Tilapia (Oreochromis mossambicus) antimicrobial peptide, hepcidin 1–5, shows antitumor activity in cancer cells

Peptides 32 (2011) 342–352

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Tilapia (Oreochromis mossambicus) antimicrobial peptide, hepcidin 1–5, shows antitumor activity in cancer cells Wang-Ting Chang a,1 , Chieh-Yu Pan b,1 , Venugopal Rajanbabu b , Chun-Wen Cheng a , Jyh-Yih Chen b,∗ a b

Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Rd., Jiaushi, Ilan 262, Taiwan

a r t i c l e

i n f o

Article history: Received 19 June 2010 Received in revised form 6 November 2010 Accepted 8 November 2010 Available online 18 November 2010 Keywords: Antimicrobial peptide Hepcidin 1–5 Antitumor activity

a b s t r a c t The inhibitory function of tilapia hepcidin (TH)1–5, an antimicrobial peptide, was not examined in previous studies. In this study, we synthesized the TH1–5 peptide and tested TH1–5’s antitumor activity against several tumor cell lines. We show that TH1–5 inhibited the proliferation of tumor cells and reduced colony formation in a soft agar assay. Scanning electron microscopy and transmission electron microscopy showed that TH1–5 altered the membrane structure similar to the function of a lytic peptide. Acridine orange/ethidium bromide staining, a wound-healing assay, and a flow cytometric analysis showed that TH1–5 induced necrosis with high-concentration treatment and induced apoptosis with low-concentration treatment. Inflammation is known to be closely associated with the development of cancer. TH1–5 showing anti-inflammatory effects in a previous publication induced us to evaluate the anti-inflammatory effects in cancer cell lines through the expressions of immune-related genes after being treated with the TH1–5 peptide. However, real-time qualitative RT-PCR indicated that TH1–5 treatment induced downregulation of the expressions of interleukin (IL)-6, IL-8, IL-12, IL-15, interferon-␥, CTSG, caspase-7, and Bcl-2, and upregulation of IL-2 and CAPN5 in HeLa cells, and upregulation of IL-8 and CTSG in HT1080 cells. These results suggest that TH1–5 possibly induces an inflammatory response in HeLa cells, but not in HT1080 cells. Overall, these results indicate that TH1–5 possesses the potential to be a novel peptide for cancer therapy. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Antimicrobial peptides (AMPs) from fish and shrimp are naturalsource agents that were shown to exert anticancer activities [4,5,21]. AMPs from marine organisms such as fish and shrimp possess a positive charge and amphipathic properties like other AMPs from terrestrial animals. AMPs are able to disrupt negatively charged membranes by electrostatic interactions. Plasma membranes of cancer cells carry a net negative charge and have elevated expressions of phosphatidylserine [8,32], O-glycosylated mucins, sialilated gangliosides [23], and heparin sulfates [15]. In contrast, surfaces of normal mammalian cell membranes are mainly composed of neutral zwitterionic phospholipids and sterols [11], which suggests a reason why AMPs have a higher specificity for neoplastic cells [14,36]. Moreover, the anticancer properties of AMPs may involve selective lysis of cancer cell membranes that may extend to the permeation and swelling of mitochondria, which results in the release of cytochrome c and induction of apoptosis [6,22].

∗ Corresponding author. Tel.: +886 920802111; fax: +886 39871035. E-mail address: [email protected] (J.-Y. Chen). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.11.003

Hepcidin was reported as three hepcidin-like AMPs (named TH1–5, TH2–2, and TH2–3) from tilapia, Oreochromis mossambicus. The complete hepcidin complementary (c)DNA sequences of TH1–5, TH2–2, and TH2–3 are respectively composed of 478, 533, and 583 base pairs containing a translated region of 88, 86, and 91 amino acids. Synthesized TH1–5 and TH2–3 peptides showed antimicrobial activities against several bacteria, while the synthesized TH2–2 peptide did not [12]. In vivo studies of TH1–5 treatment showed that systemic administration of the TH1–5 peptide was effective against nervous necrosis virus infection in medaka, but that was not found with TH2–3 treatments [34]. Moreover, TH2–3 showed potent antitumor activity against human fibrosarcoma cells, suggesting a mechanism of cytotoxic action of TH2–3, and indicates that TH2–3 may be a promising chemotherapeutic agent [4]. We concluded from those observations described above that TH1–5 and TH2–3 may have different antitumor abilities similar to their antiviral abilities. A previous publication mentioned that the main regulator of plasma iron concentrations is hepcidin. An injection of hepcidin into mice resulted in a dramatic drop in serum iron within just 60 min [28]. An increased serum hepcidin25 level increases tumor expression of hepcidin messenger (m)RNA and is associated with renal cell carcinoma metastasis [13]. On the other hand, iron is a pivotal nutrient for cell growth and cell-cycle

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regulation, so iron chelators are potentially useful for treating cancer [19]. As mentioned above, those results warrant reconsideration of the role of iron in cancer and suggest that fine control of body iron stores by hepcidin might be a novel strategy for cancer therapy and prevention [31]. The major aim of this study was to evaluate the anticancer potential of the TH1–5 AMP. TH1–5 is active against both grampositive and -negative bacteria and is more potent than either TH2–2 or TH2–3. We were interested in the effects that TH1–5 exerted upon specific tumor cells in culture, and TH1–5 cytotoxicity was examined against several tumor cell lines. We further investigated the anticancer interactions of TH1–5 in HT1080 and HeLa cells. Our results indicated that TH1–5 selectively killed cancer cells as determined by a soft-agar assay and electron microscopic assay to document morphologic changes. Furthermore, the results of the necrosis inhibition test and real-time polymerase chain reaction (PCR) suggested that TH1–5 possesses cytolytic activity and may also have an immune-related function in cancer cells, specifically HeLa cells.

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Table 1 Primer sequences, and gene names and functions used in this paper. Gene

Primer sequence 

Function

JNK

F:5 -TGGACTTGGAGGAGAGAACCA R:5 -CGACGATGATGATGGATGCT

Apoptosis

NF-␬B

F:5 -CCAGACGCCCTTGCACTT R:5 -TTACCCAAGCGGTCCAGAAG

Inflammation

P53

F:5 -GGGTTAGTTTACAATCAGCCACATT

Tumor suppressor



R:5 -GGCCTTGAAGTTAGAGAAAATTCA Bcl-2

F:5 -ACCTGCACACCTGGATCCA R:5 -AGAGACAGCCAGGAGAAATCAAA

Anti-apoptosis

Caspase 7

F:5 -AATGAAGATTCAGTGGATGCTAAGC R:5 -GGATCGCATGGTGACATTTTT

Apoptosis

Calpain 5

F:5 -CAGGTCCTCTCAGAGGCAGATAC R:5 -CCTCTCCAGGGACCTTAACG

Necrosis

CathepsinG

F:5 -TCAAGTTTCCTGCCCTGGAT R:5 -CCTGTGTCCCCGAGAAGAAG

Necrosis

TGF␤1

F:5 -AACGAAATCTATGACAAGTTCAAGA

Control proliferation

2. Materials and methods



R:5 -AGAGCAACACGGGTTCAGGTA

2.1. Peptide

IL-2

F:5 -CTGCTGGATTTACAGATGATTTTGA R:5 -TGGCCTTCTTGGGCATGT

Inflammation

TH1–5 was synthesized with an amidated C-terminus (GIKCRFCCGCCTPGICGVCCRF-NH2 ) by GL Biochemistry (Shanghai, China) at >95% purity. Synthetic peptides were reconstituted in phosphate-buffered saline (PBS; pH 7.4) for the experiments.

IL-6

F:5 -CCTGACCCAACCACAAATGC R:5 -CCTTAAAGCTGCGCAGAATGA

Inflammation

IL-8

F:5 -CTTTCCACCCCAAATTTATCAAAG R:5 -AGAGCTCTCTTCCATCAGAAAGCT

Inflammation

IL-10

F:5 -CTGGGTTGCCAAGCCTTGT R:5 -AGTTCACATGCGCCTTGATG

Inflammation

IL-12

F:5 -CCTGGACCACCTCAGTTTGG R:5 -ACGGCCCTCAGCAGGTT

Inflammation

IL-13

F:5 -GCCTCATGGCGCTTTTGTT R:5 -AGCTCCCTGAGGGCTGTAGAG

Inflammation

IL-15

F:5 -TCGTATTGTATTGTAGGAGGCATTG R:5 -TCAAAGCCACGGTAAATCCTTAA

Inflammation

IFN-␥

F:5 -TGGCTTAATTCTCTCGGAAACG R:5 -TTTTACATATGGGTCCTGGCAGTA

Inflammation

Mcl-1

F:5 -CATGTTTGGCCTTCGGAGAA R:5 -GCATGTAGTTGGTGGCTGGAG

Anti-apoptosis

Bcl-xL

F:5 -GACTGGTTGAGCCCATCTCTA R:5 -GTGAGTGGACGGTCAGTGTCT

Anti-apoptosis

Caspase 3

F:5 -ATACCAGTGGAGGCCGACTTC R:5 -CAAAGCGACTGGATGAACCA

Apoptosis

GAPDH

F:5 -ACACCCACTCCTCCACCTTT

Housekeeping gene, as control

2.2. Cell culture The HeLa (human cervix adenocarcinoma cell), HepG2 (human hepatocellular carcinoma cell), HT1080 (human fibrosarcoma cell), COS-7 (Cercopithecus aethiops kidney cell), and WS-1 (human kidney cell) cell lines were obtained from American Type Culture Collection (ATCC; Rockville, MD). Cells were cultured using ATCCsuggested medium and conditions. 2.3. Cell viability assays To test the cell viability after treatment with TH1–5, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were run. Cells were plated at a density of 5000 cells/well in 96-well plates for 24 h, and treated with different concentrations of TH1–5 or PBS alone for 24 h. The other experimental condition was treatment with 100 ␮g/ml of TH1–5 for 24, 48, 72, and 96 h. This was followed by the addition of 100 ␮l of the tetrazolium compound, MTT, and 270 ␮l of fresh culture medium for 4 h at 37 ◦ C. The optical density was measured spectrophotometrically at 563 nm on a microtiter plate reader. Experiments were run in triplicate. Results are expressed as a percentage of the inhibition rate for viable cells, and values of the PBS-treated group were subtracted. 2.4. Colony formation in the soft-agar assay Five thousand HepG2, HeLa, and HT1080 cells were resuspended in 1 ml of a 0.5% agar solution containing cell culture medium (2× modified Eagle medium (MEM) and 20% fetal bovine serum (FBS)) at a final concentration of 100 ␮g/ml TH1–5, and layered on top of a 0.5% agar layer in 6-well plates. PBS was used as the control group without adding TH1–5 to the agar. Plates were incubated for 10 days at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Cell colonies were visualized following treatment with 0.5 ml piodonitrotetrazolium violet (Sigma, Steinheim, Germany) for 16 h and were observed by light microscopy. Colony growth in TH1–5-

R:5 -TAGCCAAATTCGTTGTCATACC

or PBS-treated wells was expressed as a count number. Colonies of >50 ␮m were counted 10 days after plating. These experiments were repeated three times, and at least five wells were replicated each time for each condition.

2.5. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) HepG2 (3 × 105 cells/well), HeLa (2 × 105 cells/well), and HT1080 (3 × 105 cells/well) cells were seeded in 6-well roundbottom trays and treated with TH1–5 (which was administered at 100 ␮g/ml) for 0, 24, 48, and 72 h or left untreated. Samples were analyzed and prepared according to a published report and then analyzed by SEM and TEM [25].

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Fig. 1. Effects of tilapia hepcidin (TH)1–5 on cell proliferation. Tumor cells were treated with different doses of TH1–5 for 24 h, followed by an MTT assay. Each concentration was repeated in eight wells of three independent experiments (upper panel). TH1–5 treatment affected cell viability in a dose-dependent fashion (lower panel). There were significant differences between treatment groups (Student’s t-test, p < 0.05).

2.6. Detection of cell variations due to TH1–5 by acridine orange (AO)/ethidium bromide (EtBr) staining HepG2 (3 × 105 cells/well), HeLa (2 × 105 cells/well), and HT1080 (3 × 105 cells/well) cells were grown to confluence in each well. For AO/EtBr staining, cultures were treated with 100 ␮g/ml of the synthesized TH1–5 peptide which was administered for 0, 24, 48, and 72 h and processed for staining using cold PBS containing 1 ␮g/ml EtBr and 1 ␮g/ml AO. After treatment, excess dye was removed from cells by washing with cold PBS. Observations were made, and pictures were taken under a fluorescence microscope. Experiments were performed in triplicate. 2.7. Wound-healing assay An in vitro wound-healing assay for HeLa and HT1080 cells was performed as previously described [4]. HT1080 and HeLa cells were cultured in cell culture medium and replaced with fresh medium containing TH1–5 (0, 50, and 100 ␮g/ml). Wound closure was monitored by microscopy for 0, 4, 24, and 48 h, and the width was calculated. 2.8. Flow cytometric analysis of the effects of TH1–5 on the cell cycle and cell damage HeLa (2 × 105 cells/well) and HT1080 (3 × 105 cells/well) cells were seeded in 6-well plates. After incubation to allow for cell attachment, cells were treated with TH1–5 (50 or 100 ␮g/ml) for 0, 24, 48, and 72 h. Then cells were harvested and fixed with 70% ice-cold ethanol at −20 ◦ C overnight. After fixation, the analysis followed our previous publication [21]. 2.9. Lactate dehydrogenase (LDH) release assay Cell membrane damage and cell death cause the release of LDH. Based on this concept, we treated cells with TH1–5. HT1080 (2 × 104 cells/well) and HeLa cells (2 × 104 cells/well) were cultured in 96-well plates at 37 ◦ C in the absence or presence of TH1–5 (50

or 100 ␮g/ml) for 24 and 48 h. This assay was performed according to the manufacturer’s instructions (Cytotoxicity Detection Kit plus, Roche, Germany). Absorbance values at 490 nm were determined photometrically with a 96-well plate reader. Leakage of enzymes was expressed as the percentage of LDH activity of the total LDH activity of cells. 2.10. Real-time reverse-transcription (RT)-PCR A real-time RT-PCR analysis was used to analyze gene expressions of JNK, nuclear factor (NF)-␬B, p53, Bcl-1, caspase-3, caspase-7, calpain-5, cathepsin G, tumor growth factor (TGF)␤1, interleukin (IL)-2, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, interferon (IFN)-␥, Mcl-1, Bcl-xL, and GAPDH by HT1080 cells and HeLa cells following treatment with TH1–5 (50 or 100 ␮g/ml) or with no TH1–5 as the control. The primers used are shown in Table 1. The SYBR® Green PCR Master Mix (ABI, USA) and specific primer pairs were used for selected genes, and the primer pair for GAPDH was used as the reference gene. A quantitative PCR was performed as previously described [4,5,21]. A real-time RT-PCR was performed in triplicate for each experimental group. 3. Results 3.1. Dose- and time-dependent effects of TH1–5 on inhibiting tumor cell growth The dose- and time-dependent effects of TH1–5 against cancer cell lines are shown in Fig. 1. TH1–5 exhibited higher activity against the HT1080 cell line between treatment concentrations of 30 and 60 ␮g/ml compared to the HeLa and HepG2 cell lines. No higher cytotoxic activity against the human kidney cell line was observed using the TH1–5 peptide (Supplementary Fig. 1). However, treatment of HeLa and HT1080 cells with TH1–5 at either 50 or 100 ␮g/ml after 48 h induced a significant increase in LDH release (Supplementary Fig. 2), indicating that treatment with TH1–5 disrupted the integrity of the plasma membrane. Therefore, the release of LDH into the cell culture media occurred due to disruption

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Fig. 2. Effects of tilapia hepcidin (TH)1–5 on cell membranes of HT1080, HeLa, and HepG2 tumor cells using scanning electron microscopy (a) and transmission electron microscopy (b). Untreated cells (0 h) showed a normal surface, while cells treated with TH1–5 (100 ␮g/ml) for 24, 48, and 72 h revealed disrupted cell membranes.

of plasma membrane integrity and cytosolic protein leakage in necrotic cells, which indicated that TH1–5 functions like a lytic peptide. 3.2. TH1–5-induced cell membrane variations in HepG2, HeLa, and HT1080 cells To investigate the mode of action underlying the cytotoxic activity of TH1–5, HepG2, HeLa, and HT1080 cells were incubated with the peptide for 24, 48, and 72 h for electron microscopic studies and AO/EtBr staining. Representative micrographs by SEM of untreated and TH1–5-treated tumor cells revealed that pore formation and cell swelling were evident in HepG2, HeLa, and HT1080 cell membranes after treatment with TH1–5 (Fig. 2a). TEM studies revealed undamaged nuclei and membranes in untreated cells. However, TH1–5-treated tumor cells showed condensed and hollow nuclei which caused leakage of the intracellular contents (Fig. 2b). There-

fore, TH1–5 induced drastic changes in the cellular morphology, and microscopic observations of tumor cells revealed that they were green, while nuclei containing condensed or fragmented chromatin appeared orange (Fig. 3). After 72 h of treatment with TH1–5, HeLa (Fig. 3a), HepG2 (Fig. 3b), and HT1080 (Fig. 3c) cells exhibited significant lytic activity, the cellular or nuclear membrane had been destroyed, and the nuclei showed an orange color in morphological observations indicating that TH1–5 may have induced lysis of tumor cell membranes. These results suggested that TH1–5 inhibited tumor cell growth by a lytic mode of action.

3.3. Effects of TH1–5 on clonal growth, wound-healing, and G0 /G1 phase arrest in tumor cells The soft-agar clonal growth assay showed that tumor cell growth decreased after treatment with TH1–5 (100 ␮g/ml) indicat-

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Fig. 3. Membrane disruption of HeLa (a), HepG2 (b), and HT1080 (c) cells after treatment with tilapia hepcidin (TH)1–5 (100 ␮g/ml) for 24, 48, and 72 h followed by incubation without (0 h) or with TH1–5. Cells showed a necrotic pattern. AO/EtBr staining produced green nuclei in live cells and orange nuclei in apoptotic cells which contained condensed or fragmented chromatin.

ing that TH1–5 exhibited potential antineoplastic activity in tumor cells (Fig. 4a). To examine the effect of TH1–5 on cell migration, we performed a wound-healing assay on tumor cells. After making the wound with a pipette tip, cells were cultured in the presence of 0, 50, or 100 ␮g/ml TH1–5, and pictures were taken with a digital camera. It was observed that TH1–5 effectively inhibited the migration of cells in dose- and time-dependent manners compared to untreated control HeLa (Fig. 4b) and HT1080 cells (Fig. 4c). Exposure to TH1–5 at the concentrations of 50 and 100 ␮g/ml resulted in a time- and concentration-dependent inhibition of cell growth. The effects of TH1–5 upon cell cycle profiles were analyzed. Exposure to 50 and 100 ␮g/ml TH1–5 at 24–72 h caused an increase in the subG1 -phase population compared to control HeLa (Fig. 5) and HT1080 cells (Fig. 5). This result demonstrated that TH1–5 induced growth-inhibitory effects in subG1 -phase arrest. 3.4. Effects of TH1–5 on induction of immune-related gene expressions in tumor cells To further characterize expressions of immune-related genes induced by TH1–5, we determined gene expressions in HeLa (Fig. 6)

and HT1080 cells (Fig. 7) after TH1–5 treatment for 24, 48, and 72 h. In addition, TH1–5 treatment showed time-dependent effects in downregulating the expressions of IL-6, IL-8, IL-12, IL-15, IFN␥, CTSG, caspase-7, and Bcl-2, and upregulating IL-2 and CAPN5 in HeLa cells (Fig. 6) and upregulating IL-8 and CTSG in HT1080 cells (Fig. 7). These results suggest that TH1–5 possibly induces an inflammatory response in HeLa cells, but not in HT1080 cells. 4. Discussion In our previous study, we demonstrated that TH2–3, an AMP cloned from fish, possesses a cell-penetrating ability and kills cancer cells [4]. In this study, we demonstrated that the TH gene named TH1–5 also possesses a cell-penetrating ability and kills cancer cells. As stated before, TH1–5 has broad and strong microbicidal activity [12]. The interesting observation in this study is that TH1–5 has more-potent lytic activity toward cancer cells than toward non-cancer cells. In fact, its antitumor activity was unlike that of chemotherapeutic agents, because they cannot discriminate between normal and cancer cells [26]. These results support our assumption that TH1–5 may act on and disintegrate cell mem-

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Fig. 4. Clonal assay for the HT1080, HepG2, and HeLa cell lines treated with 100 ␮g/ml tilapia hepcidin (TH)1–5 indicated reduced colony formation compared to the control (not treated with TH1–5). (a) Data are from five separate experiments. Effects of a TH1–5 peptide on HeLa (b) and HT1080 (c) cell migration. Cell migration was analyzed by a wound-healing assay.

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Fig. 5. Tilapia hepcidin (TH)1–5 treatment led to the accumulation of cells in the subG1 phase. HeLa (a) and HT1080 (b) cells were treated with TH1–5 for different times, then analyzed by FACS for the cell cycle analysis.

branes by its ability to bind and permeate phospholipid membranes such as the 9-mer peptide of LTX-302 [1]. Its dramatic effect of disintegrating cell membranes means that it will be difficult for cancer cells to develop resistance to AMPs. SEM and TEM were utilized to directly observe the morphologic effects of TH1–5 on HT1080, HeLa, and HepG2 tumor cells.

Tumor cells treated with TH1–5 showed potent disruption of their cell membranes, which is thought to have been due to pore formation with short-term treatment. However, after epinecidin-1 (cloned from Epinephelus coioides) was used to treat cancer cells, it caused pore formation in a carpet effect identified by SEM technology [21], suggesting that similar results might be possi-

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Fig. 6. Quantification of transcript levels by comparative real-time RT-PCR. RNA from HeLa cells was co-treated with 0 (control), 50, or 100 ␮g/ml tilapia hepcidin (TH)1–5. The group not treated with the peptide served as the control. Samples were collected after treatment for 24, 48, and 72 h. Transcript abundance, normalized to GAPDH expression, is expressed as the relative expression and graphed on a rational scale. Each bar represents the mean value from three determinations with the standard error (SE). Data (mean ± SE) with different letters significantly differ (p < 0.05) between treatments.

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Fig. 7. Quantification of transcript levels by comparative real-time RT-PCR. RNA from HT1080 cells was co-treated with 0 (control), 50, or 100 ␮g/ml tilapia hepcidin (TH)1–5. The control group was not treated with the peptide. Samples were collected after treatment for 24, 48, and 72 h. Transcript abundance, normalized to GAPDH expression, is presented as the relative expression and is graphed on a rational scale. Each bar represents the mean value from three determinations with the standard error (SE). Data (mean ± SE) with different letters significantly differ (p < 0.05) between treatments.

ble with TH1–5. In addition to disrupting the surface membrane of tumor cells, which induces cytolysis/necrosis, certain subgroups of AMPs are able to cause disruption of mitochondrial membranes which subsequently leads to activation of apoptotic pathways [30]. Compared to pore-forming AMPs, intracellular-targeted AMPs are translocated across plasma membranes and do not act in a lytic manner, but only distort the structure. To visualize the association

of TH1–5 with cancer cells, we incubated cancer cells with the FITClabeled TH1–5 peptide and then observed its cellular localization by confocal microscopy (Supplementary Fig. 3). The FITC-labeled TH1–5 peptide penetrated the cell membrane and accumulated inside cells (COS-7, HT-1080, and HeLa cells). This finding indicates that the targeted site of action of the TH1–5 peptide may be the membrane.

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TH1–5 was able to selectively induce blockage of cell cycle progression in cancer cells according to our flow cytometric assay. However, technical limitations prevented the direct demonstration of free peptides in targeted cells. Therefore, the precise mechanism of how cells are killed remains unknown at present, although clear evidence of blockage of cell cycle progression was obtained by demonstrating PI staining. In agreement with this observation, treatment with A12S4 led to mitotic arrest and induction of caspase-dependent apoptosis in U937 cells [3]. Actually, TH1–5 caused cell cycle arrest at the G0 /G1 phase and triggered apoptosis (Supplementary Fig. 4) as displayed by the externalization of annexin V-targeted phosphatidylserine and accumulation of a sub-G1 peak. Thus, the same important findings were reported for tocotrienols, which inhibited growth and induced apoptosis in human HeLa cells through a cell cycle signaling pathway at a concentration of 3 ␮M. They induced alphaT3 downregulation of the expressions of cyclin D3, p16, and CDK6, while having no effect on cyclin D1, p15, p21, p27, or CDK4 expressions [35]. Indeed, many cancer cells are enriched with O-glycosylated mucines, which are high-molecular-weight glycoproteins composed of oligosaccharides attached to serine or threonine groups [27]. After accumulation on the cell surface, the peptides bind to and permeate zwitterionic membranes, an action that is probably essential for their anticancer activity [17,24]. Thus, the membranolyic mode of action of AMPs is not limited to cell membranes, and may also be extended to mitochondria, which causes the release of cytochrome c and induces apoptosis [20,22]. Our TEM and SEM data support the membranolyic mode of action of TH1–5. Cathelicidins are known to have chemoattractive properties, and angiogenic peptides were suggested to influence tumor growth [7,9,16]. The antiangiogenic activity of TH1–5 was observed in HeLa cells. The angiogenic activities are related to infection and wound healing because they promote inflammation and induce chemokine or cytokine expressions [2]. We therefore concluded that TH1–5 is capable of causing cell death either by apoptosis at low doses or by necrosis at high doses. Necrosis will induce proinflammatory cytokine expressions. Since inflammation is now well known to have a close relationship with the onset and development of cancer, we expect that TH1–5 which possesses anti-inflammatory activity will in turn participate in anticancer activity. In order to test this hypothesis, the influence of TH1–5 treatment on cancer cell-associated inflammatory gene expressions was evaluated by examining the expressions of various immune-related genes. However, in vitro studies revealed that TH1–5 was highly active against HeLa cells. The efficacy was selective since the peptide displayed 2–5-fold lower activities against WS-1 cells. Our results regarding TH1–5 treatment showed time-dependent effects in downregulating the expressions of IL-6, IL-8, IL-12, IL-15, IFN-␥, CTSG, caspase-7, and Bcl-2, and upregulating IL-2 and CAPN5 in HeLa cells and upregulating IL-8 and CTSG in HT1080 cells. These results indicated that TH1–5 increased levels of IL-8 gene expression in HeLa cells; thus, an inflammatory process was triggered with TH1–5 treatment. The exception was primarily the HeLa cell-specific production of IL6, IL-8, IL-12, IL-15, and IFN-␥, which were variably constitutively produced in detectable to high concentrations in comparison to HT1080 cells treated with TH1–5. Expressions of genes encoding proinflammatory cytokines, including TNF-␣ and IL-6 were markedly enhanced during treatment of HeLa cells, which suggested a possible increase in phagocytosis. Interestingly, primary oral and vaginal epithelial cells constitutively produced considerable levels of IL-6, IL-12, and IFN-␥ in response to a Candida albicans infection [10,29]. However, data from this study suggest that as with Th1 and Th2 cytokines, cancer cells and not epithelial cells are responsible for the presence of TH1–5. Chemokines are considered critical for the migration of cells to sites of pathogenic insults such as TH1–5 treatment of cervical cancer. On the other hand, down-

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regulated gene expression of caspase-7 (the protease involved in apoptosis and inflammation [18]) suggested that after treating HeLa cell with TH1–5, apoptosis and inflammation may have been induced. Nevertheless, certain substrates such as cochaperone p23 are prone to proteolytic processing by caspase-7 or caspase-3 after TH1–5 treatment [33]. From these results, we concluded that TH1–5 has the potential for development as a new type of anticancer agent. The current in vitro studies suggested that TH2–3 is less toxic to normal cells than other AMPs such as epinecidin-1 and may be a more-appropriate candidate for development as an antitumor agent. Acknowledgments This work was supported by a grant from the Marine Research Station (Jiaushi, Ilan), Institute of Cellular and Organismic Biology, Academia Sinica to Dr. Jyh-Yih Chen. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.peptides.2010.11.003. References [1] Berge G, Eliassen LT, Camilio KA, Bartnes K, Sveinbjørnsson B, Rekdal O. Therapeutic vaccination against a murine lymphoma by intratumoral injection of a cationic anticancer peptide. Cancer Immunol Immunother 2010;59: 1285–94. [2] Büchau AS, Morizane S, Trowbridge J, Schauber J, Kotol P, Bui JD, et al. The host defense peptide cathelicidin is required for NK cell-mediated suppression of tumor growth. J Immunol 2010;184:369–78. [3] Cerella C, Scherer C, Cristofanon S, Henry E, Anwar A, Busch C, et al. Cell cycle arrest in early mitosis and induction of caspase-dependent apoptosis in U937 cells by diallyltetrasulfide (Al2S4). Apoptosis 2009;14: 641–54. [4] Chen JY, Lin WJ, Lin TL. A fish antimicrobial peptide, tilapia hepcidin TH2–3, shows potent antitumor activity against human fibrosarcoma cells. Peptides 2009;30:1636–42. [5] Chen JY, Lin WJ, Wu JL, Her GM, Hui CF. Epinecidin-1 peptide induces apoptosis which enhances antitumor effects in human leukemia U937 cells. Peptides 2009;30:2365–73. [6] Cruciani RA, Barker JL, Zasloff M, Chen HC, Colamonici O. Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation. Proc Natl Acad Sci USA 1991;88:3792–6. [7] Yang D, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J, et al. LL37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 2000;192: 1069–74. ´ [8] Dobrzynska I, Szachowicz-Petelska B, Sulkowski S, Figaszewski Z. Changes in electric charge and phospholipids composition in human colorectal cancer cells. Mol Cell Biochem 2005;276:113–9. [9] Elssner A, Duncan M, Gavrilin M, Wewers MD. A novel P2X7 receptor activator, the human cathelicidin-derived peptide LL37, induces IL-1 beta processing and release. J Immunol 2004;172:4987–94. [10] Hedges SR, Agace WW, Svanborg C. Epithelial cytokine responses and mucosal cytokine networks. Trends Microbiol 1995;3:266–70. [11] Hoskin DW, Ramamoorthy A. Studies on anticancer activities of antimicrobial peptides. Biochim Biophys Acta 2008;1778:357–75. [12] Huang PH, Chen JY, Kuo CM. Three different hepcidins from tilapia, Oreochromis mossambicus: analysis of their expressions and biological functions. Mol Immunol 2007;44:1922–34. [13] Kamai T, Tomosugi N, Abe H, Arai K, Yoshida K. Increased serum hepcidin-25 level and increased tumor expression of hepcidin mRNA are associated with metastasis of renal cell carcinoma. BMC Cancer 2009;9: 270. [14] Kawsar HI, Ghosh SK, Hirsch SA, Koon HB, Weinberg A, Jin G. Expression of human beta-defensin-2 in intratumoral vascular endothelium and in endothelial cells induced by transforming growth factor beta. Peptides 2010;31:195–201. [15] Kleeff J, Ishiwata T, Friess H, Büchler MW, Israel MA, Korc M. The helix-loophelix protein Id2 is overexpressed in human pancreatic cancer. Cancer Res 1998;58:3769–72. [16] Koczulla R, von Degenfeld G, Kupatt C, Krötz F, Zahler S, Gloe T, et al. An angiogenic role for the human peptide antibiotic LL-37/hCAP-18. J Clin Invest 2003;111:1665–72.

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[17] Ladokhin AS, White SH. Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin. J Mol Biol 1999;285:1363– 9. [18] Lamkanfi M, Kanneganti TD. Caspase-7: a protease involved in apoptosis and inflammation. Int J Biochem Cell Biol 2010;42:21–4. [19] Le NT, Richardson DR. The role of iron in cell cycle progression and the proliferation of neoplastic cells. Biochim Biophys Acta 2002;1603:31– 46. [20] Li SH, Lam S, Cheng AL, Li XJ. Intranuclear huntingtin increases the expression of caspase-1 and induces apoptosis. Hum Mol Genet 2000;9:2859– 67. [21] Lin MC, Lin SB, Chen JC, Hui CF, Chen JY. Shrimp anti-lipopolysaccharide factor peptide enhances the antitumor activity of cisplatin in vitro and inhibits HeLa cells growth in nude mice. Peptides 2010;31:1019–25. [22] Mai JC, Mi Z, Kim SH, Ng B, Robbins PD. A proapoptotic peptide for the treatment of solid tumors. Cancer Res 2001;61:7709–12. [23] Müthing J, Meisen I, Kniep B, Haier J, Senninger N, Neumann U, et al. Tumor-associated CD75s gangliosides and CD75s-bearing glycoproteins with Neu5Acalpha2-6Galbeta1-4GlcNAc-residues are receptors for the anticancer drug rViscumin. FASEB J 2005;19:103–5. [24] Oren Z, Shai Y. Selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure–function study. Biochemistry 1997;36:1826– 35. [25] Pan CY, Chen JY, Lin TL, Lin CH. In vitro activities of three synthetic peptides derived from epinecidin-1 and an anti-lipopolysaccharide factor against Propionibacterium acnes, Candida albicans, and Trichomonas vaginalis. Peptides 2009;30:1058–68. [26] Papo N, Shai Y. Can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes? Peptides 2003;24:1693–703.

[27] Ropolo M, Daga A, Griffero F, Foresta M, Casartelli G, Zunino A, et al. Comparative analysis of DNA repair in stem and nonstem glioma cell cultures. Mol Cancer Res 2009;7:383–92. [28] Rivera S, Liu L, Nemeth E, Gabayan V, Sorensen OE, Ganz T. Hepcidin excess induces the sequestration of iron and exacerbates tumor-associated anemia. Blood 2005;105:1797–802. [29] Steele C, Fidel Jr PL. Cytokine and chemokine production by human oral and vaginal epithelial cells in response to Candida albicans. Infect Immun 2002;70:577–83. [30] Suttmann H, Retz M, Paulsen F, Harder J, Zwergel U, Kamradt J, et al. Antimicrobial peptides of the Cecropin-family show potent antitumor activity against bladder cancer cells. BMC Urol 2008;8(March):5. [31] Toyokuni S. Role of iron in carcinogenesis: cancer as a ferrotoxic disease. Cancer Sci 2009;100:9–16. [32] Utsugi T, Nii A, Fan D, Pak CC, Denkins Y, van Hoogevest P, et al. Comparative efficacy of liposomes containing synthetic bacterial cell wall analogues for tumoricidal activation of monocytes and macrophages. Cancer Immunol Immunother 1991;33:285–92. [33] Walsh JG, Cullen SP, Sheridan C, Lüthi AU, Gerner C, Martin SJ. Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc Natl Acad Sci USA 2008;105:12815–9. [34] Wang YD, Kung CW, Chen JY. Antiviral activity by fish antimicrobial peptides of epinecidin-1 and hepcidin 1–5 against nervous necrosis virus in medaka. Peptides 2010;31:1026–33. [35] Wu SJ, Ng LT. Tocotrienols inhibited growth and induced apoptosis in human HeLa cells through the cell cycle signaling pathway. Integr Cancer Ther 2010;9:66–72. [36] Wu WK, Sung JJ, To KF, Yu L, Li HT, Li ZJ, et al. The host defense peptide LL37 activates the tumor-suppressing bone morphogenetic protein signaling via inhibition of proteasome in gastric cancer cells. J Cell Physiol 2010;223:178–86.