IbACP, a sixteen-amino-acid peptide isolated from Ipomoea batatas leaves, induces carcinoma cell apoptosis

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IbACP, a sixteen-amino-acid peptide isolated from Ipomoea batatas leaves, induces carcinoma cell apoptosis Vincent H.-S. Chang a , Diane H.-A. Yang b , Hsin-Hung Lin c , Gregory Pearce d , Clarence A. Ryan d,1 , Yu-Chi Chen b,∗ a

Program for Translation Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung, Taiwan c Biodiversity Research Center, Academia Sinica, Taipei, Taiwan d Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, United States b

a r t i c l e

i n f o

Article history: Received 26 December 2012 Received in revised form 8 February 2013 Accepted 8 February 2013 Available online xxx Keywords: Sweet potato Anti-cancer peptide Alkalinization assay Apoptosis

a b s t r a c t A 16-amino-acid peptide was isolated from the leaves of sweet potato. The peptide caused a rapid alkalinization response in tomato suspension culture media, a characteristic of defense peptides in plants. No post-translational modification was observed on the peptide according to MALDI-MS analysis. We have named the peptide Ipomoea batatas anti-cancer peptide (IbACP). IbACP also was shown with the ability to dose-dependently inhibit Panc-1, a pancreatic cancer line, cell proliferation. The morphological observations of the Panc-1 cells by phase contrast microscopy showed significant changes after treatment with IbACP. Moreover, caspase-3 and PARP [poly(ADP-ribose) polymerase] were activated by IbACP treatment, followed by cell death. An increase in the levels of cleaved caspase-3 and -9 was also detected by an immunoblot assay after treatment with IbACP. In addition, genomic DNA fragmentation and decreased cellular proliferation were induced when IbACP was supplied to the Panc-1 cells, further demonstrating its biological relevance. The combined data indicates that IbACP peptide may have an important role in the regulation of cellular proliferation by inducing and promoting apoptosis through the mitochondrial apoptotic pathway. This report also showed that IbACP peptide contains potent anti-cancer effects and may play an important role in herbal medicine development. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Numerous plant peptides have been identified with biological activities such as anti-cancer cell proliferation and resistance to herbivores or pathogens. In addition to a simple protein structure and with low antigenicity, these peptides are easily absorbed and adaptable to a variety of routes of administration [22,39,40]. The search for natural components from plants with potent anti-tumor activity and low toxicity has thus become an important and active research area [20]. To this date, several small peptides from Cycas revoluta seeds and soybean were derived using newly developed purification processes and were identified and characterized as anti-cancer peptides [21,38]. A 9-amino acid peptide purified from C. revoluta seeds showed effects against human epidermoid cancer and colon carcinoma cells [21]. A 43-amino acid peptide named Lunasin was derived from soybean 2S albumin seed protein with anti-cancer and anti-inflammatory activities [38]. Except linear

∗ Corresponding author. Tel.: +886 7 717 2930x7319; fax: +886 7 605 1353. E-mail address: [email protected] (Y.-C. Chen). 1 Deceased October 7, 2007.

peptides, cyclic peptides (cyclopeptides) also have been identified with potential therapeutic effects [6,8,42]. Several small peptide products, which are potential sources of anti-cancer agents are processed from precursor proteins through natural homeostatic mechanisms in both animals and plants. Another important bioactivity of plant small peptides is in its function of self-defense from attackers. A number of defenserelated peptides have been isolated and demonstrated with functions in activating resistance to both herbivores and pathogens in plants. Systemin, isolated from tomato, was the first peptide signal in this category. Systemin is 18-amino acids in length and is processed from the C-terminal of its precursor protein, prosystemin [31]. Hydroxyproline-rich systemin (HypSys), a small defenserelated signaling glycopeptide found within the Solanaceae family of plants including tomato, tobacco, petunia, and nightshade, were also found in sweet potato (Ipomoea batatas) of the Convolvulaceae family [5]. All of the HypSys glycopeptides were isolated based on the ‘alkalinization assay’, a quick bioassay method that exploits the increase in pH of the media of suspension cells when a bioactive peptide binds to its receptor. HypSys glycopeptides induce defense genes and are part of an arsenal of plant compounds designed to avert herbivore attack [27]. A 23-amino acid peptide from

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Please cite this article in press as: Chang VH-S, et al. IbACP, a sixteen-amino-acid peptide isolated from Ipomoea batatas leaves, induces carcinoma cell apoptosis. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.02.005

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Arabidopsis, AtPep1, also was isolated utilizing the alkalinization assay [12] as screening method. Like systemin, AtPep1 is processed from the C-terminal of its precursor protein. AtPep1 induces defense genes, such as pdf1.2, and pr-1 and has been found to protect the plant from pathogen attack [13]. The AtPep family of peptides is not restricted to Arabidopsis as homologs were found in other species as well, including rice and maize. ZmPep1, encoded by the ZmPROPEP1 gene from maize (Zea mays), induces the expression of genes encoding the defense proteins endochitinase A, PR-4, PRs and SerPIN [11]. In addition to the HypSys and AtPep families of defense peptides, two other peptide signals were recently isolated from soybean (Glycine max) leaves, a 12-amino acids peptide, GmSubPep [30,32] and an 8-amino acid peptide, GmPep914 [43]. Both peptides are capable of inducing the expression of defense-related genes, such as Cyp93A1, Chib-1b, PDR12, and achs. Anthocyanins [1], red, blue and purple pigments from sweet potatoes at high concentrations all showed anti-cancer property. [24,25]. Furthermore, sweet potato leaves contain polyphenolics [19] which exhibits physiological functions of radical scavenging, anti-mutagenic, anti-cancer, anti-diabetes, and anti-bacterial activities either in vitro or in vivo [7,10,17]. Sweet potato leaves have been demonstrated to suppress the oxidation of low density lipoprotein (LDL) in vitro in human subjects [23]. It is well known that the oxidative modification of lipids by reactive oxygen species plays a role in mammalian aging and diseases related to cardiovascular system, neurodegeneration and inflammation. Although some studies have been conducted on I. batatas peptides, none were identified with anti-cancer activity so far. In our effort of isolating peptides from sweet potato leaves using the alkalinization assay [5], an alkalinizing peptide that had no homology to the HypSys peptides or to any other previously isolated defense peptide was identified. Most importantly, this peptide possesses anti-proliferation capabilities in carcinoma cells. Using serial purification and cell assay experiments, we demonstrated that a linear 16-amino-acid peptide, which we called I. batatas anti-cancer peptide (IbACP), significantly inhibits cell proliferation of carcinoma cells by inducing DNA fragmentation via a mitochondria-dependent pathway of activating apoptotic markers, and eventually reduces the survival rate of cancer cells. This is the first report of anti-cancer peptides identified in sweet potato leaves using the screening technique of alkalinization assay.

2. Materials and methods 2.1. Peptide isolation The sweet potato (I. batatas cf. Georgia Jet) plants were grown under greenhouse conditions. Six weeks after planting, the aerial parts of plants were sprayed with a 1.25 mM methyl jasmonate solution in 0.1% Triton X-100. The treated leaves and stems were collected after 15 h, frozen in liquid N2 and stored at −20 ◦ C until use. The frozen material (1.2 kg of wet weight) was homogenized in a blender for 5 min in 2.5 liters of 1% TFA (trifluoroacetic acid), and squeezed through four layers of cheesecloth and two layers of Miracloth (Calbiochem). The filtered solution was centrifuged at 10,000 × g for 60 min. 10 N NaOH was slowly added to the supernatant liquid with stirring to adjust the solution to a pH of 4.3. After re-centrifuging the mixture at 10,000 × g, the clear supernatant was collected and readjusted to a pH of 2.2 with 1 N HCl. After centrifugation at 10,000 × g for 20 min, the clear supernatant solution was applied to a 40-␮m 3 × 25-cm, C18 reversed-phase flash column (Bondesil, Varian Analytical Instruments, Walnut Creek, CA) equilibrated with 0.1% TFA/H2 O. An elution was performed at 8 p.s.i. with compressed nitrogen gas. The bound material was eluted with 40% methanol, 0.1% TFA and dried by lyophilization. The dried powder

(12 g) was dissolved in 30 ml of 0.1% TFA/H2 O and centrifuged at 10,000 × g for 15 min to clarify the solution. The supernatant was added to a Sephadex G-25 column (5.5-cm × 35-cm) equilibrated with 0.1% TFA/H2 O, and the eluent, collected in 8 ml fractions, was assayed for alkalinization activity in tomato suspension cells. In the assay, 10 ␮l of each collected fractions was used for the assay with 1 ml of the cells. The active fractions were pooled and lyophilized. The dried powder was dissolved in TFA/H2 O and centrifuged at 10,000 × g for 15 min to clarify. The pellet was discarded, and the clear supernatant was filtered through a Millex-HV PVDF membrane filter (Millipore, Bedford, MA) and loaded onto a reversed-phase C18-HPLC column (218TP1022, 10-␮m, 2.2-cm ID × 25-cm, Vydac, Hesperia, CA) with a flow rate of 4 ml/min in three sequential runs. After 5 min, an elution gradient was applied from 0 to 40% acetonitrile/0.1%TFA over 90 min. The absorbance was monitored at 225 nm. The alkalinizing activity was determined using 10 ␮l aliquots from each 1 min fraction, using 1 ml of the tomato cells per assay. The fractions from three separations containing the major activity peaks were combined and lyophilized. The dried material was dissolved in 5 mM potassium phosphate (pH 3) in 25% acetonitrile buffer and loaded onto a polySULFOETHYL AspartamideTM strong cation exchange column (5-␮m, 4.6-mm ID × 200-mm, The Nest Group, Southborough, MA). After 2 min, a gradient from 0 to 100% with the elution buffer (5 mM potassium phosphate, 0.5 M potassium chloride, pH 3, in 25% acetonitrile) was performed over 90 min. The absorbance was monitored at 225 nm. Ten-␮l aliquots from each 1-min fraction were combined with 1 ml of the tomato cells to determine the alkalinizing activity. The lyophilization of the active fractions yielded a dried powder that was dissolved in 10 mM potassium phosphate buffer (pH 6) for further purification. To further purify the active peptide, the sample was loaded onto an analytical reversed-phase C18-HPLC column (218TP54, 5-␮m, 4.6-mm ID × 250-mm, Vydac, Hesperia, CA), with a 90-min gradient from 0 to 40% with 10 mM potassium phosphate (pH 6) in 50% acetonitrile as the elution solvent at a flow rate of 1 ml/min. The absorbance was monitored at 214 nm. One-minute fractions were collected, and 10-␮l aliquots were used with 1 ml of the tomato cells to determinate the bioactivity. The collected sample was injected onto a narrow bore reversed-phase C18-HPLC column (218TP52, 5-␮m, 2.1-mm ID × 250-mm, Vydac, Hesperia, CA) equilibrated with 0.1%TFA/H2 O at a flow rate of 0.25 ml/min. Two min after sample injection, a 90-min gradient from 0 to 40% methanol/0.1%TFA was performed, and 0.25 ml fractions were collected at 1-min intervals. The absorbance was monitored at 214 nm, and the alkalinizing activity was assayed after adding 2-␮l of each fraction to 1 ml of the cells. The active fractions were pooled, and the methanol was removed by vacuum evaporation. The pooled sample was applied to a polySULFOETHYL AspartamideTM column for re-chromatography as described above, except that the 90-min gradient was performed from 0 to 75% of the elution buffer. The lyophilization of the active fractions, determined by the alkalinization assay, yielded a driedsalt powder that was dissolved in 0.1% TFA/H2 O. As a final purification step, a narrow bore reversed-phase C18-HPLC column (218TP52, 5-␮m, 2.1-mm ID × 250-mm, Vydac, Hesperia, CA) equilibrated with 0.1%TFA/H2 O at a flow rate of 0.25 ml/min was used to separate the peptide mixture and remove salts. The purification conditions were the same as above, except that the 90-min gradient was performed from 0 to 30% acetonitrile/0.1% TFA. 2.2. Alkalinization assay Tomato suspension cells were maintained in the dark, using Murashige and Skoog medium as described [36]. The medium was

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adjusted to pH 5.6 with KOH. Forty ml of cells were grown in a 125 ml flask with shaking at 160 rpm, and 5 ml of 7-day-old cell cultures were transferred into 35 ml of media. The cultures were utilized 4–7 days after transfer. One milliliter of cells was aliquoted into each well of a 24-well cell culture plate and equilibrated on an orbital shaker at 160 rpm for 1 h. Aliquots of HPLC fractions were added to the cells, and the pH was recorded after 15 or 30 min. 2.3. Peptide sequence analysis and synthesis Peptide N-terminal sequencing was performed by Edman chemistry on an Applied Biosystems (Foster City, CA) Procise model 492 protein sequencer. MALDI-MS were performed on an Applied Biosystems model 4800 tandem time-of-fight mass spectrometer equipped with the 200-Hz neodymium-doped yttrium aluminum garnet laser. The peptide sample was mixed with an equal volume of matrix solution (␣-cyano-4-hydroxycinnamic acid, 6 mg/ml in 50:50 acetonitrile: 0.25% TFA/H2 O) and air dried. Peptide synthesis was performed by N-(9-fluorenyl) methoxycarbonyl chemistry by solid-phase techniques using an Applied Biosystems model 431 synthesizer and the peptide was further purified by reversed-phase C18-HPLC. 2.4. Cell culture and exposure to IbACP peptides The PANC-1 human pancreatic adenocarcinoma cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37 ◦ C in a 5% CO2 humidified atmosphere. The lung carcinoma cell line H1299 was used in this study. The human carcinoma cell lines were purchased from the American type culture collection (ATCC) and cultured with an optimal medium following manipulation guides. The human epithelial lung cell line A549 was grown in RPMI-1640 supplemented with 10% fetal calf serum (FCS) and antibiotics. The IbACP peptides were dispersed in DMSO (with a final concentration of 1%) by pipetting for 10 min and were added to serum-free cell culture medium and analyzed by three different assays: the MTT cell viability assay, DNA fragmentation and Western blot analysis. 2.5. Cell death assays The cells were exposed to different concentrations of IbACP for 24 h. The cell viability was quantified using MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole]. MTT is soluble in ethanol (20 mg/ml) and stable for at least 6 months when stored at 0 ◦ C. The samples were supplemented with 5 mg/ml MTT (15 ␮l/well). After 4 h, 40 ␮l of a stop solution (10% SDS, 0.01 N HCl) was added. The plates were shaken for 24 h and then examined with a microplate reader at the test wavelength of 570 nm with a reference filter of 620 nm. To visualize apoptotic chromosomal condensation, the cells were stained with the chromatin dye Hoechst-33258 (2.5 ␮g/␮1) for 15 min and observed by fluorescent microscopy (Axioskop 20, Zeiss). The viable cells displayed a normal nuclear size, while the apoptotic cells displayed nuclear condensations.

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buffer [5% fat-free milk in TBST (150 mM NaCl, 10 mM Tris pH 8.0, 0/1% Tween 20) buffer]. After blocking, an anti-caspase 3 (1:3000, #9661, Cell Signaling) primary antibody, an anti-PARP antibody (1:2000, ab6079, Abcam), and a mouse anti-beta-actin antibody (1:5000, NB600-532, Novus Biologicals) were added and the membranes were incubated for 12 h. An HRP-conjugated goat anti-rabbit IgG and an HRP-conjugated rabbit anti-mouse IgG (Abcam) were used as the secondary antibodies. The protein bands were detected by ECL autoradiography. The images were detected using an image system (LAS 3000; Fujifilm) and quantified using multi gauge software V. 3.0 (Fujifilm). 2.7. DNA fragmentation assay The purified Panc-1 cells (1 × 106 ), obtained by centrifugation after transfection for 48 h, were lysed in 0.5 ml of a solution containing 20 mM Tris (pH 7.4), 0.4 mM EDTA, and 0.25% Triton X-100. After 15 min of incubation at room temperature, nuclei were removed by centrifugation (RCF = 16,000). The supernatant was transferred to a new tube, and the nuclear DNA was precipitated overnight at −20 ◦ C using 55 ␮1 of 5 M NaCl and 550 ␮1 of isopropanol. After centrifugation for 10 min, the pellet was washed with 70% ethanol and resuspended in 20 ␮1 of a solution containing 10 mM Tris (pH 8.0), 1 mM EDTA, and 0.1 mg/ml RNase. The DNA preparations were separated by 1.6% Tris borate EDTA agarose gel electrophoresis and visualized using ethidium bromide staining. 2.8. Cell immunocytochemistry The Panc-1 cells were grown on 1 mm glass coverslips for 36 h in DMEM containing 10% FBS. The cells were induced by IbACP (1 nM). After fixation (methanol/acetone), the glass coverslips were airdried and incubated with anti-cytochrome c antibodies (ab13575) for 2 h at 37 ◦ C in a humidified chamber. The secondary antibody, a high-fluorescence FITC-labeled goat anti-mouse antibody, was added and incubated for 30 min at room temperature in the dark. The coverslips were then rinsed with PBS for 3 min and incubated for 1 min with PBS containing DAPI (4 -6-diamidino-2phenylindole, 1 mg/ml). Finally, the coverslips were mounted and analyzed using a Zeiss LMS 410 inverted laser scanning microscope. 2.9. Cell proliferation assays The Panc-1 cancer cells (5 × 103 cells per well) were seeded in 96-well plates in 100 ␮l of DMEM supplemented with 10% FBS and cultured for 24 h. The cells were treated for 24 h with serial IbACP concentrations. The cell proliferation was determined by the Alamar Blue assay (invitrogen) following the manufacturer’s manual. The plates were read at dual wavelengths (570 and 595 nm) in an ELISA plate reader. Each treatment was repeated 6 times. 3. Results and discussion 3.1. Isolation and characterization of IbACP from sweet potato leaves

2.6. Western blotting analysis The Western blot method developed by Burnette et al. and Towbin et al. [2,41] was modified to detect specific proteins in the given sample of tissue extract. The protein samples (50 ␮g) were boiled in a buffer (10% SDS, 0.25 M Tris-base, 30% glycerol, pH 8.8), separated on a 12.5% SDS-PAGE gel, and then electrophoretically transferred to a PVDF membrane (NENTM Life Science) for 15 min at 120 V, 1 h at 160 V, and followed by 1 h at 0.4 V, all at 4 ◦ C. The membranes were then incubated for 1 h at room temperature with a blocking

Systemin and hydroxyproline-rich glycopeptide systemin (HypSys), peptides isolated from several species of the Solanaceae family, have been shown to be plant defense signals [28,29,31]. To expand the knowledge of defense signaling peptides outside of the Solanaceae family, sweet potato (I. batatas) which belongs to the Convolvulaceae family was chosen because of the close phylogenetic proximity to Solanaceae. The protein extracts were prepared from sweet potato leaves using the same protocols utilized for the isolation and purification of the HypSys peptides from

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Fig. 1. The isolation of IbACP from the crude extracts of sweet potato leaves. (A) Upper panel, the crude extract was eluted from a preparative reversed-phase C-18 flash chromatography column in 0.1% TFA/water with a 90-min acetonitrile gradient as described in Section 2. Lower panel, ten microliters from each fraction was added to 1 ml of tomato suspension culture to assay for alkalinization activity. (B) A final narrow-bore HPLC purification of the activity peak from the lower panel of A, eluting at 45–47 min labeled with a star, was pooled and further purified by HPLC as described in Section 2. Fractions were assayed as in (A). The active peak is identified with an arrow. (C) Aliquots of the purified peptide (10 ␮l) from the final purification in B were assayed for alkalinization activity. The change in pH was recorded after 15 and 30 min. The data represent the average of three separate experiments.

the Solanaceae family. An alkalinization assay, utilizing plant suspension cultured cells, had been developed to detect the defense signaling peptides by their ability to elevate the pH of the suspension culture medium [29]. IbHypSys, a hydroxyproline-rich glycopeptide defense signal, was isolated from the sweet potato by utilizing the alkalinization assay [5]. In addition to IbHypSys, another peak found within the sweet potato HPLC column fractions had the ability to increase the pH of plant suspension cell media. To detect the pH changes, ten microliter aliquots from each HPLC-elution fraction were added to 1 ml of the tomato suspension cultured cells. This combination had been found to be suitable for the recognition of the HypSys peptides from other species. Multiple weak peaks with increased alkalinizing activity were detected (Fig. 1A). Fractions eluted between 45 and 47 min (with a star label) were pooled and further purified by HPLC as described in Section 2. A strong alkalinizing response from this fraction was detected throughout a series of HPLC purification steps, with the final activity peak from a narrow-bore C18 HPLC, as shown in Fig. 1B. Two microliter aliquots of the purified fraction were added to 1 ml of the tomato suspension cultured cells, and a strong alkalinizing response was induced with the tomato cell media after 30 min (Fig. 1C). A MALDI-MS analysis revealed a mass peak at 1597.8 Da. Nterminal sequencing analysis of the peak component revealed a16-amino-acid peptide: AASTPVGGGRRLDRGQ (Fig. 2A). A search of the GenBank database identified a very similar and sometimes identical sequence in close proximity to the N-terminus of many proteins including the thaumatin-like proteins (PepTLP) in pepper (GenBank accession number AAK97184) [16], osmotin in petunia (GenBank accession number AAK55411) [15], the osmotin-like protein (OSM) in potato (GenBank accession number AAU95237) [4], PR-5x in tomato (GenBank accession number AAM23272) [34], the pathogenesis-related protein PR P23 in tomato (GenBank accession number CAA50059) [35], and a protein from black nightshade (GenBank accession number AAL87640) [3] (Table 1). This result suggests that IbACP peptide maybe the fragments of a larger plant protein, such as thaumatin-like proteins, osmotin, or some other PR proteins and its release from the precursor occurred when the precursor protein was exposed to proteolytic enzymes. Plant genes encoding pathogenesis-related (PR) proteins are induced by

responding to various abiotic stresses or infection by pathogens. The PR-5 proteins, defined as thaumatin-like proteins because of their sequence similarities to thaumatin from Thaumatococcus daniellii [33], are divided into three subgroups: acidic (PR-S), basic (Osmotin), and neutral proteins (Osmotin-Like-Protein, OLP). The pepper thaumatin-like protein, which belongs to the PR-5 protein group, was used as a molecular marker to monitor disease resistance, ripening, and sugar accumulation in fruits [16]. The peptide, IbACP, we isolated from the sweet potato leaf extracts is the first example of a small proteolyzed fragment from the thaumatin-like protein family with distinct bioactivity. In plants, inceptin and GmSupPep are other examples of small fragments of larger, functional proteins whose proteolyzed fragments, however, carry totally different functions [30,37]. In viewing of these reports, however, we cannot exclude the possibility that IbACP is a simple fragmentation of a larger protein due to hard extraction applied to leaves. For further characterization, IbACP peptide was synthesized and applied to all subsequent studies. Molecular mass was confirmed to be the same as the native peptide by MALDI-MS (data not shown). The synthetic peptide exhibited a half-maximal alkalinizing activity at concentrations between 1 and 10 ␮M (Fig. 2B). Compared with peptide hormones, such as systemin, HypSys, or AtPep, the half-maximal alkalinizing activity of IbACP was 1000–10,000-fold lower, suggesting that IbACP may have lower affinity for its receptor or have a completely different mechanism inducing the alkalinization of the medium of suspension cultures. 3.2. IbACP inhibited cellular proliferation of Panc-1 cells via induction of cell apoptosis There is an increasing interest in the use of herbal and plant extracts for treating human diseases, including cancer. Sweet potato is consumed as a fresh vegetable in many parts of the world such as the islands of the Pacific Ocean and Asian and African countries. The bioactive compounds contained within the sweet potato leaves have been demonstrated to play a role in promoting health by improving immune functions, reducing oxidative stress and free radical damage, reducing cardiovascular disease risk, and suppressing cancer cell growth. The results from Kurata

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Fig. 2. Amino acid sequence and bioactivity of IbACP. (A) A MALDI MS analysis of the activity peak from Fig. 1B. (B) The increase in pH of the suspension cell medium in response to increasing concentrations of the synthetic IbACP peptide was recorded 30 min after the addition of the peptide. The change in pH was compared with distilled H2 O control of an equal volume. The data represent the average of three separate experiments. Table 1 Comparison of the amino acids sequence of isolated IbACP with similar sequences from different plants. IbACP peptide was aligned with similar sequences from pepper (thaumatin-like proteins; PepTLP), petunia (osmotin), potato (OSM), tomato (PR-5x and PR P23), and black nightshade (osmotin-like protein precursor). Amino acid differences are shown in red. Protein IbACP PepTLP Osmotin OSM PR-5x PR P23 OLPP

Protein sequences

Accession

1

AASTPVGGGRRLDRGQ16

36

51

AASTPVGGGRRLDRGQ

36

51

36

51

36

51

AASTPIGGGRRLDRGQ AASTPIGGGRRLDRGQ AASTPIGGGRRLDRGQ

23

AASTPIGGGRRLDRGQ38

36

AASTPIGGGRRLDRGQ

51

Source

Ipomoea batatas AAK97184

Capsicum annuum

AAK55411

Petunia hybrida

AAU95237

Solanum phureja

AAM23272

Solanum lycopersicum

CAA50059

Solanum lycopersicum

AAL87640

Solanum nigrum

OLPP: osmotin-like protein precursor.

et al. indicated that 3,4,5-tri-O-caffeoylquinic acid (3,4,5-triCQA), purified from the sweet potato leaves, may have a potential role in cancer prevention. Moreover, data suggests that polyphenolics, especially 3,4,5-triCQA, from the sweet potato leaves may suppress not only the mutation of normal cells but also the growth of cancer

cells by apoptosis induction [19]. The objective of this study is to investigate the effect of a purified bioactive sweet potato peptide, IbACP, on the suppression of cancer cell growth. Serial doses of IbACP were added to the Panc-1 cells and then cell viability from various groups was observed by the MTT test. IbACP inhibited

Fig. 3. Effects of IbACP concentration on the viability of Panc-1 cells. (A) The cell growth was measured after treatment with IbACP peptides for 48 h by the MTT assay. The survival rate of cells grown in the control medium with serum but without IbACP was normalized to 100%. The data represent the average of five separate experiments. Error bars indicate standard error of the mean. (B) The morphological changes. The Panc-1 cells were treated with 100 ␮M IbACP or DMSO (control group; con) for 48 h, followed by a morphological assessment. Images were obtained using a phase-contrast microscope at 40× magnification (Zeiss).

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Fig. 4. Subcellular distribution of both caspase-3 and PARP by immunocytochemistry. The cells were cultured with 10−6 and 10−9 M IbACP for 24 h and incubated with anti-activated caspase-3 (A) or anti-PARP (B) antibodies. The nuclear staining was detected with DAPI.

about 40% of Panc-1 cell proliferation in a dose-dependent manner (Fig. 3A). The same anti-proliferation effect (20–30% of inhibition) of IbACP also can be detected, although a slightly less, in two other cancer cell lines, H1299 (lung cancer) and A 549 (epidermoid cancer) (data not shown). Although the inhibitory activity was less in comparison to other peptides, the advantage of IbACP is that it is a nature peptide from plant and shows no significant cytotoxicity to normal mice (C57/B6 strain) pancreas cells (data not shown). It indicates that IbACP caused a specific deleterious activity to cancer cell survival but did not show much effect in normal mammalian cell killing. To explain the mechanism of cell growth suppression by IbACP, morphological changes to the Panc-1 cells were examined with phase contrast microscopy (Fig. 3B). Compared with the control cells, the peptide-treated cells showed cellular blebbing, membrane shrinkage, lossing of membrane asymmetry and attachment and chromatin condensation. 3.3. IbACP induced cellular apoptosis through a mitochondria-dependent pathway One of the earliest observed features of apoptosis is the induction of a series of cytosolic proteases such as mitochondrial caspase-3, a protease whose activation is crucial during the early stages of apoptosis. The Panc-1 cells were incubated with IbACP for 24 h, and then incubated with either anti-caspase-3 (Fig. 4A) or anti-PARP antibodies (Fig. 4B) and finally stained with DAPI. Nucleus granulation (blue color) is one of the morphological changes observed during cellular apoptosis, and these results suggest that IbACP induced apoptosis in the Panc-1 cells. In addition, both caspase-3 and PARP were detected when IbACP was added to the Panc-1 cells. As shown in Fig. 4, caspase-3 (activated form) and PARP were activated after treatment with IbACP for 24 h. The PARPs are a family of proteins involved in a number of cellular processes such as DNA repair and programmed cell death. Activated PARP can deplete cellular ATP in an attempt to repair the damaged DNA. In addition, sequential activation of caspases plays

a central role in the execution-phase of cell apoptosis. Caspase3, a member of the cysteine-aspartic acid protease family, exists as an inactive proenzyme (uncleaved caspase-3) that undergoes proteolytic processing at conserved aspartic residues to produce two subunits that dimerize to form an active enzyme (cleaved caspase-3). Caspase-3 is processed and activated by caspases-8, -9, and -10, and the protein itself cleaves and activates caspases6 and -7. To know whether IbACP induces cell apoptosis, we attempted to detect the level of cleaved caspase-3. The expression level of cleaved caspase-3 was assayed by Western blot analysis with a specific antibody that detected cleaved caspase-3 but not uncleaved caspase-3. In the same treatment, caspase-9 was also observed. As shown in Fig. 5A, adding IbACP to serum-free media increased the expression of cleaved caspases-3 and -9, indicating that IbACP may promote cancer cell apoptosis. To confirm the finding that IbACP induced apoptosis via a mitochondria-dependent process, the cellular localization of cytochrome c was examined by confocal immunofluoresence microscopy in Panc-1 cells. In the control cells, cytochrome c displayed punctate cytoplasmic staining, which is in agreement with its localization in the mitochondria (Fig. 5B). After induction of apoptosis by IbACP, the cells exhibited diffused cytoplasmic cytochrome c staining, consistent with the translocation of cytochrome c from the mitochondria to the cytoplasm. 3.4. IbACP further induced cellular DNA fragmentation and decreased the cell proliferation It has been shown that introduction of activated caspase-3 results in DNA fragmentation and cellular blebbing in various cells [14]. Therefore, we investigated whether DNA fragmentation is increased by IbACP. Systemin (Sys), an 18-amino-acid nontargeting peptide with alkalinization bioactivity in plants, was synthesized and used as a negative control. The DNA fragmentation assay was adopted to confirm that IbACP not only induced cell apoptosis but also activated mitochondria-dependent genome

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Fig. 5. IbACP induced cellular apoptosis. (A) IbACP induced cleavage of caspases-3 and -9. The Panc-1 cells were treated with (1 pM–100 ␮M) or without IbACP (Con). The immunoblot membranes of whole cell extracts were analyzed for cleavage of caspases-3 and -9 after 24 h. Immunoblot membranes were re-probed with ␤-actin antibody, demonstrating equal loading. Two independent experiments showed similar results. (B) The cytochrome c release was visualized by confocal immunofluorescence microscopy. The cells were grown for 24 h on glass coverslips and incubated with IbACP (1 nM), fixed 4 h after treatment and immuno-stained with anti-cytochrome c monoclonal antibodies (ab13575, abcam).

fragmentation. Genomic DNA fragmentation was detected when the cells were treated with serial doses of IbACP for 48 h (Fig. 6A). A DNA agarose gel electrophoresis demonstrated that the Panc-1 cells displayed a typical DNA ladder pattern for apoptosis after treatment with 10 nM–100 ␮M IbACP peptides in the cultured media. Finally, we used alamarBlue, a redox indicator that yields a colorimetric

change and a fluorescent signal in response to a metabolic activity, to verify the effects of cell proliferation after IbACP treatment with the incorporation of tritiated thymidine (Fig. 6B). Based on these results, we conclude that IbACP is a cell proliferation regulator that induces and promotes apoptosis through the mitochondrial apoptotic pathway.

Fig. 6. IbACP induced DNA fragments. (A) IbACP increases the levels of low molecular weight DNA fragments. DNA fragments were isolated and then separated by horizontal electrophoresis on 1.6% agarose gel with Tris borate EDTA as running buffer. The concentrations of IbACP are as indicated. Systemin, an 18-amino-acids non-targeting peptide, was used as a negative control. (B) The dose-response effects of IbACP in a Panc-1 cell proliferation assay. The cells were incubated for 24 h with a serial concentration of IbACP. The cell proliferation was determined by the alamarBlue assay. The results in the histogram are presented as the mean ± SEM and are the results of at least six independent experiments.

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3.5. IbACP as a potential biotechnological tool for cancer treatment Our data presented here and other unpublished results support the notion that IbACP exhibited cytotoxicity to pancreatic cancer cells but not so much to the normal cells. Current prodrug design strategies are focused on bioavailability and tissue targeting thereby avoiding their cytotoxic effects on noncancerous cells. Such probable drugs are designed by conjugating to low or high molecular carriers that can transport the drugs to tumors and subsequently release it outside or inside the tumor cells [26]. Besides the commonly considered carriers such as sugars, cytokines, vitamins, antibodies, peptides and synthetic polymers etc., albumin is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptideor protein-based drugs. Kratz described that albumin accumulates in malignant and inflamed tissue, in which tumor uptake can be easily visualized by injecting the dye, which binds to albumin and makes subcutaneously grown tumors turn blue within a few hours post-injection [18]. Furthermore, versatile applications of albumin as a drug carrier have been proposed; from extending the half-life of therapeutically active proteins and peptides (such as alb-IFN, Insulin Detemir and Exendin-4) to drug targeting (such as albmethotrexate, INNO-206 or Abraxane) [9]. The carrier conjugation will be our next objective of study in the process of cancer therapeutic development. In this study we identified IbACP as a new member of anticancer peptides. We demonstrated that IbACP inhibits cellular proliferation of Panc-1 cells via induction of cell apoptosis in a dosedependent manner; caspase-3 and poly (ADP-ribose) polymerase were activated, and the levels of cleaved caspase-3 and -9 were increased by IbACP treatment. In addition, genomic DNA fragmentation and a decrease in cellular proliferation were induced when IbACP was added to Panc-1 cells. Our findings herein not only indicat that IbACP can regulate cellular proliferation and has anticancer effects, but also show that IbACP deserves further exploration as a novel anti-cancer agent. Acknowledgments This work was supported by the National Science Council under a grant (grant number NSC98-2311-B-017-001-MY3 to Y.-C. Chen, grant number NSC 99-2311-B-006-003 to H.-S. Chang). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides. 2013.02.005. References [1] Bovell-Benjamin AC. Sweet potato: a review of its past, present, and future role in human nutrition. Adv Food Nutr Res 2007;52:1–59. [2] Burnette WN. ’Western blotting’: electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981;112:195–203. [3] Campos MA, Ribeiro SG, Rigden DJ, Monte DC, Grossi de Sa MF. Putative pathogenesis-related genes within Solanum nigrum var. americanum genome: isolation of two genes coding for PR5-like proteins, phylogenetic and sequence analysis. Physiol Mol Plant Pathol 2002;61:205–16. [4] Castillo Ruiz RA, Herrera C, Ghislain M, Gebhardt C. Organization of phenylalanine ammonia lyase (PAL), acidic PR-5 and osmotin-like (OSM) defence-response gene families in the potato genome. Mol Genet Genomics 2005;274:168–79. [5] Chen Y-C, Siems WF, Pearce G, Ryan CA. Six peptide wound signals derived from a single precursor protein in Ipomoea batatas leaves activate the expression of the defense gene sporamin. J Biol Chem 2008;283:11469–76.

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Please cite this article in press as: Chang VH-S, et al. IbACP, a sixteen-amino-acid peptide isolated from Ipomoea batatas leaves, induces carcinoma cell apoptosis. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.02.005