Photo-activated 5-hydroxyindole-3-acetic acid induces apoptosis of prostate and bladder cancer cells

Journal of Photochemistry and Photobiology B: Biology 103 (2011) 50–56

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Photo-activated 5-hydroxyindole-3-acetic acid induces apoptosis of prostate and bladder cancer cells Yun-Mi Jeong a, Hailan Li a, Su Yeon Kim a, Woo-Jae Park a, Hye-Young Yun a, Kwang Jin Baek a, Nyoun Soo Kwon a, Ji Hoon Jeong b, Soon Chul Myung c,d, Dong-Seok Kim a,d,⇑ a

Department of Biochemistry, Chung-Ang University College of Medicine, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Korea Department of Pharmacology, Chung-Ang University College of Medicine, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Korea Department of Urology, Chung-Ang University College of Medicine, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Korea d Research Institute for Translational System Biomics, Chung-Ang University College of Medicine, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 7 October 2010 Received in revised form 21 December 2010 Accepted 10 January 2011 Available online 21 January 2011 Keywords: 5-Hydroxyindole-3-acetic acid Ultraviolet B Prostate cancer Bladder cancer Apoptosis

a b s t r a c t 5-Hydroxyindole-3-acetic acid (5-HIAA), an indole derivative, is the main metabolite of serotonin in the human body. We determined whether or not ultraviolet B (UVB)-activated 5-HIAA (5-HIAAUVB) affects the viability of human prostate (LnCaP and PC-3) and bladder cancer cells (TCCSUP). While 5-HIAA alone had no cytotoxic effect at <1 mM, 5-HIAAUVB induced LnCaP, PC-3, and TCCSUP cell death in a time- and dose-dependent manner. Cell cycle analysis showed that 5-HIAAUVB markedly increased the sub-G0/G1 phase and resulted in cell cycle disruption. To elucidate the death mechanism by 5-HIAAUVB, we examined the signal transduction pathways related to apoptosis using Western blot analysis. 5-HIAAUVB led to phosphorylation of stress-activated signaling proteins, such as c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK). Furthermore, 5-HIAAUVB activated caspase-8, -9, and -3 and cleaved poly(ADP-ribose) polymerase (PARP), which are indicators of apoptosis. From these findings, the present study demonstrated that 5-HIAAUVB induces apoptotic cell death of prostate and bladder cancer cells via stress-mediated signaling and apoptotic pathways. Therefore, we suggest that 5-HIAA might be used as a new photosensitizer for photodynamic cancer therapy. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Genitourinary cancer consists of prostate, bladder, renal, and testicular cancers [1]. Prostate and bladder cancers have emerged as major public health problems in industrialized countries [2–4]. A major cause of morbidity and mortality in prostate and bladder cancers is the high incidence of metastases and the high rate of local recurrence after current therapies, including surgery, biological therapy, chemotherapy, or radiotherapy [3,4]. In the early stage, prostate and bladder cancers are highly curable by current therapies. Despite advances in their treatment, prostate and bladder cancers are difficult to treat with current therapies because both cancers metastasize to the head, neck and cervical lymph nodes [2–4]. Furthermore, prostate and bladder cancers have an increased risk of recurrence after initial treatment [3,5]. Nevertheless, successful management of patients with prostate or bladder cancers are dependent on current therapies. ⇑ Corresponding author at: Department of Biochemistry, Chung-Ang University College of Medicine, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Korea. Tel./fax: +82 2 820 5768. E-mail address: [email protected] (D.-S. Kim). 1011-1344/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2011.01.011

Photodynamic therapy (PDT) is a promising local treatment modality based on the selective activation of a photosensitizer by irradiation with the light of a specific wavelength in malignant tissues [6]. Recently, PDT has become a popular treatment because of the development of new photosensitizers and lasers [6]. Malignant and abnormal cells are destroyed by a light-activated photosensitizer [7]. In this process, singlet oxygen (1O2) is generated by energy transfer from the exposure of a photosensitizer to light. Singlet oxygen and other reactive oxygen species (ROS) induce diverse cellular responses, such as oxidative DNA damage and apoptosis [7]. PDT has merits because of selective targeting, minimal invasiveness, and reduced severe side effects [7]. There have been many approaches to develop new photosensitizers, including 5-aminolevulinic acid [8], hexaminolevulinate [9], protoporphyrin IX [10], and indole-3-acetic acid (IAA) [11]. For example, IAA is a plant growth hormone that plays a key role in plant cell division, elongation, and differentiation [12,13]. Previously, we reported that IAA alone exhibits no cytotoxic effects. IAA is activated by horseradish peroxidase (HRP) or ultraviolet radiation B (UVB) exposure and induces apoptosis in several cancer cells, suggesting that IAA is a new potential photosensitizer for the PDT [13]. 5-hydroxyindole-3-acetic acid (5-HIAA) is the most

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abundant end product of both central and peripheral serotonin metabolism [14]. Because the chemical structure of 5-HIAA and IAA is very similar, we investigated whether or not 5-HIAA could be activated by light. In the present study, we examined the effects of the 5-HIAA and 5-HIAAUVB on the death of androgen-sensitive LnCaP human prostate cancer cells, androgen-insensitive PC-3 human prostate cancer cells, and TCCSUP human urinary bladder carcinoma cells. We also investigated the effects of 5-HIAAUVB on stress-activated signaling pathways, as well as on apoptotic pathways. 2. Materials and methods 2.1. Materials 5-HIAA, Trolox, and 2,7-dichlorofluorescin diacetate (DCFH-DA) were obtained from Sigma (St. Louis, MO, USA). Antibodies that recognize phospho-specific JNK (CST-9251), total JNK (CST-9258), phospho-specific p38 (CST-9211), and total p38 (CST-9212) were obtained from Cell Signaling (Danvers, MA, USA). Antibodies against caspase-9 (sc-8355), caspase-8 (sc-7890), and caspase-3 (sc-7272) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-PARP (556,362) antibody was obtained from BD Pharmingen (San Diego, CA, USA). 2.2. Cell cultures LnCaP cells were obtained from the Korean Cell Line Bank (Seoul, Korea). PC-3 and TCCSUP cells were obtained from the ATCC (Rockville, MD, USA). The LnCaP cells were grown in RPMI-1640 supplemented with 10% FBS, 50 lg/ml of streptomycin, and 50 lg/ml of penicillin at 37 °C in 5% CO2. The PC-3 cells were grown in Nutrient Mixture F-12 Ham Kaighn’s Modification medium (Ham’s F-12 K) supplemented with 10% FBS, 50 lg/ml of streptomycin, and 50 lg/ml of penicillin at 37 °C in 5% CO2. The TCCSUP cells were grown in minimum essential medium (MEM) supplemented with 10% FBS, 50 lg/ml of streptomycin, and 50 lg/ml of penicillin at 37 °C in 5% CO2. 2.3. Cell viability Cells were observed under a phase contrast microscope (Olympus Optical Co., Tokyo, Japan) and photographed using a DCM300 digital camera for a microscope (Scopetek, Inc., Hangzhou, China), which was supported by ScopePhoto software (Scopetek, Inc.). Cell viability was assessed using the crystal violet staining assay [15]. After treating cells with 5-HIAA for 24 h, the culture medium was removed. Cells were stained with 0.1% crystal violet in 10% ethanol for 5 min at room temperature, then rinsed four times with distilled water. The crystal violet retained by the adherent cells was extracted with 95% ethanol, and the absorbance was determined in lysates at 590 nm using an ELISA reader (VERSAMax; Molecular Devices, Sunnyvale, CA, USA).

2.5. Cell cycle analysis To determine 5-HIAAUVB-induced cell death, PC-3 cells were exposed to UVB (100 mJ/cm2)-activated 5-HIAA (0.5 mM). After 24 h, cells were trypsinized, adjusted to 5  105–1  106 cells/tube, washed with ice cold phosphate-buffered saline (PBS), and re-suspended in 2 ml of ethanol. After incubation at 4 °C for 1 h, the ethanol was removed, and 100 ll of ribonuclease solution (10 mg/ml) was added to each test tube. The tubes were then re-incubated at room temperature for 30 min, and 500 ll of analysis solution (37 mM EDTA and 0.1% Triton X-100 in PBS) and 100 ll of propidium iodide solution (400 lg/ml) were added. Samples were stored in the dark at 4 °C and analyzed by flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA, USA). The proportion of cells in the subG0/G1 phases with low DNA content was regarded as apoptotic cells. 2.6. Western blotting Cells were grown in 60 mm culture dishes, starved of serum for 24 h, and treated with the test substances at the indicated time points. Cell lysates were prepared in M-PER mammalian protein reagent (Pierce, Rockford, IL, USA) containing a complete protease inhibitor mixture (Roche, Mannheim, Germany). Samples were separated on 12% SDS–polyacrylamide gels and were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% dried milk in PBS containing 0.4% Tween 20. The blots were incubated with the appropriate primary antibodies at a dilution of 1:1000. Membrane-bound primary antibodies were detected using secondary antibodies conjugated with HRP and chemiluminescent substrate (Pierce). The images of the blotted membranes were obtained using a LAS-1000 lumino-image analyzer (Fuji Film, Tokyo, Japan). 2.7. Detection of free radical Free radical formation was determined using DCFH-DA, which is oxidized by free radicals to dichlorofluorescein [12]. Briefly, to activate DCFH-DA, 350 ll of a 1 mM stock solution of DCFH-DA in ethanol was mixed with 1.75 ml of 0.01 N NaOH and allowed to stand for 20 min before adding 17.9 ml of 25 mM sodium phosphate buffer (pH 7.2). Reaction mixtures contained activated DCFH-DA solution and 5-HIAA (0, 0.1, 0.5, and 1 mM), and were irradiated with UVB (100 mJ/cm2). Absorbance was determined at room temperature at 490 nm using an ELISA reader (VERSAMax; Molecular Devices). 2.8. Statistics The statistical significance of the differences between groups was assessed by analysis of variance (ANOVA), followed by Student’s t-test. P values <0.05 were considered significant.

2.4. Treatment of 5-HIAAUVB The source of UVB was from Sankyo Denki Company Ltd. (Hiratsuka, Kanagawa, Japan). To determine the cytotoxicity of 5-HIAAUVB, LnCaP, PC-3, and TCCSUP cells (5  104 cells per well) were prepared on 6-well plates. For treatment with 5-HIAAUVB, 5-HIAA was irradiated with UVB (100 or 200 mJ/cm2), then 5-HIAAUVB (0.5 mM or 1 mM) was added immediately to 6-well plates containing each cell line. At the indicated time points, cell viabilities were measured by the crystal violet staining assay.

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Fig. 1. The structure of 5-HIAA.

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3. Results 3.1. 5-HIAA alone is not cytotoxic in prostate and bladder cancer cells The chemical structure of 5-HIAA is shown in Fig. 1. To examine the cytotoxicity of 5-HIAA on cell viability, LnCaP, PC-3, and TCCSUP cells were treated with 5-HIAA at 0–1 mM for 24 h. Cell viability was assessed by the crystal violet staining. 5-HIAA did not exhibit any cytotoxic effect on LnCaP, PC-3, and TCCSUP cells (data not shown). 3.2. 5-HIAAUVB induces cell death in prostate and bladder cancer cells We next examined whether or not 5-HIAAUVB affected the cell viability of prostate and bladder cancer. After 24 h of 5-HIAAUVB

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treatment, cell viability was measured using the crystal violet assay. Phase contrast microscopic images showed cell morphology of 5-HIAAUVB-treated LnCaP, PC-3, and TCCSUP cells. We observed that many cells were detached from the culture dish after 5HIAAUVB treatment, indicating that the cells were not viable (Fig. 2A). Consistent with the morphologic observations, 5-HIAAUVB treatment caused cell death of LnCaP, PC-3, and TCCSUP cells (Fig. 2B–D). To further confirm the cytotoxic effect of 5-HIAAUVB on cancer cells, PC-3 and TCCSUP cells were treated with 5HIAAUVB in a time-course experiment. After 8 h of 5-HIAAUVB treatment, PC-3 and TCCSUP cells showed a reduction in cell viability (Fig. 3A and B). Previously, we reported that UVB-activated IAA (IAAUVB) produces reactive oxygen species (ROS) [12]. Thus, we measured free radical production by 5-HIAAUVB using the DCFH-DA assay. As

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Fig. 2. Effects of 5-HIAAUVB on cell viability. (A) Morphology of LnCaP, PC-3, and TCCSUP cells after 5-HIAA or 5-HIAAUVB treatment. Phase contrast photographs were taken using a digital video camera. (B) After serum starvation, LnCaP (left panel), PC-3 (middle panel), and TCCSUP (right panel) cells were treated with UVB (100 or 200 mJ/cm2)activated 5-HIAA (0.5 or 1 mM), as described in Section 2. Cell viabilities were measured using crystal violet assay after 24 h of treatment. Data represent the means ± SD of triplicate assays expressed as percentages of the control.

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Fig. 3. Effects of 5-HIAAUVB on cell viability in a time-course experiment. After serum starvation, PC-3 (left panel) and TCCSUP (right panel) cells were treated with UVB (100 mJ/cm2)-activated 5-HIAA (0.5 mM). Cell viabilities were measured using crystal violet assays at the indicated time points. Data represent the means ± SD of triplicate assays expressed as percentages of the control.

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Fig. 4. Free radical production by 5-HIAAUVB. (A) The formation of free radicals was determined using DCFH-DA, which is oxidized by free radicals to DCF, as described in Section 2. Increasing amounts of 5-HIAA were added at a UVB dose of 100 mJ/cm2. IAA was used as a positive control. (B) To further determine the involvement of free radicals on 5-HIAAUVB–induced cell death, PC-3 (left panel) and TCCSUP (right panel) cells were treated with 5-HIAAUVB in the absence or presence of Trolox (40 lg/ml) after serum starvation. After 24 h, a crystal violet assay was performed. Data represent the means ± SD of triplicate assays expressed as percentages of the control.

shown in Fig. 4A, 5-HIAAUVB, unlike IAAUVB, did not generate free radicals. We further determined whether ROS is involved in 5HIAAUVB-induced cell death. The results showed that pre-treatment with Trolox, a water-soluble vitamin E, did not block the cytotoxic effects of 5-HIAAUVB on PC-3 and TCCSUP cells (Fig. 4B and C).

3.3. 5-HIAAUVB increases the sub-G0/G1 phase in cell cycle distribution To determine whether or not 5-HIAAUVB-induced cell death is caused by apoptosis, cell cycle assay was performed with 5-HIAAUVB-treated cells. The cell cycle assay was assessed using a flow cytometry as described in Section 2. As shown in Fig. 5,

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Con

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Fig. 5. Effects of 5-HIAAUVB on cell cycle. PC-3 cells were treated with UVB (100 mJ/cm2)-activated 5-HIAA (0.5 mM). After 24 h, cell cycle analysis of the cells was performed by flow cytometry, as described in Section 2; con, untreated control.

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Fig. 6. Effects of 5-HIAAUVB on the JNK, p38 MAPK, and apoptotic pathways. (A) After serum starvation, PC-3 cells were treated with UVB (100 mJ/cm2)-activated 5-HIAA (0.5 mM) for 4 h. Cells were harvested and subjected to Western blot analysis with antibodies against phospho-specific JNK or p38 MAPK. Equal protein loadings were confirmed by Western blotting with phosphorylation-independent JNK or p38 MAPK antibodies, respectively. (B) To determine the caspase activation and PARP cleavage, PC3 cells were treated with UVB (100 mJ/cm2)-activated 5-HIAA (0.5 mM) for 24 h. Cells were harvested. Western blot analysis of caspase-9, -8, and -3, and PARP was performed, as described in Section 2.

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5-HIAA alone or UVB-activated vehicle did not affect the cell cycle. However, 5-HIAAUVB treatment resulted in a significant increase in the sub-G0/G1 phase in cell cycle distribution (Fig. 5). 3.4. 5-HIAAUVB-induced cell death involves the stress-activated and apoptotic signaling pathways Although the crystal violet assay demonstrated that 5-HIAAUVB induced cell death, it did not distinguish between apoptosis and necrosis. Previous studies have reported that c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK) activation is required for apoptosis [12,16]. To determine whether 5-HIAAUVB activates the JNK and/or p38 MAPK pathway in PC-3 cells, JNK and p38 MAPK phosphorylation was detected by Western blotting. As shown in Fig. 6A, 5-HIAA alone did not stimulate phosphorylation of JNK and p38 MAPK. After 5-HIAAUVB treatment for 4 h, JNK and p38 MAPK was activated markedly. To explore the mechanisms of 5-HIAAUVB-induced cell death, the activation of caspase-9, -8, and -3 and PARP cleavage were assessed using Western blotting. It is well-known that caspase-8, -9, and -3 play essential roles in apoptotic cell death. Caspases are known to become active when cleaved into fragments [17]. We used anti-caspase-8 and anti-caspase-9 antibodies directed against the precursor forms and anti-caspase-3 antibody directed against the cleaved form. After 24 h, a reduction in the precursor form of caspase-8 and -9 was observed in 5-HIAAUVB-treated PC-3 cells, indicating that caspase-9 and -8 were activated (Fig. 6B). We also detected a clear increase in caspase-3 activation (Fig. 6B). Consistent with the activation of caspases, the 116-kDa full-length PARP was also cleaved to the apoptotic 85-kDa fragment after 5-HIAAUVB treatment (Fig. 6B). 4. Discussion In the present study, we demonstrated that 5-HIAA is a potent photosensitizer for the treatment of prostate and bladder cancers using PDT. At present, hematoporphyrin derivatives and their analogues are most frequently used for PDT of prostate and bladder cancers [18]. However, these photosensitizers are generally expensive and sometimes induce side effects to normal tissues. To develop a new photosensitizer, the following two basic qualifications are required: (i) low cytotoxicity to normal cells and (ii) superior efficacy and selective ability against cancer cells [19]. Prostate and bladder cancer arise as a result of complex problems in endocrine signaling pathways. However, most hormonedependent prostate cancer becomes hormone-independent prostate cancer after 1–3 years [20,21]. It is reported that 70% of bladder cancer patients present superficial tumors, and 30% of patients present muscle-invasive disease associated with a high risk of death from distant metastases [22]. We showed that 5-HIAA alone showed no cytotoxic effects. When 5-HIAA was activated by UVB, 5-HIAAUVB induced apoptosis of LnCaP, PC-3, and TCCSUP cells (Fig. 2). From these results, it is proposed that 5-HIAA showed anti-cancer effects in androgen-dependent or -independent prostate and bladder cancer cells. Therefore, these results serve as compelling evidence that 5-HIAA could be considered a new possible photosensitizer for the selective cancer therapy. Bladder cancer would be easily accessible to 5-HIAA and a light source for PDT. Although prostate cancer would pose serious obstacles to clinical application, 5-HIAA-based techniques could be developed for intraoperative photodynamic diagnosis of prostate cancer. In the present study, the DCF assay showed that 5-HIAAUVB did not produce ROS. In addition, Trolox did not inhibit the apoptosis of 5-HIAAUVB-treated cells (Fig. 4). These results were unexpected, because our previous study showed that IAAUVB generates hydro-

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gen peroxide [12]. Thus, it remains to be determined how 5-HIAA is changed by light and further investigations are needed for the relationship between the activated structure of 5-HIAA and 5HIAAUVB-induced cell death. It is accepted that the activation of the apoptotic pathway leads to the phosphorylation of the stress-activated kinase pathways and caspase activation [16,23]. In particular, the death-receptor and mitochondrial pathways are two key points in the apoptotic processes [23]. For activation of the death-receptor pathway, Fas ligand or TNF-a contacts the cell surface death receptors, which induces the activation of caspase-8 [23]. To activate the mitochondrial pathway, a variety of death stimuli induces the release of cytochrome c, which binds to Apaf-1 and activates caspase-9 [24]. Western blotting revealed that 5-HIAAUVB led to the activation of p38 and JNK (Fig. 6). In addition, 5-HIAAUVB induced the activation of caspase-8, -9, and -3 as well as PARP cleavage (Fig. 6). Therefore, we suggest that 5-HIAAUVB-induced cell death is a result of the apoptotic pathway activation. In summary, the present study demonstrated that 5-HIAAUVB induces apoptosis in prostate and bladder cancer cells through the stress signaling and apoptotic pathways. Consequently, this finding provides a possible candidate for a novel photosensitizer of PDT. 5. Abbreviations DCFH-DA 2,7-dichlorofluorescein diacetate 5-HIAA 5-hydroxyindole-3-acetic acid 5-HIAAUVB UVB-activated 5-hydroxyindole-3-acetic acid HRP horseradish peroxidase IAA indole-3-acetic acid JNK c-Jun N-terminal kinase MAPK mitogen-activated protein kinase PDT photodynamic therapy PARP poly(ADP-ribose) polymerase ROS reactive oxygen species UVB ultraviolet B

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