Radiosensitivity of human prostate cancer cells can be modulated by inhibition of 12-lipoxygenase

Cancer Letters 335 (2013) 495–501

Contents lists available at SciVerse ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Radiosensitivity of human prostate cancer cells can be modulated by inhibition of 12-lipoxygenase J. Lövey a, D. Nie b, J. Tóvári c, I. Kenessey d, J. Tímár d, M. Kandouz e,f,g, K.V. Honn e,f,g,⇑ a

Departments of Radiotherapy and Experimental Therapeutics, National Institute of Oncology, Budapest, Hungary Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, United States c Department of Experimental Pharmacology, National Institute of Oncology, Budapest, Hungary d 2nd Department of Pathology, Semmelweis University, Budapest, Hungary e Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48202, United States f Karmanos Cancer Institute, Detroit, MI 48202, United States g Bioactive Lipids Research Program, Detroit, MI 48202, United States b

a r t i c l e

i n f o

Article history: Received 10 January 2013 Received in revised form 12 March 2013 Accepted 13 March 2013

Keywords: 12-Lipoxygenase 12(S)-HETE Prostate cancer Radiation therapy Radioresistance

a b s t r a c t Nearly 30% of prostate cancer (PCa) patients treated with potentially curative doses relapse at the sites of irradiation. How some tumor cells acquire radioresistance is poorly understood. The platelet-type 12-lipoxygenases (12-LOX)-mediated arachidonic acid metabolism is important in PCa progression. Here we show that 12-LOX confers radioresistance upon PCa cells. Treatment with 12-LOX inhibitors baicalein or BMD122 sensitizes PCa cells to radiation, without radiosensitizing normal cells. 12-LOX inhibitors and radiation, when combined, have super additive or synergistic inhibitory effects on the colony formation of both androgen-dependent LNCaP and androgen-independent PC-3 PCa cells. In vivo, the combination therapy significantly reduced tumor growth. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Arachidonic acid is metabolized by cyclooxygenases (COX) and lipoxygenases (LOX) into various bioactive lipids which were shown to regulate cell survival, cell cycle control, invasion and the angiogenic phenotype [1]. The mammalian LOX family contains a number of lipid peroxidizing enzymes, which are distinguished by their regional specificity of arachidonic acid oxygenation. Accordingly 5-, 8-, 12-, and 15-LOX isoforms are known, which produce 5(S)-, 8(S)-, 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE) metabolites respectively [2]. 12-LOX has two isoforms, the platelet- and the leukocyte-types sharing 65% DNA homology [3,4]. In prostate cancer a number of studies outlined the pattern of changes in the expression of AA-metabolizing enzymes. Based on preclinical and clinicopathological studies it is evident that in prostate cancer COX-2, TxS, platelet-type 12-LOX and 15-LO1 (leukocyte-type 12-LOX) are upregulated, while the 15-LOX2 is downregulated, resulting in a characteristic pattern of AA metabolites rich in PGE2, TX, 12-S-HETE, 15-S-HETE and 13-S-HODE [1]. It is ⇑ Corresponding author. Address: Department of Pathology, 431 Chemistry Bldg., Wayne State University, Detroit, MI 48202, United States. Tel.: +1 313 577 1018; fax: +1 313 577 0798. E-mail address: [email protected] (K.V. Honn). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.03.012

interesting, that the ectopic expression of 12-lipoxygenases can be considered as the manifestation of stem cell characteristrics since platelet- or leukocyte-type 12-LOX (15-LO1) are constitutively expressed in bone marrow progenitors [5]. Previous data indicated that 12-S-HETE activates antiapoptotic-, motility- and angiogenic signaling cascades [6–8]. 12-S-HETE also regulates gene expression including VEGF [8]. 12-LOX levels positively correlated with the stage and grade of prostate cancer [9]. Overexpression of 12-LOX also enhances tumor growth through the induction of angiogenesis [10]. Inhibition of 12-LOX activity with specific inhibitors induced apoptosis and decreased the metastatic potential of DU-145 prostate cancer cells in a lung colonization assay [11]. As screening methods for prostate cancer improve, more patients with localized disease are diagnosed. The curative treatment choice of localized prostate cancer can be both surgery and radiotherapy [12]. Recently, due to patients’ preference, the lower morbidity and the comparable result achieved with radical radiotherapy, the number of prostate radiotherapy treatments both with external irradiation and brachytherapy is increasing [13]. However, radiosensitivity of prostate cancer is variable due to mostly unknown factors among which the geno- and phenotype of the tumor must be important [14]. Therefore, potentiation of the effect of radiotherapy may have clinical relevance in this tumor

496

J. Lövey et al. / Cancer Letters 335 (2013) 495–501

type as well. Since signaling pathways involving 12-LOX play an important role in the progression of various cancers including prostate cancer [1], we have postulated that 12-LOX derived bioactive lipids may modulate the cytotoxic effect of ionizing radiation as well. 2. Materials and methods 2.1. Cell lines Human prostate cancer cell lines PC-3, DU 145 and LNCaP were obtained from ATCC (Manassas, VA) and maintained in humidified atmosphere of 5% CO2 at 37 °C and cultured in RPMI 1640 or DMEM supplemented with 10% FBS and 100 lg/ml penicillin–streptomycin. 2.2. In vitro treatments Prior to treatment, prostate cancer cells were plated in 10 cm diameter tissue culture plates in serum containing medium and allowed 12 h to attach. Cells were treated with irradiation, 12-LOX inhibitors Baicalein (Biomol, Plymouth, PA) [15] or N-benzyl-N-hydroxy-5-phenylpentamide (BMD122) (Biomide Corp., Grosse Pointe Farms, MI) [11], 5-LOX inhibitor Rev-5901 (Cayman Chemicals, Ann Arbor, MI) and with irradiation in combination with the above mentioned LOX inhibitors. Add-back experiments were carried out using 12(S)-, 5(S) - and 15(S)-HETE (Cayman Chemicals). Irradiation was carried out with a Cs-137 scientific gamma irradiator with a dose rate of 85 cGy/min. LOX inhibitors and different HETEs were diluted in serum-free medium. Treatment schedules were as follows. Single treatment with BMD122 and Rev-5901 was carried out with concentrations of 10– 50 lM, and of Baicalein with 3–15 lM. Doses of irradiation as single treatment were 25–500 cGy. During combined treatment 25 lM BMD122, 25 lM Rev-5901 or 7.5 lM Baicalein was used 1 h prior irradiation. Doses of irradiation were the same as above described. During the add-back experiments, 100 ng/ml of 5(S)-, 12(S)- or 15(S)-HETE was applied just before administering BMD122 or Baicalein or delivering irradiation. 2.3. Clonogenic survival Prostate carcinoma cells were placed in 6-well culture plates and were allowed to attach for 24 h in the presence of serum. The number of cells per well was set to produce around 100–200 surviving colonies after treatment. Plating efficiency ranged 45–70%. Cells were treated according to the schedules described above. Plates were incubated for 14 d (except fast growing LNCaP for 7 d) at 37 °C in 10% FBS (Gibco) containing medium. Cells then were fixed in 4% buffered formaldehyde and stained with crystal violet. Colonies containing at least 50 cells were counted under a light microscope and survival was normalized to untreated controls. 2.4. Apoptosis Apoptosis was assessed by the incorporation of fluorescein-12-dUTP mediated by terminal deoxynucleotidyl transferase into fragmented DNA (TUNEL-assay Roche Diagnostics GmbH). Cells were seeded onto 10 cm diameter Petri-dishes and treated as described previously in the treatment section. Throughout the experiment cells were maintained in the presence of serum. Following treatment 24 h, cells were digested by trypsin–EDTA, washed in PBS and fixed in ice cold ethanol for 1 h. Permeabilization was carried out applying ice-cold 0.1% Triton-X100 and 0.1% sodium-citrate for 3 min, then cells were washed in PBS and working solution of the TUNEL reaction was added. Samples were incubated for 1 h at 37 °C, washed and resuspended in PBS. The apoptotic fraction of the cells was quantified by laser flow cytometry. 2.5. Western blot analysis Forty-eight hours following the above described treatments, total cell lysates were prepared. Proteins (20 lg) were fractionated on precast SDS–PAGE and transferred onto nitrocellulose membranes. Following incubation in blocking buffer containing 5% skimmed milk dissolved in 1 mM TRIS–HCl, 100 mM NaCl and 0.1% Tween 20 for 1 h, blots were probed overnight at 4 °C with primary antibodies against Bcl-2, Bax, and survivin (R&D Systems, MN, USA) with a dilution of 1:1000. 2.6. In vivo growth PC3 cells (106) were injected s.c. into SCID mice. Tumors were grown till 5 mm diameter for 2 weeks. BMD122 was administered i.p. (100 mg/kg/day) for 4 consecutive days. Irradiation was completed on day 16, with a single dose of 5 Gy. Animals were followed afterwards for another 2 weeks. Tumor was measured each third day by measuring 2 diameters to calculate tumor volume. Control animals lost more than 20% of their body weight on day 28 therefore they were terminated with Nembutal overdose.

2.7. Statistical analysis For the analysis of potential treatment interactions (synergism or antagonism), a combination index (CI) was calculated with the Calcusyn software, which uses the Chou–Talalay equation, based on the median effect principle [16]. First, clonogenic survival curves are created. For the analysis of additivity of the combined treatments, additive curves were created by multiplying the survival fractions of the groups treated with the compounds alone with the survival fractions of the different doses of radiation alone. This theoretical additive curve represents the maximal possible additive effect of the two modalities. Therefore if survival curves of combined modality treatments result in a lower survival than the theoretical additive curve, then combined treatment are super-additive or synergistic. For the analysis of the different curves, linear regression of all curves was performed. When the survival curves of treatments containing radiation are non-linear, curves of logarithms of survival are used for regression and compared to each other pairwise. Curve (line) comparisons were carried out by Graphpad Prism software (GraphPad Software, Inc. San Diego, CA). When comparing two lines using Prism, slopes are compared first. It calculates a P value testing the null hypothesis that the slopes are all identical in the overall populations. If the P value is less than 0.05, Prism concludes that the lines are significantly different. In that case, there is no point in comparing the intercepts. The P value is two-sided. If the P value for comparing slopes is greater than 0.05, Prism concludes that the slopes are not significantly different and calculates an overall (pooled) slope. Since the slopes are assumed to be identical, there are two possibilities. Either the lines are identical, or they are different but parallel. Prism calculates a second P value testing the null hypothesis that the lines are identical. If this P value is low, it can be concluded that the lines are not identical, but rather that they are distinct but parallel. If this second P value is high, we can conclude that there is no evidence that the lines are not identical. For the animal experiments we used the non-parametric Kruskal–Wallis test with post hoc analysis. Statistical analysis was performed by Statistica 9.0 software (StatSoft, Tulsa, OK).

3. Results 3.1. Effect of 12-LOX inhibitors and irradiation on the clonogenic survival of prostate cancer cells Treatment of PC-3, LNCaP and DU145 human prostate cancer cells with different concentrations of 12-LOX inhibitors (Baicalein and BMD122, Fig. 1A and B) or different doses of radiation (Fig. 1C) resulted in a dose dependent decrease in the clonogenic survival of the cells. The less sensitive prostate cancer cell line for 12-LOX inhibition was DU145, while the three cell lines were similarly sensitive to irradiation, although at lower doses PC3 seemed to be moderately resistant. Human prostate cancer cell lines were treated with the combination of radiation (0–500 cGy doses) and 25 lM BMD122 and clonogenic survival was analyzed. The synergism between radiation and 12-LOX inhibition was evaluated by combination indexes calculated employing mutually exclusive assumption and median effect principle based on Chou–Talalay equation using the Calcusyn software. In PC-3 cells the combined effect of radiation and 12LOX inhibitor proved to be supra-additive as shown by the combination indexes calculated employing the median effect principle (Table 1). In the case of LNCaP cells the radiation enhancing effect of 12-LOX inhibitor was apparent only from higher – over 100 cGy – radiation doses. In contrast to PC-3 and LNCaP cells, the combined radiation and 12-LOX inhibitor treatment of DU145 showed only a simple additive effect (Table 1). We also examined the effect of baicalein, a selective inhibitor of 12-LOX, on radioresponse of LNCaP and PC3 cells. These cells were treated with 7.5 lM baicalein for 2 h before initiation of radiation. As shown in Fig. 2A, baicalein and radiation, when combined, have super additive or synergistic inhibitory effect on the colony formation of LNCaP cells. Regression analysis indicates that combined treatment of LNCaP cells with radiation and baicalein has significant super-additive or synergistic effect (P < 0.05). Similarly, as shown in Fig. 2B, baicalein and radiation, when combined, have super additive or synergistic inhibition on the colony formation of PC3 cells (P < 0.01). Taken together, these data suggest that 12-LOX

497

J. Lövey et al. / Cancer Letters 335 (2013) 495–501

1.00 0.75

DU-145

0.50 0.25

0

20

40

60

1.25

0.01

0

200

400

600

PC-3 DU-145

0.50 0.25

5

10

Measured additive curve

1

0.75

0

PC3

B

LnCAP

1.00

15

20

Baicalein concentration (µM)

Survival fraction

Survival fraction

0.1

Radiation dose (cGy)

0.00

Survival fraction

Measured additive curve Calculated additive curve

0.001

BMD122 concentration (µM)

C

1

LnCAP

0.00

B

LNCaP

A

PC-3

Survival fraction

Survival fraction

A

Calculated additive curve 0.1 0.01 0.001 0.0001

0

200

400

600

Radiation dose (cGy)

1.00 PC-3

Fig. 2. Synergism between irradiation and 12-LOX inhibition in prostate cancer cells. (A) Radiosensitization of LNCaP cells by a 12-LOX inhibitor, baicalein. (A) 12LOX inhibitor baicalein sensitizes LNCaP cells to radiation as indicated by colony formation assay. P = 0.02688 < 0.05. (B) Radiosensitization of androgen independent PC-3 cells by baicalein. 12-LOX inhibitor baicalein sensitizes PC3 cells to radiation as indicated by colony formation assay. P = 0.0086 < 0.01.

LnCAP

0.75

DU-145 0.50 0.25 0.00 0

200

400

600

Radiation dose (cGy) Fig. 1. Sensitivity of prostate cancer cells to radiation and 12-LOX inhibitors. 14 d clonogenic survival of human prostate cancer cells PC-3, LNCaP and DU145 treated with various concentrations of BMD122 (A), Baicalein (B) for 1 h or various doses of radiation (C). Each point represents the mean of a triplicate (SD < 10%).

Table 1 Analysis of synergism between 12-LOX inhibition and irradiation on clonogenic survival of human prostate cancer cell lines. Radiation dose (cGy)

PC-3 25 lM BMD122

LNCaP 25 lM BMD122

DU 145 25 lM BMD122

25 50 100 200 500

0.87 0.66 0.64 0.61 0.50

1.23 0.89 0.76 0.56 0.62

1.12 1.05 1.36 1.54 1.23

CI = 1 represents simple additive, while CI > 1 represents sub-additive, CI < 1 represents supra-additive effect.

inhibition sensitizes both androgen-dependent (LNCaP) and independent (PC-3) cells to radiation. 3.2. 12-LOX inhibitors do not sensitize normal cells to radiation. To study whether 12-LOX inhibitors can also sensitize normal prostate epithelial cells to radiation, we treated human normal prostate epithelial cells (purchased from Clonetics, San Diego, CA) with 7.5 lM baicalein 2 h before radiation (800 cGy). The cells

are harvested 36 h after radiation for evaluation of apoptosis using a TUNEL assay. We used apoptosis, rather than colony formation assays, as the end point for potential radiosensitization of normal prostate epithelial cells by 12-LOX inhibitors, because unlike prostate cancer cells, normal prostate cells have limited ability to proliferate and form colonies. As shown in Fig. 3A, the presence of baicalein did not potentiate radiation-elicited apoptosis in normal prostate epithelial cells. A similar result was observed in human normal skin fibroblast (Fig. 3B). The lack of radiosensitization by 12-LOX inhibitor in normal prostate epithelial cells may be due to the low or absence of 12-LOX expression [17]. 3.3. Radiosensitization by 12-LOX inhibition depends on the 12(S)-HETE metabolic product To prove that 12(S)-HETE, the sole 12-LOX metabolic product from arachidonic acid, mediated the effect on irradiation, PC3 cells were treated either with 12-LOX inhibitor, BMD122 (Fig. 4A), radiation (Fig. 4B) or the combination of both (Fig. 4C) in the presence of exogenous 12(S)-HETE applied at a concentration of 100 ng/ml for 1 h prior to treatments. 12(S)-HETE suspended the inhibitory effect of BMD122 on clonogenic survival (Fig. 4A) but did not affect radiation efficacy (Fig. 4B). 12(S)-HETE also increased clonogenic survival of cells subject to a pre-treatment with BMD122 (7.5 lM; 2 h pre-irradiation) and subsequently to different doses of irradiation (Fig. 3C). In order to prove that the 12-LOX pathway is exclusively responsible for these effects, experiments were repeated with a 5-LOX inhibitor, Rev-5901, and add-back experiments were carried out with 5- and 15-HETE. Treatment of the cells with the 5-LOX inhibitor or pretreatment of the cells with 5(S)- or 15(S)-HETE did not have any significant effect on the clonogenic survival of the PC-3 cells (data not shown). A further

498

J. Lövey et al. / Cancer Letters 335 (2013) 495–501

1.00

Survival fraction

A

12-HETE CONTROL

0.75 0.50 0.25 0.00 0

20

40

60

BHPP concentration (µM)

B

1.00

Survival fraction

12-HETE CONTROL

0.75 0.50 0.25 0.00 0

200

400

600

Radiation dose (cGy)

Fig. 3. Lack of radiosensitization of baicalein, a 12-LOX inhibitor, in normal prostate epithelial cells. (A) Human normal prostate epithelial cells were treated with 7.5 lM baicalein 2 h prior to radiation (800 cGy). The cells were harvested 36 h after radiation for evaluation of apoptosis using a flow cytometric assay kit based on TUNEL staining (APO-DIRECT, Pharmingen, San Diego, CA). (B) The same experiment was performed on human normal skin fibroblasts. Note the increase in apoptosis after radiation (800 cGy) and the absence of effect of baicalein treatment on apoptosis, regardless of radiation.

confirmation of this conclusion is that, when we used PC3 cells which overexpress the platelet-type 12-LOX and are capable of higher 12(S)-HETE biosynthesis [18], the 12-LOX-overexpressing nL8 cells presented strong radioresistance when compared to the vector control, neo-r, as indicated by enhanced clonogenic survival (Fig. 5). 3.4. Effect of 12-LOX inhibitors and radiation on apoptosis induction Since 12-LOX is involved in apoptosis sensitivity of prostate cancer cells [6,19,20], the combined effect of the 12-LOX inhibitors and irradiation was tested on apoptosis induction in PC3 cells as determined by TUNEL assay and measured by flow cytometry. An irradiation dose of 2 Gy alone resulted in a small but significant increase in apopotosis reaching 8% while following 12-LOX inhibition (BMD122, 25 lM) the apoptosis rate was doubled (15%) and the combination of both resulted in a maximal effect (25%) (Fig. 6A). To prove that this increase was a consequence of 12-LOX inhibition, the experiment was repeated in the presence of exogenous 12(S)-HETE (30 min before radiation, 100 ng/ml). 12-HETE or 2 Gy irradiation alone did not affect apoptosis. On the other hand, 12-HETE decreased the apoptosis-inducing effect of BMD122 to 10% and the combination modality to 16.5% from 25% (Fig. 6A). To explore the molecular mechanism of the effect of 12-LOX inhibitors and radiation we analyzed their effects on expression of proteins regulating apoptosis in PC3 cells using immunoblotting. The level of Bcl-2 was unchanged following radiotherapy. After 12LOX inhibition by BMD122 and combined treatments the level of Bcl-2 protein decreased significantly (Fig. 6B,C), while the proapoptotic protein, Bax, did not change significantly after treatments. On the other hand, the level of survivin, a member of the apoptosis

Survival fraction

C

1

12-HETE CONTROL

0.1

0.01

0.001 0

200

400

600

Radiation dose (cGy) Fig. 4. Rescue of the radiosensitizing effect of 12-LOX inhibitors by the 12(S)-HETE metabolite. Effect of 100 ng/ml 12(S)HETE on the clonogenic survival of PC-3 cells treated with various concentrations of BMD122 (A) for 1 h, irradiation (B) or their combinations (C). Data represent mean of 3 parallel samples where SD is <10%.

inhibitor family, was unchanged following 2 Gy radiation but significantly decreased after BMD122 or combined treatments (Fig. 6B and C). 3.5. Effect of 12-LOX inhibitor administration on the antitumoral effect of irradiation in vivo Finally, to assess the radiosensitizing effect of 12-LOX inhibitors in vivo, we used a model of prostate cancer xenografts. PC3 cells (106) were inoculated s.c. into SCID mice and were left to grow to a size of 100 mm3 (day 16). The mice were then treated with the 12-LOX inhibitor, BMD122 i.p. (100 lg/kg) for 4 consecutive days or irradiated on day 16 or the two modalities were combined. Although the physiologically relevant dosing in humans remains to be determined, the maximum tolerated dose (MTD) for BMD122 in animals is 50 mg/kg. The changes in tumor volume were monitored till the euthanasia of control animals (day 28). Data indicated that 4 days BMD122 administration alone did not affect s.c. growth of PC3 cells; a single administration of 5 Gy irradiation caused shrinkage of tumor growth, however this result was not statistically significant. However, the most effective growth inhibition was obtained with the combination treatment, whereby the inhibitory effect was manifest at day 28 (Fig. 7). Therefore, we

499

J. Lövey et al. / Cancer Letters 335 (2013) 495–501

Fig. 5. 12-LOX enhances radioresistance of PC3 cells as indicated by colony formation assay. (A) Increased clonogenic survival by enhanced expression of 12LOX in PC3 cells. nL8, a 12-LOX overexpressing clone of PC3 cells; neo-r, vector control. (B) Regression analysis. P < 0.01.

conclude that 12-LOX inhibition is effective at radiosensitizing prostate cancer cells in vivo. 4. Discussion Prostate cancer is the most common cancer in men in the United States and the second leading cause of cancer death. Therapeu-

tic interventions include mainly: radical prostatectomy or radiation therapy (RT), with or without androgen deprivation therapy (ADT). However, nearly 30% of patients treated with potentially curative radiation doses relapse at the sites of irradiation and there is a need for new strategies to radiosensitize prostate cancer cells [21,22]. Eicosanoids, the metabolites of arachidonic acid (AA), are potent lipid mediators involved in a number of homeostatic biological functions and inflammation [23]. They are also important players in cancer [24]. Arachidonic acid is metabolized by the cyclooxygenase pathway to prostaglandins and thromboxane [25], by the lipoxygenase (LOX) pathway to leukotrienes [26], lipoxins [27,28], hepoxilins [29], and hydroxyeicosatetraenoic acids (HETE) [2,30], and by the epoxygenase pathway to epoxy eicosatetraenoic acids (EpETrE) [31–33]. Oxidative metabolism of AA by LOXs, especially 12-LOX, plays an important role in cancer pathophysiology [34,35]. Many studies have demonstrated that 12-LOX and its metabolite 12(S) HETE regulate a plethora of biological functions such as cell survival, matrix adhesion [36,37], cell motility [7] and secretion of matrix degrading enzymes [34]. Subsequently, in several cancer types where 12-LOX is active, it is involved in tumor progression and metastasis [34,38,39]. The work from our lab and others’ has identified 12-LOX as a regulator of the cell survival and cell cycle machineries in prostate cancer cells. We have elucidated the underlying mechanisms that affect cell cycle control by 12-LOX inhibitors. In particular, we have shown that 12-LOX inhibition results in cell cycle arrest in G0–G1, which subsequently induces caspase- and Bcl-2- dependent apoptosis [19]. Cell cycle and apoptosis are critical determinants of the response to various therapies, including radiation therapy [40–44]. As radiotherapy plays a paramount role in the treatment of prostate cancer, there is a great interest in enhancing the effect of radiation. Radiosensitivity of prostate cancer depends on the apoptotic propensity and hypoxia of cancer cells. It was shown that expression of the antiapoptotic

B

BCL-2

p≤0.01

*

30

BAX

25

Survivin

10

RX+BMD122+12HETE

RX+BMD122

BMD122+12HETE

BMD122

RX+12HETE

RX

0

Control+12HETE

5

RX+BMD122

15

BMD122

CTR

*

RX

p≤0.05 20

Control

Apoptosis (%FITC-dUTPcells)

A

C

Fig. 6. Effect of radiation and 12-LOX inhibition on apoptosis induction in PC-3 cells. (A) Cells were treated by the 12-LOX inhibitor BMD122 (25 lM) for 1 h or irradiated (2 Gy) or both, cultured for 24 h and harvested. Apoptosis was measured using the TUNEL assay. CTR = Control, RX = Irradiated. Data are mean ±SD, n = 5. Statistical comparison of apoptosis induction in PC3 cells was performed with ANOVA and Duncan post hoc test, and all conditions except 12HETE were significantly different in comparison with the control (p 6 0.05). (B) Western blot analysis of apoptosis regulating proteins in PC-3 cells treated with irradiation and 12-LOX inhibitors 1 h or in combination. Proteins were isolated and immunoblotts were prepared for BCL2, BAX and Survivin. (C) The densities of the appropriate bands were measured by densitometry. The western blots were performed three times and representative blots and densitometry measurements are shown.

500

J. Lövey et al. / Cancer Letters 335 (2013) 495–501

tumor volume (mm3)

700 600

Control

500

BMD122

400 300

Irrad.

200 BMD122 + Irrad.

100 0 16

18

21

25

28

Days after tumor inoculation Fig. 7. Effect of 12-LOX inhibitor (BMD122) administration on the antitumoral effect of irradiation on PC3 prostate carcinoma xenograft tumor. 106 PC3 cells were inoculated into SCID mice sc. and primary tumor was established within 16 days. Tumors were irradiated on day 16 or were treated i.p. with 100 mg/kg BMD122 for 4 d or a combination of the two procedures was applied. Changes in tumor volume were followed by daily caliper measurements till the end of the experiment (day 28). Data are expressed as mean tumor volume (±SD, n = 8, p < 0.05, Kruskal–Wallis test). Only BMD122 + Irrad is statistically different than the control (p < 0.05).

significant increase in biosynthesis of 12(S)-HETE in B16 melanoma cells as early as 5 min. The increased 12(S)-HETE formation led to an enhanced adhesion of B16 melanoma cells to fibronectin in vitro and metastasis in vivo [54]. The results suggest that lowdose radiation, at levels comparable to those used in fractionated or hyper-fractionated radiotherapy, caused a rapid increase in 12-LOX metabolism of arachidonic acid [54]. Taken together, these data emphasize the importance of 12-LOX metabolic activity in cellular response to radiation therapy. The molecular mechanisms regulated by 12-LOX influencing the radiosensitivity of human prostate cancer cells involved the Bcl2/Bax apoptotic pathway. Interestingly, an earlier report emphasized the role of the Bcl-2/Bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer [45]. It would be interesting to explore the selective targeting of this pathway by the use of other apoptosis inducers in combination of 12-LOX inhibition. Conflict of Interest The authors declare no conflict of interest.

proteins BCL2, BCL-XL (the BCL2/BAX ratio), clusterin and caspase-1 were associated with radiosensitivity [45–47]. Furthermore, BCL2 inhibitors have been shown to have radiosensitizing effects [48]. Building on the above-mentioned findings, we hypothesized that inhibiting the activity of 12-LOX in prostate cancer cells could be effective at promoting radiation-induced cytotoxicity. In this preclinical study we have shown that inhibition of 12-LOX activity by various selective inhibitors promoted the effect of radiation on human prostate cancer cells. All three human prostate cancer cell lines have been shown earlier to express 12-LOX and 15LO1 at various levels [38] and to produce 12-HETE [49]. The difference between these cell lines is that DU-145 showed only additive effect upon 12-LOX inhibition while PC-3 and LNCaP, showed synergistic effects. It is interesting that both hormone refractory and sensitive cell lines are radiosensitized by 12-LOX inhibition. Hormone sensitivity plays an important role in the treatment of prostate cancer and hormone-refractory cancer is known to have a worse prognosis [50]. Moreover, to apply curative radiotherapy for prostate cancer currently, it is advised to employ a 2–3 months hormonal treatment prior to radiotherapy, in order to sensitize cancer cells to radiation [51]. The add-back experiments where 12(S)-HETE could suspend the effect of 12-LOX inhibition when administered alone or in conjunction with radiation, further confirm that this radiosensitization effect is mediated through the 12-LOX pathway. Using a cellular model of PC3 cells transfected with the platelettype 12-LOX, we found that the latter increased the radioresistance significantly. These data demonstrate the translational validity of the 12-LOX inhibitors as radiosensitizers in prostate cancer. This is further confirmed by using a xenograft model, whereby 12LOX inhibitors could efficiently potentiate the radioresponse of prostate cancer cells in vivo. The clinical impact of our findings is also supported by our earlier report that the level of 12-LOX expression was correlated with the tumor stage and grade [17,52] and that overall, 38% of patients demonstrated elevated levels of 12-LOX in prostate cancer tissue compared with their matching normal tissues. Thus, increased 12-LOX levels might be important in determining the radioresistance in a large sub-population of prostate cancer patients. It is interesting to note that increased levels of free and esterified 12(S)-HETE were detected in cleanup workers from the Chernobyl accident who were exposed to radiation compared to unexposed individuals, indicating an increased accumulation of 12(S)-HETE in irradiated persons [53]. In vitro, we have shown that low-dose c radiation stimulates 12-LOX activity and biosynthesis of 12(S)-HETE [54]. An exposure to 0.5 Gy c-radiations caused a

Acknowledgements This work was supported by a DOD IDEA Award W81XWH-111-0519 (K.V.H. and M.K), the Fund for Cancer Research (K.V.H), TAMOP 4.2.1.B-09/1-KMR-2010-001 (J.T.) and OTKA K84173 (J.Tó). References [1] D. Nie, M. Che, D. Grignon, K. Tang, K.V. Honn, Role of eicosanoids in prostate cancer progression, Cancer Metastasis Rev. 20 (2001) 195–206. [2] A.R. Brash, Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate, J. Biol. Chem. 274 (1999) 23679–23682. [3] C.D. Funk, L. Furci, G.A. FitzGerald, Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase, Proc. Natl. Acad. Sci. USA 87 (1990) 5638–5642. [4] T. Yoshimoto, H. Suzuki, S. Yamamoto, T. Takai, C. Yokoyama, T. Tanabe, Cloning and sequence analysis of the cDNA for arachidonate 12-lipoxygenase of porcine leukocytes, Proc. Natl. Acad. Sci. USA 87 (1990) 2142–2146. [5] J. Timar, J. Tovari, E. Raso, L. Meszaros, B. Bereczky, K. Lapis, Platelet-mimicry of cancer cells: epiphenomenon with clinical significance, Oncology 69 (2005) 185–201. [6] D.G. Tang, Y.Q. Chen, K.V. Honn, Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis, Proc. Natl. Acad. Sci. USA 93 (1996) 5241–5246. [7] J. Timar, S. Silletti, R. Bazaz, A. Raz, K.V. Honn, Regulation of melanoma-cell motility by the lipoxygenase metabolite 12-(S)-HETE, Int. J. Cancer 55 (1993) 1003–1010. [8] D. Nie, S. Krishnamoorthy, R. Jin, K. Tang, Y. Chen, Y. Qiao, A. Zacharek, Y. Guo, J. Milanini, G. Pages, K.V. Honn, Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells, J. Biol. Chem. 281 (2006) 18601– 18609. [9] X. Gao, D.J. Grignon, T. Chbihi, A. Zacharek, Y.Q. Chen, W. Sakr, A.T. Porter, J.D. Crissman, J.E. Pontes, I.J. Powell, Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer, Urology 46 (1995) 227–237. [10] D. Nie, J. Nemeth, Y. Qiao, A. Zacharek, L. Li, K. Hanna, K. Tang, G.G. Hillman, M.L. Cher, D.J. Grignon, K.V. Honn, Increased metastatic potential in human prostate carcinoma cells by overexpression of arachidonate 12-lipoxygenase, Clin. Exp. Metastasis 20 (2003) 657–663. [11] D.G. Tang, L. Li, Z. Zhu, B. Joshi, C.R. Johnson, L.J. Marnett, K.V. Honn, J.D. Crissman, S. Krajewski, J.C. Reed, J. Timar, A.T. Porter, BMD188, A novel hydroxamic acid compound, demonstrates potent anti-prostate cancer effects in vitro and in vivo by inducing apoptosis: requirements for mitochondria, reactive oxygen species, and proteases, Pathol. Oncol. Res. 4 (1998) 179–190. [12] E.A. Klein, P.A. Kupelian, Localized prostate cancer: radiation or surgery?, Urol Clin. North Am. 30 (2003) 315–330 (xi). [13] W.R. Lee, J. Moughan, J.B. Owen, M.J. Zelefsky, The patterns of care study of radiotherapy in localized prostate carcinoma: a comprehensive survey of prostate brachytherapy in the United States, Cancer 98 (2003) (1999) 1987– 1994. [14] W.J. Ellis, Prostate brachytherapy, Cancer Metastasis Rev. 21 (2002) 125–129. [15] K. Sekiya, H. Okuda, Selective inhibition of platelet lipoxygenase by baicalein, Biochem. Biophys. Res. Commun. 105 (1982) 1090–1095. [16] T.C. Chou, R.J. Motzer, Y. Tong, G.J. Bosl, Computerized quantitation of synergism and antagonism of taxol, topotecan, and cisplatin against human

J. Lövey et al. / Cancer Letters 335 (2013) 495–501

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25] [26] [27]

[28]

[29] [30] [31]

[32]

[33]

[34]

[35] [36]

teratocarcinoma cell growth: a rational approach to clinical protocol design, J. Natl Cancer Inst. 86 (1994) 1517–1524. X. Gao, D.J. Grignon, T. Chbihi, A. Zacharek, Y.Q. Chen, W. Sakr, A.T. Porter, J.D. Crissman, J.E. Pontes, I.J. Powell, K.V. Honn, Elevated 12-lipoxygenase mRNA expression correlated with advanced stage and poor differentiation of human prostate cancer, Urology 46 (1995) 1995. D. Nie, G.G. Hillman, T. Geddes, K. Tang, C. Pierson, D.J. Grignon, K.V. Honn, Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth, Cancer Res. 58 (18) (1998) 9–15. G.P. Pidgeon, M. Kandouz, A. Meram, K.V. Honn, Mechanisms controlling cell cycle arrest and induction of apoptosis after 12-lipoxygenase inhibition in prostate cancer cells, Cancer Res. 62 (2002) 2721–2727. G.P. Pidgeon, J. Lysaght, S. Krishnamoorthy, J.V. Reynolds, K. O’Byrne, D. Nie, K.V. Honn, Lipoxygenase metabolism: roles in tumor progression and survival, Cancer Metastasis Rev. 26 (2007) 503–524. G.W. Allen, A.R. Howard, D.F. Jarrard, M.A. Ritter, Management of prostate cancer recurrences after radiation therapy-brachytherapy as a salvage option, Cancer 110 (2007) 1405–1416. A. Heidenreich, J. Bellmunt, M. Bolla, S. Joniau, M. Mason, V. Matveev, N. Mottet, H.P. Schmid, T. van der Kwast, T. Wiegel, F. Zattoni, EAU guidelines on prostate cancer. Part I: Screening, diagnosis, and treatment of clinically localised disease, Actas Urol. Esp. 35 (2011) 501–514. J.Z. Haeggstrom, A. Rinaldo-Matthis, C.E. Wheelock, A. Wetterholm, Advances in eicosanoid research, novel therapeutic implications, Biochem. Biophys. Res. Commun. 396 (2010) 135–139. D. Wang, R.N. Dubois, Eicosanoids and cancer, Nat. Rev. Cancer 10 (2010) 181– 193. M.T. Wang, K.V. Honn, D. Nie, Cyclooxygenases, prostanoids, and tumor progression, Cancer Metastasis Rev. 26 (2007) 525–534. R.C. Murphy, M.A. Gijon, Biosynthesis and metabolism of leukotrienes, Biochem. J. 405 (2007) 379–395. C.N. Serhan, B. Samuelsson, Lipoxins: a new series of eicosanoids (biosynthesis, stereochemistry, and biological activities), Adv. Exp. Med. Biol. 229 (1988) 1–14. S.J. O’Meara, K. Rodgers, C. Godson, Lipoxins: update and impact of endogenous pro-resolution lipid mediators, Rev. Physiol. Biochem. Pharmacol. 160 (2008) 47–70. S. Nigam, M.P. Zafiriou, R. Deva, R. Ciccoli, M.R. Roux-Van der, Structure, biochemistry and biology of hepoxilins: an update, FEBS J. 274 (2007) 3503–3512. T. Yoshimoto, Y. Takahashi, Arachidonate 12-lipoxygenases, Prostaglandins Other Lipid Mediat. 68–69 (2002) 245–262. F.A. Fitzpatrick, R.C. Murphy, Cytochrome P-450 metabolism of arachidonic acid: formation and biological actions of ‘‘epoxygenase’’-derived eicosanoids, Pharmacol. Rev. 40 (1989) 229–241. B. Fisslthaler, R. Popp, L. Kiss, M. Potente, D.R. Harder, I. Fleming, R. Busse, Cytochrome P450 2C is an EDHF synthase in coronary arteries, Nature 401 (1999) 493–497. R. Kaspera, R.A. Totah, Epoxyeicosatrienoic acids: formation, metabolism and potential role in tissue physiology and pathophysiology, Expert. Opin. Drug Metab. Toxicol. 5 (2009) 757–771. K.V. Honn, D.G. Tang, X. Gao, I.A. Butovich, B. Liu, J. Timar, W. Hagmann, 12lipoxygenases and 12(S)-HETE: role in cancer metastasis, Cancer Metastasis Rev. 13 (1994) 365–396. D. Nie, Cyclooxygenases and lipoxygenases in prostate and breast cancers, Front Biosci. 12 (2007) 1574–1585. J. Timar, Y.Q. Chen, B. Liu, R. Bazaz, J.D. Taylor, K.V. Honn, The lipoxygenase metabolite 12(S)-HETE promotes alpha IIb beta 3 integrin-mediated tumorcell spreading on fibronectin, Int. J. Cancer 52 (1992) 594–603.

501

[37] G.P. Pidgeon, K. Tang, Y.L. Cai, E. Piasentin, K.V. Honn, Overexpression of platelet-type 12-lipoxygenase promotes tumor cell survival by enhancing alpha(v)beta(3) and alpha(v)beta(5) integrin expression, Cancer Res. 63 (2003) 4258–4267. [38] J. Timar, E. Raso, B. Dome, L. Li, D. Grignon, D. Nie, K.V. Honn, W. Hagmann, Expression, subcellular localization and putative function of platelet-type 12lipoxygenase in human prostate cancer cell lines of different metastatic potential, Int. J. Cancer 87 (2000) 37–43. [39] E. Raso, B. Dome, B. Somlai, A. Zacharek, W. Hagmann, K.V. Honn, J. Timar, Molecular identification, localization and function of platelet-type 12lipoxygenase in human melanoma progression, under experimental and clinical conditions, Melanoma Res. 14 (2004) 245–250. [40] A.R. Cuddihy, R.G. Bristow, The p53 protein family and radiation sensitivity: Yes or no?, Cancer Metastasis Rev 23 (2004) 237–257. [41] T. Shimura, Acquired radioresistance of cancer and the AKT/GSK3beta/cyclin D1 overexpression cycle, J. Radiat. Res. 52 (2011) 539–544. [42] R.G. Bristow, S. Benchimol, R.P. Hill, The p53 gene as a modifier of intrinsic radiosensitivity: implications for radiotherapy, Radiother. Oncol. 40 (1996) 197–223. [43] D. Eriksson, T. Stigbrand, Radiation-induced cell death mechanisms, Tumour. Biol. 31 (2010) 363–372. [44] S. Fulda, Inhibitor of Apoptosis (IAP) proteins as therapeutic targets for radiosensitization of human cancers, Cancer Treat. Rev. 38 (2012) 760–766. [45] T.J. Mackey, A. Borkowski, P. Amin, S.C. Jacobs, N. Kyprianou, Bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer, Urology 52 (1998) 1085–1090. [46] T. Zellweger, K. Chi, H. Miyake, H. Adomat, S. Kiyama, K. Skov, M.E. Gleave, Enhanced radiation sensitivity in prostate cancer by inhibition of the cell survival protein clusterin, Clin. Cancer Res. 8 (2002) 3276–3284. [47] R.N. Winter, J.G. Rhee, N. Kyprianou, Caspase-1 enhances the apoptotic response of prostate cancer cells to ionizing radiation, Anticancer Res. 24 (2004) 1377–1386. [48] J. An, A.S. Chervin, A. Nie, H.S. Ducoff, Z. Huang, Overcoming the radioresistance of prostate cancer cells with a novel Bcl-2 inhibitor, Oncogene 26 (2007) 652–661. [49] Y.Q. Chen, Z.M. Duniec, B. Liu, W. Hagmann, X. Gao, K. Shimoji, L.J. Marnett, C.R. Johnson, K.V. Honn, Endogenous 12(S)-HETE production by tumor cells and its role in metastasis, Cancer Res. 54 (1994) 1574–1579. [50] C. Nabhan, B. Parsons, E.Z. Touloukian, W.M. Stadler, Novel approaches and future directions in castration-resistant prostate cancer, Ann. Oncol. 22 (2011) 1948–1957. [51] M. Bolla, L. Collette, L. Blank, P. Warde, J.B. Dubois, R.O. Mirimanoff, G. Storme, J. Bernier, A. Kuten, C. Sternberg, J. Mattelaer, T.J. Lopez, J.R. Pfeffer, C.C. Lino, A. Zurlo, M. Pierart, Long-term results with immediate androgen suppression and external irradiation in patients with locally advanced prostate cancer (an EORTC study): a phase III randomised trial, Lancet 360 (2002) 103–106. [52] J. Timar, E. Raso, B. Dome, L. Li, D. Grignon, D. Nie, K.V. Honn, W. Hagmann, Expression, subcellular localization and putative function of platelet-type 12lipoxygenase in human prostate cancer cell lines of different metastatic potential, Int. J. Cancer 87 (2000) 37–43. [53] A. Chumak, C. Thevenon, N. Gulaya, M. Guichardant, V. Margitich, D. Bazyka, A. Kovalenko, M. Lagarde, A.F. Prigent, Monohydroxylated fatty acid content in peripheral blood mononuclear cells and immune status of people at long times after the Chernobyl accident, Radiat. Res. (2001) 476–487. [54] J.M. Onoda, S.S. Kantak, M.P. Piechocki, W. Awad, R. Chea, B. Liu, K.V. Honn, Inhibition of radiation-enhanced expression of integrin and metastatic potential in B16 melanoma cells by a lipoxygenase inhibitor, Radiat. Res. 140 (1994) 410–418.