with the final DMSO concentration kept 60.2%.
0006-291X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.022
P. Gao et al. / Biochemical and Biophysical Research Communications 402 (2010) 308–311
2.2. Cell lines and cell culture The human colon cancer HT-29 cells and human prostate cancer PC-3 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Chinese Academy of Sciences). PC-3 cells were cultured in F12 media, and HT-29 cells in RPMI 1640 media, which were supplemented with 10% FBS plus 100 units/ml penicillin and 100 lg/ml streptomycin. For experiments, cells were fed with fresh medium every third day and digested at a confluence of about 80%. 2.3. Cell Viability Cells were seeded onto 96-well plates at 5 103 cells per well and each group consisted of six parallel wells. After reaching 50% confluence, they were incubated in fresh medium with or without the appropriate treatments. After the required period of culture, cell viability was determined by MTT assay. During the treatment, the percentage of cells floating in the medium increased over time. Both adherent and floating cells were collected for the assessment. 2.4. Measurement of PGE2 and LTB4 levels Levels of PGE2 and LTB4 released in media were measured using PGE2 and LTB4 ELISA kits, respectively. Medium was sampled, centrifuged to remove floating cells and frozen immediately at 70 °C until assay. The assay was performed according to the manufacturer’s instructions, following dilution to ensure that readings were within the limits of accurate detection by the assay. 2.5. Statistical analysis All quantitative variables are presented as means ± SD (standard deviation). The difference between two groups was assessed with the use of an independent t-test. We compared the differences of three or more groups with a oneway ANOVA. P < 0.05 was considered statistically significant. 3. Results 3.1. Celecoxib induces cell death of HT-29 and PC-3 cells Celecoxib with various concentrations was used to treat HT-29 and PC-3 cells for 72 h. Under the phase contrast microscope, the cells exhibited a dramatic morphologic change as well as inhibition of cell survival after drug treatment. The cell viability in response to celecoxib treatment was assayed by MTT assay. As shown in Fig. 1, significant induction of cell death was observed in both cell
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lines in a dose-dependent manner, whereas, this effect was obviously higher in HT-29 cells (IC50 = 40 lM) than in PC-3 cells (IC50 = 70 lM) under concentrations less than 80 lM. 3.2. Effect of celecoxib on production of PGE2 and LTB4 Amounts of PGE2 and LTB4 in medium were evaluated after celecoxib treatment for 72 h. All doses of celecoxib significantly reduced LTB4 secretion in both cell lines (Fig. 2A and B), indicating that celecoxib is a potent inhibitor of 5-LOX, which induces LTB4 production. In contrast with LTB4, the influence of celecoxib on PGE2 was different in HT-29 and PC-3 cells. As shown in Fig. 2, celecoxib treatment obviously inhibited PGE2 production in HT-29 cells (Fig. 2A), however, the level of PGE2 was not changed in PC3 cells after celecoxib treatment (Fig. 2B). 3.3. Exogenous LTB4 only reverses celecoxib-mediated inhibitory effect in HT-29 cells Because celecoxib caused cell inhibition in both cell lines and inhibited LTB4 secretion, we hypothesized that this inhibitory effect was LTB4 dependent. To determine whether celecoxibmediated inhibitory effect could be reversed by exogenous LTB4, LTB4 was added to cultures of HT-29 and PC-3 cells treated with constant dose (80 lM) of celecoxib. Varying amounts of LTB4 (0.001–10 nM) were added to the medium in order to take into account the fact that some of the LTB4 may degrade or be internalized into cells. In PC-3 cells, cell inhibition induced by 80 lM celecoxib could not be prevented by exogenous LTB4 (Fig. 3B), suggesting that celecoxib-induced anticancer effect in PC-3 cells may be independent of LTB4 level. However, addition of 10 nM LTB4 almost reversed the inhibitory effect induced by 80 lM celecoxib in HT-29 cells (Fig. 3A), suggesting that celecoxib-mediated anticancer effect in this cell line may be dependent on the level of LTB4. 3.4. Effects of LTB4 and NDGA on viability of HT-29 and PC-3 cells In order to confirm the results depicted above, we investigated the role of exogenous LTB4 alone in HT-29 and PC-3 cells. As shown in Fig. 4, LTB4 promoted the viability of HT-29 cells, but inhibited that of PC-3 cells (Fig. 4A and B, respectively). However, these effects of LTB4 on cancer cell survival were mild in our study. As LTB4 is a critical component of 5-LOX pathway, we also explored the importance of LOX pathway in survival of both cell lines by using a potent LOX inhibitor NDGA. Results showed that NDGA obviously inhibited the viability of HT-29 cells in a dose-dependent manner, whereas, NDGA inhibited that of PC-3 cells in a limited extent. 4. Discussion
Vi a b i l i t y ( % o f c o n t r o l )
120 PC-3 HT-29
100 80 60 40 20 0
0
20
40 60 Celecoxib (µM)
80
100
Fig. 1. Inhibitory effect of celecoxib on cell viability. HT-29 and PC-3 cells were treated with celecoxib at increasing concentrations and harvested 72 h later for MTT assay, as described in Section 2. Values are average of duplicates, standard deviation (SD) were within 10%.
Celecoxib is a selective COX-2 inhibitor possessing anticancer effects for many cancers. Although various COX-2-independent chemo-preventive and tumor-regressive activity have been suggested, the respective non-COX-2 targets of celecoxib are still a matter of research. The results presented here clearly show that down-regulation of LTB4 synthesis is the primary mechanism of celecoxib-mediated anticancer effect in human colon cancer HT29 cells. Several investigators have suggested that celecoxib has anticancer activity via COX-2-dependent and COX-2-independent pathways. It is reported that inhibition of PGE2 synthesis is an early, but not sufficient, step in the mechanism of celecoxib-mediated cell growth inhibition [12]. As shown in our work, celecoxib suppressed the survival of both COX-2-positive HT-29 and COX-2deficient PC-3 cells, whereas, celecoxib had a higher inhibitory
P. Gao et al. / Biochemical and Biophysical Research Communications 402 (2010) 308–311
Concentration (pg/ml)
A
B
HT-29
250 200 150 100
LTB 4 PGE2
50
PC-3
100
Concentration (pg/ml)
310
0
80 60 40
LTB 4 PGE2
20 0
0
20
40 60 80 Celecoxib (µM)
100
0
20
40 60 80 Celecoxib (µM)
100
Fig. 2. Effects of celecoxib on production of PGE2 and LTB4 in HT-29 and PC-3 cells. Cells were treated with celecoxib for 72 h at increasing concentrations. PGE2 and LTB4 levels in the culture medium were measured by ELISA.
**
100
**
B 120
HT-29 ** **
Viability (%ofcontrol)
Viability (%ofcontrol)
A120
100
80 60 40 20 0
80
NS
+ _
+ 0.001
+ 0.1
+ 10
NS
*
60 40 20 0
_ _
PC-3
**
_ _
Celecoxib(80 µ M) LTB4 (nM)
+ _
+ 0.001
+ 0.1
+ 10
Celecoxib(80 µ M) LTB4 (nM)
Fig. 3. Effect of LTB4 on celecoxib-mediated inhibition in HT-29 and PC-3 cells. Cells were incubated for 72 h in absence or presence of 80 lM celecoxib. In some groups the cells were incubated with 80 lM celecoxib and LTB4 at the concentrations indicated above. *P < 0.05, **P < 0.01. NS represents no sense.
48 h 72 h 0.001 0.1 LTB 4 (nM)
0
C 120
0
20
40 60 80 NDGA ( µ M )
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48 h 72 h 0.001 0.1 LTB 4 (nM)
0
D
H T- 2 9
100 80 60 40 20 0
PC-3
120 100 80 60 40 20 0
10
% Viability
% Viability
B
H T- 2 9
100 80 60 40 20 0
% Viability
% Viability
A 120
10
PC-3
120 100 80 60 40 20 0 0
20
40 60 80 NDGA ( µ M )
100
Fig. 4. Effects of LTB4 and NDGA on cell viability. HT-29 and PC-3 cells were treated with LTB4 or NDGA at increasing concentrations and harvested 48 and/or 72 h later for MTT assay. Values are average of duplicates, SD were within 10%.
effect on the former. Consistent with these results, celecoxib only suppressed PGE2 synthesis in HT-29 cells. However, we unexpectedly observed that celecoxib could inhibit LTB4 synthesis in both cell lines. Although celecoxib has recently been reported as a direct inhibitor of 5-LOX in vitro and in vivo [15], to our knowledge, this is the first study to show that celecoxib inhibits LTB4 synthesis in tumor cells cultured in vitro. Several studies have reported that inhibition of the 5-LOX pathway suppresses the proliferation of human colon cancer cells [16] and prostate cancer cells [17,18]. As the major metabolite in the 5-LOX pathway, LTB4 has also been indicated to be involved in carcinogenesis in these organs [4]. Additionally, LTB4-mediated proliferation-promoting effects in colon cancer cells and prostatic
cancer cells are also reported [2,13]. Based on these reports and our findings, we hypothesized that LTB4 may be involved in celecoxib-mediated anticancer effect, at least in certain cancers. To clarify this hypothesis, we studied the effect of LTB4 on HT29 and PC-3 cells. Our results showed that LTB4 itself promoted the survival of HT-29 cells and nearly completely reversed celecoxibmediated inhibitory effect at high concentration. However, the stimulating effect of LTB4 on HT-29 cells was not so dramatic as other reported [13,19]. The possible cause is that the endogenous LTB4 level in HT-29 cells is already high enough to stimulate the proliferation. In contrast, in PC-3 cells, LTB4 not only suppressed cell survival but also had no influence on celecoxib-mediated inhibitory effect. Although growth-inhibitory effect of LTB4 on hu-
P. Gao et al. / Biochemical and Biophysical Research Communications 402 (2010) 308–311
man mammary cancer MCF-7 cells has been reported [20], until now LTB4 mainly exhibits stimulating cell growth [13,14,19]. Further research is needed to elucidate these findings in our study. The different response of HT-29 and PC-3 cells to LTB4 was also observed when cells were treated with LOX inhibitor NDGA, indicating that for survival LOX pathway is more important in HT-29 cells than PC-3 cells. Alterations of phosphatidylinositol 3-kinase (PI3K)-PDK-1 signaling pathway are frequently associated with the pathogenesis of many cancers, including prostate and colorectal cancers [21]. Recent years, this pathway has been reported as one major COX2-independent target for celecoxib in HT-29 cells as celecoxib can directly inhibit PDK-1 activity and PDK-1 mutation impairs celecoxib-induced inhibition [6]. This observation is consistent with our results as PI3K-PDK-1 signaling has also been suggested as one pathway by which LTB4 stimulates proliferation of cancer cells [14]. Overexpression of Akt, a PDK-1 downstream kinase, does not efficiently protect HT-29 cells from celecoxib-induced apoptosis [6], we propose that reversion of celecoxib-mediated inhibition by LTB4 is mainly due to activation of other PDK-1 downstream kinases except Akt, such as p70S6-kinase (p70S6-K) and protein kinase C (PKC) [22]. Additionally, most signaling pathways affected by both celecoxib and LTB4 converge to mammalian target of rapamycin (mTOR) signaling [6,9,14,23,24], suggesting a potential target for further investigations. In summary, our results suggest that anticancer effect of celecoxib is COX-2-independent in PC-3 and HT-29 cells. However, only in HT-29 cells, celecoxib exerts anticancer effect primarily via down-regulating LTB4 production. Additional studies are warranted to elucidate the above mechanisms in detail. Acknowledgments This work was supported by the National Natural Science Foundation of China (No.30540049) and the Scientific Research Foundation of Graduate School of Southeast University (No.YBJJ0915). References [1] D. Divisi, S. Di Tommaso, S. Salvemini, M. Garramone, R. Crisci, Diet and cancer, Acta Biomed. 77 (2006) 118–123. [2] D. Wang, R.N. Dubois, Eicosanoids and cancer, Nat. Rev. Cancer 10 (2010) 181– 193. [3] A. Greenhough, H.J. Smartt, A.E. Moore, H.R. Roberts, A.C. Williams, C. Paraskeva, A. Kaidi, The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment, Carcinogenesis 30 (2009) 377–386. [4] S. Larré, N. Tran, C. Fan, H. Hamadeh, J. Champigneulles, R. Azzouzi, O. Cussenot, P. Mangin, J.L. Olivier, PGE2 and LTB4 tissue levels in benign and cancerous prostates, Prostaglandins Other Lipid Mediat. 87 (2008) 14–19.
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[5] R.S. Pruthi, E.M. Wallen, Cyclooxygenase-2: a therapeutic target for prostate cancer, Clin. Genitourin. Cancer 4 (2005) 203–211. [6] S. Arico, S. Pattingre, C. Bauvy, P. Gane, A. Barbat, P. Codogno, E. Ogier-Denis, Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT-29 cell line, J. Biol. Chem. 277 (2002) 27613–27621. [7] M.V. Swamy, C.R. Herzog, C.V. Rao, Inhibition of COX-2 in colon cancer cell lines by celecoxib increases the nuclear localization of active p53, Cancer Res. 63 (2003) 5239–5242. [8] M.I. Patel, K. Subbaramaiah, B. Du, M. Chang, P. Yang, R.A. Newman, C. CordonCardo, H.T. Thaler, A.J. Dannenberg, Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism, Clin. Cancer Res. 11 (2005) 1999–2007. [9] T.J. Maier, A. Janssen, R. Schmidt, G. Geisslinger, S. Grösch, Targeting the betacatenin/APC pathway: a novel mechanism to explain the cyclooxygenase-2independent anticarcinogenic effects of celecoxib in human colon carcinoma cells, FASEB J. 19 (2005) 1353–1355. [10] T.J. Maier, K. Schilling, R. Schmidt, G. Geisslinger, S. Grösch, Cyclooxygenase-2 (COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells, Biochem. Pharmacol. 67 (2004) 1469–1478. [11] S. Grösch, I. Tegeder, E. Niederberger, L. Bräutigam, G. Geisslinger, COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib, FASEB J. 15 (2001) 2742–2744. [12] S. Lev-Ari, D. Kazanov, E. Liberman, R. Ben-Yosef, N. Arber, Down-regulation of PGE2 by physiologic levels of celecoxib is not sufficient to induce apoptosis or inhibit cell proliferation in human colon carcinoma cell lines, Dig. Dis. Sci. 52 (2007) 1128–1133. [13] A. Ihara, K. Wada, M. Yoneda, N. Fujisawa, H. Takahashi, A. Nakajima, Blockade of leukotriene B4 signaling pathway induces apoptosis and suppresses cell proliferation in colon cancer, J. Pharmacol. Sci. 103 (2007) 24–32. [14] W.G. Tong, X.Z. Ding, M.S. Talamonti, R.H. Bell, T.E. Adrian, LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways, Biochem. Biophys. Res. Commun. 335 (2005) 949–956. [15] T.J. Maier, L. Tausch, M. Hoernig, O. Coste, R. Schmidt, C. Angioni, J. Metzner, S. Groesch, C. Pergola, D. Steinhilber, O. Werz, G. Geisslinger, Celecoxib inhibits 5-lipoxygenase, Biochem. Pharmacol. 76 (2008) 862–872. [16] L.G. Melstrom, D.J. Bentrem, M.R. Salabat, T.J. Kennedy, X.Z. Ding, M. Strouch, S.M. Rao, R.C. Witt, C.A. Ternent, M.S. Talamonti, R.H. Bell, T.A. Adrian, Overexpression of 5-lipoxygenase in colon polyps and cancer and the effect of 5-LOX inhibitors in vitro and in a murine model, Clin. Cancer Res. 14 (2008) 6525–6530. [17] J. Ghosh, C.E. Myers, Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells, Proc. Natl. Acad. Sci. USA 95 (1998) 13182–13187. [18] J. Ghosh, Inhibition of arachidonate 5-lipoxygenase triggers prostate cancer cell death through rapid activation of c-Jun N-terminal kinase, Biochem. Biophys. Res. Commun. 307 (2003) 342–349. [19] C. Bortuzzo, R. Hanif, K. Kashfi, L. Staiano-Coico, S.J. Shiff, B. Rigas, The effect of leukotrienes B and selected HETEs on the proliferation of colon cancer cells, Biochim. Biophys. Acta. 1300 (1996) 240–246. [20] A. Przylipiak, J. Hafner, J. Przylipiak, B. Runnebaum, T. Rabe, F.M. Köhn, Influence of leukotrienes on in vitro growth of human mammary carcinoma cell line MCF-7, Eur. J. Obstet. Gynecol. Reprod. Biol. 77 (1998) 61–65. [21] J.A. Engelman, Targeting PI3K signalling in cancer: opportunities, challenges and limitations, Nat. Rev. Cancer 9 (2009) 550–562. [22] A. Toker, A.C. Newton, Cellular signaling: pivoting around PDK-1, Cell 103 (2000) 185–188. [23] J.B. Tuynman, L. Vermeulen, E.M. Boon, K. Kemper, A.H. Zwinderman, M.P. Peppelenbosch, D.J. Richel, Cyclooxygenase-2 inhibition inhibits c-Met kinase activity and Wnt activity in colon cancer, Cancer Res. 68 (2008) 1213–1220. [24] X.M. Ma, J. Blenis, Molecular mechanisms of mTOR-mediated translational control, Nat. Rev. Mol. Cell Biol. 10 (2009) 307–318.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Role of leukotriene B4 in celecoxib-mediated anticancer effect Peng Gao, Lei Guan, Jie Zheng ⇑ Department of Pathology and Pathophysiology, School of Medical Science, Southeast University, Nanjing 210009, Jiangsu, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 26 September 2010 Available online 19 October 2010 Keywords: Celecoxib Leukotriene B4 Colon cancer Prostate cancer
a b s t r a c t Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has anticancer effect on many cancers associated with chronic inflammation by both COX-2-dependent and COX-2-independent mechanisms. The non-COX-2 targets of celecoxib, however, are still a matter of research. Leukotriene B4 (LTB4) has been implicated in prostate and colon carcinogenesis, but little is known about the potential role of LTB4 in celecoxib-mediated anticancer effect. In this study, we evaluated whether LTB4 was involved in celecoxib-mediated inhibitory effect on human colon cancer HT-29 cells and human prostate cancer PC-3 cells. Our data showed that survival of both cell lines was obviously suppressed after celecoxib treatment for 72 h in a concentration-dependent manner. However, only in HT-29 cells, this inhibitory effect could be reversed by LTB4, which promoted survival of HT-29 cells rather than PC-3 cells. Consistent with these results, lioxygenase (LOX) potent inhibitor nordihydroguaiaretic acid (NDGA) had a higher inhibitory effect on HT-29 cells than PC-3 cells. Additionally, ELISA results showed that celecoxib could suppress expression of LTB4 in both cell lines, whereas, inhibition of PGE2 was only detected in HT-29 cells. These results indicate that the anticancer effect of celecoxib is COX-2-independent in HT-29 and PC-3 cells and in HT-29 cells primarily via down-regulating LTB4 production. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Epidemiological studies provide evidence that a high-fat diet can be associated with an increased risk for cancer, in particular colorectal, prostate and pancreatic cancer [1,2]. Arachidonic acid is one major ingredient of the membranes and via two major metabolic pathways, the cyclooxygenase (COX) pathway and the lipoxygenase (LOX) pathway, metabolized to prostaglandins and leukotrienes, which are represented by prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), respectively [2]. These biologically active lipids are involved in chronic inflammation and cancer [1–4]. There are two known COX isoforms, COX-1 and COX-2, with distinct expression patterns and biological activities [2]. COX-1 is constitutively expressed in most tissues, whereas, COX-2 is absent from most tissues but is highly induced by pro-inflammatory stimuli and is often actively expressed in various epithelial cancers [3,5]. COX-2-derived prostaglandins, particularly PGE2, have been associated with antiapoptotic as well as proliferation-promoting effects [2,3]. Since celecoxib is able to inhibit both cell proliferation in vitro and tumor growth in vivo, at first glance it appears that the pronounced anticancer effects of celecoxib are primarily due to its ability to inhibit COX-2. Other studies, however, have demon⇑ Corresponding author. Fax: +86 25 83324887. E-mail addresses: [email protected] (P. Gao), [email protected] (L. Guan), [email protected] (J. Zheng).
strated that non-COX-2 targets, such as 3-phosphoinositidedependent protein kinase-1 (PDK-1) [6] and p53 [7], are involved in celecoxib-mediated antiproliferative effects in prostate [8] and colon [9–12] cancer cells in vitro. Until now most of the effort has focused on the role of cyclooxygenase products in carcinogenesis, very little is known about the roles of lipoxygenase products, particularly LTB4, in this process. However, emerging data suggest that LTB4 has important roles in the prostate and colon carcinogenesis [4,13,14]. Based on these observations, we evaluated the role of LTB4 in celecoxib-mediated anticancer effect on colon cancer HT-29 cells and prostate cancer PC-3 cells in vitro. 2. Materials and methods 2.1. Materials RPMI 1640 and F12 media were purchased from Invitrogen (Green Island, NY). Fetal bovine serum (FBS) was from Sijiqing (Hangzhou, Zhejiang, China). PGE2 and LTB4 enzyme-linked immunosorbent assay (ELISA) kits were from R&D (Abingdon, UK). Celecoxib was from Pfizer (New York, NY). Nordihydroguaiaretic acid (NDGA) was purchased from Sigma Chemicals (St. Louis, MO). LTB4 was purchased from Cayman Chemicals (Ann Arbor, MI). For studies, these agents were dissolved in dimethyl sulfoxide (DMSO) and were then added to the cells in medium with the final DMSO concentration kept 60.2%.
0006-291X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.022
P. Gao et al. / Biochemical and Biophysical Research Communications 402 (2010) 308–311
2.2. Cell lines and cell culture The human colon cancer HT-29 cells and human prostate cancer PC-3 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Chinese Academy of Sciences). PC-3 cells were cultured in F12 media, and HT-29 cells in RPMI 1640 media, which were supplemented with 10% FBS plus 100 units/ml penicillin and 100 lg/ml streptomycin. For experiments, cells were fed with fresh medium every third day and digested at a confluence of about 80%. 2.3. Cell Viability Cells were seeded onto 96-well plates at 5 103 cells per well and each group consisted of six parallel wells. After reaching 50% confluence, they were incubated in fresh medium with or without the appropriate treatments. After the required period of culture, cell viability was determined by MTT assay. During the treatment, the percentage of cells floating in the medium increased over time. Both adherent and floating cells were collected for the assessment. 2.4. Measurement of PGE2 and LTB4 levels Levels of PGE2 and LTB4 released in media were measured using PGE2 and LTB4 ELISA kits, respectively. Medium was sampled, centrifuged to remove floating cells and frozen immediately at 70 °C until assay. The assay was performed according to the manufacturer’s instructions, following dilution to ensure that readings were within the limits of accurate detection by the assay. 2.5. Statistical analysis All quantitative variables are presented as means ± SD (standard deviation). The difference between two groups was assessed with the use of an independent t-test. We compared the differences of three or more groups with a oneway ANOVA. P < 0.05 was considered statistically significant. 3. Results 3.1. Celecoxib induces cell death of HT-29 and PC-3 cells Celecoxib with various concentrations was used to treat HT-29 and PC-3 cells for 72 h. Under the phase contrast microscope, the cells exhibited a dramatic morphologic change as well as inhibition of cell survival after drug treatment. The cell viability in response to celecoxib treatment was assayed by MTT assay. As shown in Fig. 1, significant induction of cell death was observed in both cell
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lines in a dose-dependent manner, whereas, this effect was obviously higher in HT-29 cells (IC50 = 40 lM) than in PC-3 cells (IC50 = 70 lM) under concentrations less than 80 lM. 3.2. Effect of celecoxib on production of PGE2 and LTB4 Amounts of PGE2 and LTB4 in medium were evaluated after celecoxib treatment for 72 h. All doses of celecoxib significantly reduced LTB4 secretion in both cell lines (Fig. 2A and B), indicating that celecoxib is a potent inhibitor of 5-LOX, which induces LTB4 production. In contrast with LTB4, the influence of celecoxib on PGE2 was different in HT-29 and PC-3 cells. As shown in Fig. 2, celecoxib treatment obviously inhibited PGE2 production in HT-29 cells (Fig. 2A), however, the level of PGE2 was not changed in PC3 cells after celecoxib treatment (Fig. 2B). 3.3. Exogenous LTB4 only reverses celecoxib-mediated inhibitory effect in HT-29 cells Because celecoxib caused cell inhibition in both cell lines and inhibited LTB4 secretion, we hypothesized that this inhibitory effect was LTB4 dependent. To determine whether celecoxibmediated inhibitory effect could be reversed by exogenous LTB4, LTB4 was added to cultures of HT-29 and PC-3 cells treated with constant dose (80 lM) of celecoxib. Varying amounts of LTB4 (0.001–10 nM) were added to the medium in order to take into account the fact that some of the LTB4 may degrade or be internalized into cells. In PC-3 cells, cell inhibition induced by 80 lM celecoxib could not be prevented by exogenous LTB4 (Fig. 3B), suggesting that celecoxib-induced anticancer effect in PC-3 cells may be independent of LTB4 level. However, addition of 10 nM LTB4 almost reversed the inhibitory effect induced by 80 lM celecoxib in HT-29 cells (Fig. 3A), suggesting that celecoxib-mediated anticancer effect in this cell line may be dependent on the level of LTB4. 3.4. Effects of LTB4 and NDGA on viability of HT-29 and PC-3 cells In order to confirm the results depicted above, we investigated the role of exogenous LTB4 alone in HT-29 and PC-3 cells. As shown in Fig. 4, LTB4 promoted the viability of HT-29 cells, but inhibited that of PC-3 cells (Fig. 4A and B, respectively). However, these effects of LTB4 on cancer cell survival were mild in our study. As LTB4 is a critical component of 5-LOX pathway, we also explored the importance of LOX pathway in survival of both cell lines by using a potent LOX inhibitor NDGA. Results showed that NDGA obviously inhibited the viability of HT-29 cells in a dose-dependent manner, whereas, NDGA inhibited that of PC-3 cells in a limited extent. 4. Discussion
Vi a b i l i t y ( % o f c o n t r o l )
120 PC-3 HT-29
100 80 60 40 20 0
0
20
40 60 Celecoxib (µM)
80
100
Fig. 1. Inhibitory effect of celecoxib on cell viability. HT-29 and PC-3 cells were treated with celecoxib at increasing concentrations and harvested 72 h later for MTT assay, as described in Section 2. Values are average of duplicates, standard deviation (SD) were within 10%.
Celecoxib is a selective COX-2 inhibitor possessing anticancer effects for many cancers. Although various COX-2-independent chemo-preventive and tumor-regressive activity have been suggested, the respective non-COX-2 targets of celecoxib are still a matter of research. The results presented here clearly show that down-regulation of LTB4 synthesis is the primary mechanism of celecoxib-mediated anticancer effect in human colon cancer HT29 cells. Several investigators have suggested that celecoxib has anticancer activity via COX-2-dependent and COX-2-independent pathways. It is reported that inhibition of PGE2 synthesis is an early, but not sufficient, step in the mechanism of celecoxib-mediated cell growth inhibition [12]. As shown in our work, celecoxib suppressed the survival of both COX-2-positive HT-29 and COX-2deficient PC-3 cells, whereas, celecoxib had a higher inhibitory
P. Gao et al. / Biochemical and Biophysical Research Communications 402 (2010) 308–311
Concentration (pg/ml)
A
B
HT-29
250 200 150 100
LTB 4 PGE2
50
PC-3
100
Concentration (pg/ml)
310
0
80 60 40
LTB 4 PGE2
20 0
0
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40 60 80 Celecoxib (µM)
100
0
20
40 60 80 Celecoxib (µM)
100
Fig. 2. Effects of celecoxib on production of PGE2 and LTB4 in HT-29 and PC-3 cells. Cells were treated with celecoxib for 72 h at increasing concentrations. PGE2 and LTB4 levels in the culture medium were measured by ELISA.
**
100
**
B 120
HT-29 ** **
Viability (%ofcontrol)
Viability (%ofcontrol)
A120
100
80 60 40 20 0
80
NS
+ _
+ 0.001
+ 0.1
+ 10
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*
60 40 20 0
_ _
PC-3
**
_ _
Celecoxib(80 µ M) LTB4 (nM)
+ _
+ 0.001
+ 0.1
+ 10
Celecoxib(80 µ M) LTB4 (nM)
Fig. 3. Effect of LTB4 on celecoxib-mediated inhibition in HT-29 and PC-3 cells. Cells were incubated for 72 h in absence or presence of 80 lM celecoxib. In some groups the cells were incubated with 80 lM celecoxib and LTB4 at the concentrations indicated above. *P < 0.05, **P < 0.01. NS represents no sense.
48 h 72 h 0.001 0.1 LTB 4 (nM)
0
C 120
0
20
40 60 80 NDGA ( µ M )
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48 h 72 h 0.001 0.1 LTB 4 (nM)
0
D
H T- 2 9
100 80 60 40 20 0
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120 100 80 60 40 20 0
10
% Viability
% Viability
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100 80 60 40 20 0
% Viability
% Viability
A 120
10
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120 100 80 60 40 20 0 0
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40 60 80 NDGA ( µ M )
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Fig. 4. Effects of LTB4 and NDGA on cell viability. HT-29 and PC-3 cells were treated with LTB4 or NDGA at increasing concentrations and harvested 48 and/or 72 h later for MTT assay. Values are average of duplicates, SD were within 10%.
effect on the former. Consistent with these results, celecoxib only suppressed PGE2 synthesis in HT-29 cells. However, we unexpectedly observed that celecoxib could inhibit LTB4 synthesis in both cell lines. Although celecoxib has recently been reported as a direct inhibitor of 5-LOX in vitro and in vivo [15], to our knowledge, this is the first study to show that celecoxib inhibits LTB4 synthesis in tumor cells cultured in vitro. Several studies have reported that inhibition of the 5-LOX pathway suppresses the proliferation of human colon cancer cells [16] and prostate cancer cells [17,18]. As the major metabolite in the 5-LOX pathway, LTB4 has also been indicated to be involved in carcinogenesis in these organs [4]. Additionally, LTB4-mediated proliferation-promoting effects in colon cancer cells and prostatic
cancer cells are also reported [2,13]. Based on these reports and our findings, we hypothesized that LTB4 may be involved in celecoxib-mediated anticancer effect, at least in certain cancers. To clarify this hypothesis, we studied the effect of LTB4 on HT29 and PC-3 cells. Our results showed that LTB4 itself promoted the survival of HT-29 cells and nearly completely reversed celecoxibmediated inhibitory effect at high concentration. However, the stimulating effect of LTB4 on HT-29 cells was not so dramatic as other reported [13,19]. The possible cause is that the endogenous LTB4 level in HT-29 cells is already high enough to stimulate the proliferation. In contrast, in PC-3 cells, LTB4 not only suppressed cell survival but also had no influence on celecoxib-mediated inhibitory effect. Although growth-inhibitory effect of LTB4 on hu-
P. Gao et al. / Biochemical and Biophysical Research Communications 402 (2010) 308–311
man mammary cancer MCF-7 cells has been reported [20], until now LTB4 mainly exhibits stimulating cell growth [13,14,19]. Further research is needed to elucidate these findings in our study. The different response of HT-29 and PC-3 cells to LTB4 was also observed when cells were treated with LOX inhibitor NDGA, indicating that for survival LOX pathway is more important in HT-29 cells than PC-3 cells. Alterations of phosphatidylinositol 3-kinase (PI3K)-PDK-1 signaling pathway are frequently associated with the pathogenesis of many cancers, including prostate and colorectal cancers [21]. Recent years, this pathway has been reported as one major COX2-independent target for celecoxib in HT-29 cells as celecoxib can directly inhibit PDK-1 activity and PDK-1 mutation impairs celecoxib-induced inhibition [6]. This observation is consistent with our results as PI3K-PDK-1 signaling has also been suggested as one pathway by which LTB4 stimulates proliferation of cancer cells [14]. Overexpression of Akt, a PDK-1 downstream kinase, does not efficiently protect HT-29 cells from celecoxib-induced apoptosis [6], we propose that reversion of celecoxib-mediated inhibition by LTB4 is mainly due to activation of other PDK-1 downstream kinases except Akt, such as p70S6-kinase (p70S6-K) and protein kinase C (PKC) [22]. Additionally, most signaling pathways affected by both celecoxib and LTB4 converge to mammalian target of rapamycin (mTOR) signaling [6,9,14,23,24], suggesting a potential target for further investigations. In summary, our results suggest that anticancer effect of celecoxib is COX-2-independent in PC-3 and HT-29 cells. However, only in HT-29 cells, celecoxib exerts anticancer effect primarily via down-regulating LTB4 production. Additional studies are warranted to elucidate the above mechanisms in detail. Acknowledgments This work was supported by the National Natural Science Foundation of China (No.30540049) and the Scientific Research Foundation of Graduate School of Southeast University (No.YBJJ0915). References [1] D. Divisi, S. Di Tommaso, S. Salvemini, M. Garramone, R. Crisci, Diet and cancer, Acta Biomed. 77 (2006) 118–123. [2] D. Wang, R.N. Dubois, Eicosanoids and cancer, Nat. Rev. Cancer 10 (2010) 181– 193. [3] A. Greenhough, H.J. Smartt, A.E. Moore, H.R. Roberts, A.C. Williams, C. Paraskeva, A. Kaidi, The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment, Carcinogenesis 30 (2009) 377–386. [4] S. Larré, N. Tran, C. Fan, H. Hamadeh, J. Champigneulles, R. Azzouzi, O. Cussenot, P. Mangin, J.L. Olivier, PGE2 and LTB4 tissue levels in benign and cancerous prostates, Prostaglandins Other Lipid Mediat. 87 (2008) 14–19.
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