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AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells Rym BenAissa a, Houcemeddine Othman a, 1, Claude Villard b, Steve Peigneur c, Saoussen Mlayah-Bellalouna a, Zaineb Abdelkafi-Koubaa a, Naziha Marrakchi a, Luis b, Najet Srairi-Abid a, * Khadija Essafi-Benkhadir d, Jan Tytgat c, Jose Universit e de Tunis El Manar, Institut Pasteur de Tunis, LR11IPT08 Venins et Biomol ecules Th erapeutiques, 1002, Tunis, Tunisia CNRS-UMR 7051, Institut de Neuro Physiopathologie (INP), Universit e Aix-Marseille, 27 Bd Jean Moulin, 13385, Marseille, Cedex 5, France Toxicology and Pharmacology, KU Leuven, Campus Gasthuisberg, O & N2, Herestraat 49, P.O. Box 922, 3000, Leuven, Belgium d Universit e de Tunis El Manar, Institut Pasteur de Tunis, LR11IPT04 Epid emiologie Mol eculaire et Pathologie Exp erimentale Appliqu ee Aux Maladies Infectieuses, 1002, Tunis, Tunisia a
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a r t i c l e i n f o
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Article history: Received 26 September 2019 Accepted 13 October 2019 Available online xxx
Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC50 value of 15 mM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Naþ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells. © 2019 Elsevier Inc. All rights reserved.
Keywords: Sodium channel subtypes Scorpion peptide Prostate cancer DU145 cells Proliferation
1. Introduction Prostate cancer is one of the most diagnosed cancer. It is associated with the highest mortality rate among men over 65 years worldwide [1]. Early detection and treatment of prostate cancer may significantly increase the progression-free survival of patients [2]. However, the diagnosis of the disease is challenging when high proportion of patients develop locally recurrent tumors or distant metastases [3]. Therefore, exploring novel bioactive molecules is needed for improving the outcomes of prostate cancer treatment. Various studies focused on pharmacological agents targeting
* Corresponding author. Institut Pasteur de Tunis, 13 Place Pasteur Tunis, 1002, de re, Tunisia. Tel.: þ21695146444; fax: þ21671791833. Tunis Belve E-mail address: [email protected] (N. Srairi-Abid). 1 Current address: Sydney Brenner Institute for Molecular Bioscience, University of the Witwatersrand, Johannesburg, South Africa.
cell metastasis and/or proliferation [4]. Recently, several reports suggested that scorpion venoms contain a rich repertory of anticancer peptides endowed with activities that cover a wide range of cancer hallmarks [5,6]. Scorpion neurotoxins have been identified as potent ligands for numerous ion channels because of their high activity and specificity [7]. Many of their natural targets are implicated in the pathophysiology of cancer [6,8]. In particular, voltage-gated sodium channels were found to affect the metabolism of cancer cells, suggesting their important status as molecular markers for prostate cancer [8]. Previous data showed that the high expression of voltage-gated sodium channels by tumor cells was positively correlated with cell proliferation and crucial in their invasion and metastasis. As a result, sodium channels have been considered as new targets in cancers and especially in prostate cancer [9]. Targeting ion channels on tumor cells is achieved by chemical Naþ blockers. For instance, in vitro
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Please cite this article as: R. BenAissa et al., AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.115
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invasiveness of PC3 and MAT-LyLu prostate tumor cells, was significantly reduced by the marine neurotoxin tetrodotoxin (TTX) [10] which, at 6 mM, blocks completely the voltage-gated Naþ channel activity. In addition, veratridine, a natural steroid extracted from plants, widely used as a sodium channel opener, reduced the proliferation of AT-2 and MAT-LyLu cell lines starting from a concentration of 50 mM [11]. In this context, we screened the negatively charged fractions of A. Australis scorpion venom to identify and characterize a peptide able to inhibit the proliferation of prostate cancer cells. We found that the most active peptide was AaHIV previously identified as a putative sodium channel modulator [12,13]. 2. Experimental 2.1. Chemicals and reagents Venom of Androctonus Australis scorpions (from BeniKhedach, Tunisia) was collected by the veterinarian service of the Pasteur Institute of Tunis and kept frozen at 20 C in its crude form until use. Chemicals (reagent grade) were purchased from Sigma Chemical Company, except indicated otherwise. 2.2. Purification Crude venom was extracted with cold water (1:4 v/v), and centrifuged at 15 000g for 15 min. The supernatant was loaded on Sephadex G-50 gel filtration chromatography column (K26/100) equilibrated with 0.1 M acetic acid, to obtain mainly 4 fractions as previously described [14]. AaHG50 containing toxins with molecular weight ranging from 3000 Da to 7000 Da was then fractionated by FPLC on a cation exchange Resource-S column pre-equilibrated using a solution buffer of 0.05 M ammonium acetate. Peptides were eluted over 40 min of linear gradient from 0.05 to 0.5 M ammonium acetate (pH 6.6), at a flow rate of 0.8 ml/min [15]. Absorbance was monitored at 280 nm. Non-retained fractions were applied to a reverse-phase high performance liquid chromatography on a C18 column (250 4.6 mm, 5 mm; Waters Spherisorb®). Peptides were eluted from the column at the rate of 0.8 ml/min using a linear gradient (45 min) from 10 to 45% of solution B [0.1% trifluoroacetic acid (TFA) in acetonitrile] in solution A (0.1%TFA in water). The concentration of purified proteins was determined by the QuantiProBCA Assay Kit. 2.3. Mass spectrometry and amino acid sequence determination MALDI MS in Source Decay (ISD) was performed using an Ultraflextreme TOF/TOF mass spectrometer controlled by the Flex Control 3.3 software (BrukerDaltonics). The laser was increased (20%) to fragment protein in the source of the mass spectrometer using a specific matrix enabling the generation of hydrogen radicals breaking the peptide backbone producing c ions and z ions from 1000 to 5000Da generating tag masses that could be used to search for proteins in databases. Spectra were acquired in positive reflectron ion mode with 2000 laser shots accumulated with a frequency of 1000 Hz. The mass spectrometer parameters were set according manufacturer’s settings for optimal acquisition performance. Sequences were analyzed on Flex Analysis software, version 3.0 (BrukerDaltonics). 2.4. In vivo toxicity assay The in vivo toxicity of AaHIV was tested on 20 ± 2 g male C57Bl/6 mice by intracerebro-ventricular injection (i.c.v). The peptide was diluted in 0.1% (w/v) BSA and 5 ml of the solution containing
increasing amounts of peptide were injected to six mice for each dose [15,16]. Two control mice were injected with 0.1% BSA in water. I.c.v administration was performed under ether anesthesia [16]. All experiments were carried out in accordance with the European Community Council Directive 2010/63/EC for experimental animal care and all procedures met with the approval of the Institutional Research Board of the Pasteur Institute of Tunis. 2.5. Cell proliferation assay DU145 cells were routinely cultured in RPMI 1640, supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum from Biochrom, Berlin, Germany. DU145 cells were seeded in a microtiter plate (5000 cells/well) and incubated overnight at 37 C in a humidified atmosphere of 5% CO2. The medium is renewed in the presence of molecules to be tested. After 72 h, DU145 cells were incubated with 100 ml of complete medium containing 1 mg/ml 3(4,5-diethylthiazoly- 2-yl)-2,5-diphenyltetrazolium bromide MTT at 37 C for 3 h then 100 ml of dimethyl-sulfoxide (Sigma) were added to solubilize it. The absorbance at 540 nm was measured with a microplate reader (MULTISKAN, Labsystems, CA). Cell proliferation was expressed as percentage of the viable cell number of the control (non-treated cells). 2.6. Cell migration assay In vitro cell migration assay was performed in modified Boyden chambers (NeuroProbe Inc., Bethesda, MD) as previously described [17], using porous membranes coated with 10 mg/ml of fibronectin and incubated overnight at 4 C. DU145 cells were harvested as a single cell suspension (106 cells/ml) and treated with molecules to be tested, then added to pre-coated membranes and allowed to migrate for 6 h at 37 C. Migrated cells were fixed with 1% glutaraldehyde, stained with crystal violet at 0.1% and solubilized with 1% SDS.Cells migration was quantified by measuring the absorbance at 600 nm. 2.7. Cell adhesion assay DU145 cells in single cell suspension (106 cells/ml) were treated with molecules of interest and then deposited on fibronectin (Fn)coated wells (10 mg/ml). Cells were allowed to adhere to the substrate for 2 h at 37 C. After washing, attached cells were fixed, stained by 0.1% crystal violet, lysed with 1% SDS and quantified by measuring the absorbance at 600 nm. 2.8. Electrophysiological effect of AaHIV For the expression of Nav channels (rNav1.2, rNav1.4, hNav1.5, mNav1.6, hNav1.7 and hNav1.8) in Xenopus laevis oocytes, the linearized plasmids were transcribed using the T7 or SP6 mMESSAGE-mMACHINE transcription kit (Ambion, Carlsbad, CA). The harvesting of stage VeVI oocytes from anesthetized female Xenopus laevis frog was previously described (Peigneur et al., 2019). Oocytes were injected with 50 nl of cRNA at a concentration of 1 ng/ nl using a micro injector (Drummond Scientific, Broomall, PA). The oocytes were incubated in a solution containing 96-mM NaCl, 2mM KCl, 1.8-mM CaCl2, 2-mM MgCl2, and 5-mM HEPES (pH 7.4), supplemented with 50 mg/l gentamycin sulfate. Two-electrode voltage-clamp recordings were performed at room temperature (18e22 C) using a Geneclamp 500 amplifier (Molecular Devices, Downingtown, PA) controlled by a pClamp data acquisition system (Axon Instruments, Union City, CA). Whole cell currents from oocytes were recorded 1e4 days after injection. Bath solution composition was 96-mM NaCl, 2-mM KCl, 1.8-mM CaCl2,
Please cite this article as: R. BenAissa et al., AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.115
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2-mM MgCl2, and 5-mM HEPES (pH 7.4). Toxin were applied directly to the bath. Resistances of both electrodes were kept between 0.8 and 1.5 MU. The elicited currents were filtered at 1 kHz and sampled at 20 kHz using a four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol. Sodium current traces were evoked by 100-ms depolarization to the voltage corresponding to maximal sodium current in control conditions. All data were analyzed using pClampClampfit 10.0 (Molecular Devices) and Origin 7.5 software (Originlab, Northampton, MA).
the voltage sensor domain (VS) was included in this study. AaHIV and the receptor sequences were aligned with needle software from EMBOSS package [20] respectively to AaHII and Nav1.7 voltage sensor from the structure of the template [21]. For each complex, we constructed 10 solutions using MODELLER 9.20 software [22] from which we selected the one with the lowest DOPE score. The complex structure was subsequently refined using HADDOCCK protein-protein docking software [23]. The binding energy of each complex was scored with PRODIGY [24].
2.9. Homology search and sequence analysis
3. Results and discussion
Target protein underwent a sequence analysis by establishing its alignment with different other homologous proteins. We started by collecting the sequences using FASTA homology search method [18]. Different peptides were selected to cover a wide range of percentage identity to the query sequence. The collected primary structures were then aligned using T-coffee [19].
3.1. Identification of the most active peptide on DU145 cell viability from negatively charged fractions of Aa venom
2.10. Molecular modeling and docking of AaHIV We used the structure of the complex between Nav1.7-NavPas chimera and AaHII scorpion peptide to establish a homologybased molecular docking solution between AaHIV and two sodium channel subtypes: Nav1.2 (Q99250), Nav1.7 (Q15858). Only
In the present study, we aimed to search for peptides with the lowest toxicity and capable to inhibit DU145 cells viability. Previously, we showed that most negative charged scorpion peptides lack of toxic activity [25]. For this reason, we screened non-retained fractions F1, F2 and F3 by the Resource-S FPLC column from toxic fraction AahG50 (Fig. 1A). We have previously identified KAaH1 and KAaH2, two peptides that target Kv1.3 and Kv1.1 potassium channels, respectively [15] and inhibited cell proliferation, migration and adhesion of U87 (glioblastoma) and MDA-MB-231 (breast cancer) cell lines [5]. Therefore, we assumed that these fractions
Fig. 1. Identification of AaHIV toxin. (A) The toxic fraction of the venom was submitted to FPLC on a Mono S column pre-equilibrated with 0.05 M ammonium acetate buffer. Proteins were eluted with a 40 min linear gradient from 0.05 to 0.5 M ammonium acetate (pH 6.6) at a flow rate of 0.8 ml/min; absorbance was monitored at 280 nm. (B) Two fractions were purified from the non-retained fraction F2 by RP-HPLC on a C18 column as described in the experimental section. (C) The most active molecule, 2F2 was obtained from fraction F2 and was eluted at 44 min. (D) Amino acid sequence determination by Maldi-Isd of the fraction 2F2 showing c ions matching the sequence of AaHIV.
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might contain other pharmacologically active peptides that could inhibit DU145 prostate cancer cell proliferation. These fractions were purified by HPLC. Six major peaks were obtained from F1 (data not shown), three major fractions from F2 (Fig. 1B) and 2 major peaks were obtained from F3 (Data not shown). A total of 11 fractions were screened (at 100 mg/ml) on the viability of DU145 cells (Data not shown). The best activity was found for 2F2 (51.5%) and 1F2 (36%) of F2 fraction (Fig. 1C). When analyzed by MALDI-ISD mass spectrometry, the result confirmed that the fraction 2F2 contains one chemical entity corresponding to different ionization states in the mass spectrum profile (Data not shown). Thus, 2F2 contains one peptide with a molecular mass of 6882 Da. 2F2 was further analyzed by In Source Decay (ISD) MALDI mass spectrometry. We were able to identify a fragment of 55 amino acids (Fig. 1D). The annotation shows 100% of local identity with sequence of the peptide AaHIV (Uniprot database accession number: P45658), a putative voltage-gated sodium channel alpha toxin. Theoretical mono-isotopic mass of this sequence corresponds to 6891.81 Da calculated using ProtParam server. Therefore, the peptide 2F2 corresponds to AaHIV toxin in its oxidized form. AaHIV was previously purified by Martin and Rochat, using a different protocol [13]. It has high sequence identity with alpha scorpion toxins [14]. However, its effect, neither on sodium channels nor on cancer cells, has never been explored. Some sodium channel scorpion toxins, such as the two analgesic peptides BmKAGAP-SUPU2 and r-BmKAGAP from Buthus martensii Karsch scorpion venom have been reported to exhibit antitumor activities against S-180 fibrosarcoma cells, SHG-44 malignant glioma cells, HepG2 hepatic cells, SW480 colon cancer cells, MCF-7 and MDA-MB-231 breast cancer cells [7,26,27]. Cn2, from the Centruroïdes noxius scorpion venom, was also capable to induce apoptosis and inhibits the proliferation of neuroblastoma cells [28]. However, AaHIV is the first scorpion peptide inhibiting DU-145 prostate cancer cells proliferation.
C57bL/6 mice of 20 g by i.c.v root which is concordant with result previously reported by Martin and Rochat (LD50 ¼ 18 ng\20 g mouse) [13]. When compared to AaHII, the most toxic scorpion peptide (0.5 ng/20 g mouse) [13], we found that AaHIV is 30 times less toxic, showing thus that it has low toxicity on animals. 3.3. AaHIV has the best activity on DU145 proliferation compared to other AaH scorpion toxins and veratridine AaHIV exhibited dose-dependent inhibition on the proliferation of DU145 cells with an IC50 of 15 mM (Fig. 2A). Compared to AaHI and AaHII toxins from the same venom, we found that at 15 mM, the two scorpion peptides inhibited cell viability by only 26% and 28%, respectively (Fig. 2B). Comparative adhesion and migration assays were also performed at 15 mM using fibronectin as extracellular matrix (ECM). After 2 h of incubation, we noted that AaHIV and AaHII partially reduced DU145 cells adhesion by only 11% and 16%, respectively, whereas AaHI had no effect (Figure SD). The three scorpion peptides, had no significant effect on DU145 cell migration (Data not shown), suggesting that sodium channels targeted by these peptides are not implicated in the migration of these prostate cancer cells. Functional inference from sequence analysis, suggests that AaHIV target’s is a voltage-dependent sodium channel. For such reason, we tested the peptides alongside with veratridine, an activator for some Nav channels, notably, Nav1.1 and Nav1.7 [29] which are expressed in DU145 cells. Our results show that veratridine had a dose-dependent effect on the viability of DU145 cells with IC50 value of 250 mM (Fig. 2C). Veratridine decreased slightly (25% inhibition at 200 mM) their adhesion (Figure SD) and migration to ECM (Data not shown). 3.4. AaHIV is active on Nav1.6 channel
3.2. In vivo toxicity of AaHIV We found that AaHIV has a LD50 of 15 ng, when injected to
Electrophysiological recordings at a concentration of 5 mM of AaHIV were investigated for its activity against 6 different voltage-
Fig. 2. Effect on DU145 proliferation. (A) Dose-effect of AaHIV on DU145 cell proliferation after 72 h (B) Effect of AaHIV, AaHI and AaHII at 15 mM (C) Dose-effect of veratridine. All data represent at least 3 independent experiments (n 3) and are presented as mean ± standard error.
Please cite this article as: R. BenAissa et al., AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.115
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Fig. 3. Electrophysiological characterization of AaHIV on Nav channels expressed in X. laevis oocytes. Representative whole-cell current traces in control and peptide conditions are shown. The dotted line indicates the zero-current level. Asterisks mark steady-state current traces after application of 5 mM AaHIV. Traces shown are representative of at least three independent experiments (n 3).
gated sodium channels, shown to be expressed in DU145 cells [30] (Fig. 3). We found that the peptide acted by slowing down the inactivation process of the Nav1.6 channel (I30 ms/Ip ¼ 0.16 ± 0.02) as can be seen by the not fully inactivating current in the presence of the toxin (Fig. 3). However, no activity was seen on Nav1.4, Nav1.5, Nav1.7, Nav1.8 nor Nav1.2. The later is reported to be highly expressed in DU145 cells [30]. Our results show that Nav1.2 might not be involved in the proliferation of DU145 cells. However, Nav1.6 could be involved in the proliferation but not the adhesion or migration of these cells.
3.5. Computational study of AaHIV structure and interaction AaHIV share a sequence identity of 42% with AaHII indicating that they present a similar structure-activity relationship. To get more insight about the interaction of the peptide and sodium channel subtypes, we generated the structure model of AaHIV, as well as its complex with Nav1.2, Nav1.6 and Nav1.7, based on the recently published structure of AaHII and a chimera construction between NavPas channel and Nav1.7. We found that AaHIV interacts with residues 1601e1613 and 1541e1548 of the two extracellular loops formed between the paddle and helices S1eS2 of the
Fig. 4. Docking of AaHIV to Nav1.7 and Nav1.2 voltage sensor domain. (A) Adjustment of AaHIV peptide to the extracellular side of the voltage sensor domain of Nav1.7 showing Helices S1, S2, S3, and S4. (B) Docking complexes of AaHIV/Nav1.7 and AaHIV/Nav1.2 showing the interactions of K83 of AaHIV with residues of the connecting loop between S1 and S2.
Please cite this article as: R. BenAissa et al., AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.115
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channel with inter-helices space being the major interacting pocket in Nav1.6 (Fig. 4). Values of the binding free energy for Nav1.6 is the most favorable evaluated at 6.5 kcal/mol. AaHIV is able to form a salt bridge bond only with Nav1.6 between K75 and D1604. The orientation of K75 side chain seems to be dictated by the presence of K1548 which repulse the former toward a close distance to D1604. In Nav1.7, the peptide relay only on hydrogen bonds which might be stable while in Nav1.2 there could be a strong electrostatic repulsion between the C-termini of AaHIV and the loop between S1eS2. 4. Conclusion In this work, we identified AaHIV as the most active peptide on DU145 cell proliferation purified from natural animal products. However, it showed a weak activity on DU145 cell adhesion and no effect on their migration. The two processes are independent from cell proliferation, relying majorly on signaling pathways involving membrane proteins interacting with the extracellular matrix. Nevertheless, several reports suggest the involvement of Nav channels in such cancerous cell processes [31]. In prostate cancer, Nav1.5 and Nav1.7 seem to be involved more than any other Nav subtypes [31e33]. However, being not active on these two channels, AaHIV highlights the importance of Nav1.6 in the proliferation of DU145 cells. We can also conclude that the Nav1.2 channel, even though highly expressed in DU145 cell, is not involved in their proliferation. Declaration of competing interest The authors declare no conflict of interest. Acknowledgments We would like to thank, Dr. Ines ElBini as well as Thouraya Chagour and AmeneAllah Ellafi (LVMT, Pasteur Institute of Tunis) for their help. Alessandra Pagano Aurrand Lions and Françoise Aix-Marseille, are also knowledged Garrouste, from INP, Universite for their help. This work was supported by the Tunisian Ministry of Higher Education and Scientific Research (LR11IPT08) and the research grant PHC-Utique [code 17G 0811]. JT was funded by GOC2319 N and GOA4919 N (F.W.O. Vlaanderen) and CELSA/17/047 (BOF, KU Leuven). SP is supported by KU Leuven funding (PDM/19/164). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.115. References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, CA Cancer J Clin 64 (2014) (2014) 9e29, https://doi.org/10.3322/caac.21208. [2] P. Colanzi, A. Santinelli, R. Mazzucchelli, R. Pomante, R. Montironi, Changes in the normal-looking epithelium in prostates with PIN or cancer, AdvClin Path 3 (1999) 129e134. [3] B.A. Hellerstedt, K.J. Pienta, The current state of hormonal therapy for prostate cancer, CA Cancer J Clin 52 (2002) 154e179. [4] S.P. Fraser, V. Salvador, E.A. Manning, J. Mizal, S. Altun, M. Raza, R.J. Berridge, M.B.A. Djamgoz, Contribution of functional voltage-gated Naþ channel expression to cell behaviors involved in the metastatic cascade in rat prostate cancer: I. Lateral motility, J. Cell. Physiol. 195 (2003) 479e487, https://doi.org/ 10.1002/jcp.10312. [5] D. Aissaoui, S. Mlayah-Bellalouna, J. Jebali, Z. Abdelkafi-Koubaa, S. Souid, W. Moslah, H. Othman, J. Luis, M. ElAyeb, N. Marrakchi, K. Essafi-Benkhadir, N. Srairi-Abid, Functional role of Kv1.1 and Kv1.3 channels in the neoplastic progression steps of three cancer cell lines, elucidated by scorpion peptides, Int. J. Biol. Macromol. 111 (2018) 1146e1155, https://doi.org/10.1016/
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AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells Rym BenAissa a, Houcemeddine Othman a, 1, Claude Villard b, Steve Peigneur c, Saoussen Mlayah-Bellalouna a, Zaineb Abdelkafi-Koubaa a, Naziha Marrakchi a, Luis b, Najet Srairi-Abid a, * Khadija Essafi-Benkhadir d, Jan Tytgat c, Jose Universit e de Tunis El Manar, Institut Pasteur de Tunis, LR11IPT08 Venins et Biomol ecules Th erapeutiques, 1002, Tunis, Tunisia CNRS-UMR 7051, Institut de Neuro Physiopathologie (INP), Universit e Aix-Marseille, 27 Bd Jean Moulin, 13385, Marseille, Cedex 5, France Toxicology and Pharmacology, KU Leuven, Campus Gasthuisberg, O & N2, Herestraat 49, P.O. Box 922, 3000, Leuven, Belgium d Universit e de Tunis El Manar, Institut Pasteur de Tunis, LR11IPT04 Epid emiologie Mol eculaire et Pathologie Exp erimentale Appliqu ee Aux Maladies Infectieuses, 1002, Tunis, Tunisia a
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2019 Accepted 13 October 2019 Available online xxx
Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC50 value of 15 mM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Naþ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells. © 2019 Elsevier Inc. All rights reserved.
Keywords: Sodium channel subtypes Scorpion peptide Prostate cancer DU145 cells Proliferation
1. Introduction Prostate cancer is one of the most diagnosed cancer. It is associated with the highest mortality rate among men over 65 years worldwide [1]. Early detection and treatment of prostate cancer may significantly increase the progression-free survival of patients [2]. However, the diagnosis of the disease is challenging when high proportion of patients develop locally recurrent tumors or distant metastases [3]. Therefore, exploring novel bioactive molecules is needed for improving the outcomes of prostate cancer treatment. Various studies focused on pharmacological agents targeting
* Corresponding author. Institut Pasteur de Tunis, 13 Place Pasteur Tunis, 1002, de re, Tunisia. Tel.: þ21695146444; fax: þ21671791833. Tunis Belve E-mail address: [email protected] (N. Srairi-Abid). 1 Current address: Sydney Brenner Institute for Molecular Bioscience, University of the Witwatersrand, Johannesburg, South Africa.
cell metastasis and/or proliferation [4]. Recently, several reports suggested that scorpion venoms contain a rich repertory of anticancer peptides endowed with activities that cover a wide range of cancer hallmarks [5,6]. Scorpion neurotoxins have been identified as potent ligands for numerous ion channels because of their high activity and specificity [7]. Many of their natural targets are implicated in the pathophysiology of cancer [6,8]. In particular, voltage-gated sodium channels were found to affect the metabolism of cancer cells, suggesting their important status as molecular markers for prostate cancer [8]. Previous data showed that the high expression of voltage-gated sodium channels by tumor cells was positively correlated with cell proliferation and crucial in their invasion and metastasis. As a result, sodium channels have been considered as new targets in cancers and especially in prostate cancer [9]. Targeting ion channels on tumor cells is achieved by chemical Naþ blockers. For instance, in vitro
https://doi.org/10.1016/j.bbrc.2019.10.115 0006-291X/© 2019 Elsevier Inc. All rights reserved.
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invasiveness of PC3 and MAT-LyLu prostate tumor cells, was significantly reduced by the marine neurotoxin tetrodotoxin (TTX) [10] which, at 6 mM, blocks completely the voltage-gated Naþ channel activity. In addition, veratridine, a natural steroid extracted from plants, widely used as a sodium channel opener, reduced the proliferation of AT-2 and MAT-LyLu cell lines starting from a concentration of 50 mM [11]. In this context, we screened the negatively charged fractions of A. Australis scorpion venom to identify and characterize a peptide able to inhibit the proliferation of prostate cancer cells. We found that the most active peptide was AaHIV previously identified as a putative sodium channel modulator [12,13]. 2. Experimental 2.1. Chemicals and reagents Venom of Androctonus Australis scorpions (from BeniKhedach, Tunisia) was collected by the veterinarian service of the Pasteur Institute of Tunis and kept frozen at 20 C in its crude form until use. Chemicals (reagent grade) were purchased from Sigma Chemical Company, except indicated otherwise. 2.2. Purification Crude venom was extracted with cold water (1:4 v/v), and centrifuged at 15 000g for 15 min. The supernatant was loaded on Sephadex G-50 gel filtration chromatography column (K26/100) equilibrated with 0.1 M acetic acid, to obtain mainly 4 fractions as previously described [14]. AaHG50 containing toxins with molecular weight ranging from 3000 Da to 7000 Da was then fractionated by FPLC on a cation exchange Resource-S column pre-equilibrated using a solution buffer of 0.05 M ammonium acetate. Peptides were eluted over 40 min of linear gradient from 0.05 to 0.5 M ammonium acetate (pH 6.6), at a flow rate of 0.8 ml/min [15]. Absorbance was monitored at 280 nm. Non-retained fractions were applied to a reverse-phase high performance liquid chromatography on a C18 column (250 4.6 mm, 5 mm; Waters Spherisorb®). Peptides were eluted from the column at the rate of 0.8 ml/min using a linear gradient (45 min) from 10 to 45% of solution B [0.1% trifluoroacetic acid (TFA) in acetonitrile] in solution A (0.1%TFA in water). The concentration of purified proteins was determined by the QuantiProBCA Assay Kit. 2.3. Mass spectrometry and amino acid sequence determination MALDI MS in Source Decay (ISD) was performed using an Ultraflextreme TOF/TOF mass spectrometer controlled by the Flex Control 3.3 software (BrukerDaltonics). The laser was increased (20%) to fragment protein in the source of the mass spectrometer using a specific matrix enabling the generation of hydrogen radicals breaking the peptide backbone producing c ions and z ions from 1000 to 5000Da generating tag masses that could be used to search for proteins in databases. Spectra were acquired in positive reflectron ion mode with 2000 laser shots accumulated with a frequency of 1000 Hz. The mass spectrometer parameters were set according manufacturer’s settings for optimal acquisition performance. Sequences were analyzed on Flex Analysis software, version 3.0 (BrukerDaltonics). 2.4. In vivo toxicity assay The in vivo toxicity of AaHIV was tested on 20 ± 2 g male C57Bl/6 mice by intracerebro-ventricular injection (i.c.v). The peptide was diluted in 0.1% (w/v) BSA and 5 ml of the solution containing
increasing amounts of peptide were injected to six mice for each dose [15,16]. Two control mice were injected with 0.1% BSA in water. I.c.v administration was performed under ether anesthesia [16]. All experiments were carried out in accordance with the European Community Council Directive 2010/63/EC for experimental animal care and all procedures met with the approval of the Institutional Research Board of the Pasteur Institute of Tunis. 2.5. Cell proliferation assay DU145 cells were routinely cultured in RPMI 1640, supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum from Biochrom, Berlin, Germany. DU145 cells were seeded in a microtiter plate (5000 cells/well) and incubated overnight at 37 C in a humidified atmosphere of 5% CO2. The medium is renewed in the presence of molecules to be tested. After 72 h, DU145 cells were incubated with 100 ml of complete medium containing 1 mg/ml 3(4,5-diethylthiazoly- 2-yl)-2,5-diphenyltetrazolium bromide MTT at 37 C for 3 h then 100 ml of dimethyl-sulfoxide (Sigma) were added to solubilize it. The absorbance at 540 nm was measured with a microplate reader (MULTISKAN, Labsystems, CA). Cell proliferation was expressed as percentage of the viable cell number of the control (non-treated cells). 2.6. Cell migration assay In vitro cell migration assay was performed in modified Boyden chambers (NeuroProbe Inc., Bethesda, MD) as previously described [17], using porous membranes coated with 10 mg/ml of fibronectin and incubated overnight at 4 C. DU145 cells were harvested as a single cell suspension (106 cells/ml) and treated with molecules to be tested, then added to pre-coated membranes and allowed to migrate for 6 h at 37 C. Migrated cells were fixed with 1% glutaraldehyde, stained with crystal violet at 0.1% and solubilized with 1% SDS.Cells migration was quantified by measuring the absorbance at 600 nm. 2.7. Cell adhesion assay DU145 cells in single cell suspension (106 cells/ml) were treated with molecules of interest and then deposited on fibronectin (Fn)coated wells (10 mg/ml). Cells were allowed to adhere to the substrate for 2 h at 37 C. After washing, attached cells were fixed, stained by 0.1% crystal violet, lysed with 1% SDS and quantified by measuring the absorbance at 600 nm. 2.8. Electrophysiological effect of AaHIV For the expression of Nav channels (rNav1.2, rNav1.4, hNav1.5, mNav1.6, hNav1.7 and hNav1.8) in Xenopus laevis oocytes, the linearized plasmids were transcribed using the T7 or SP6 mMESSAGE-mMACHINE transcription kit (Ambion, Carlsbad, CA). The harvesting of stage VeVI oocytes from anesthetized female Xenopus laevis frog was previously described (Peigneur et al., 2019). Oocytes were injected with 50 nl of cRNA at a concentration of 1 ng/ nl using a micro injector (Drummond Scientific, Broomall, PA). The oocytes were incubated in a solution containing 96-mM NaCl, 2mM KCl, 1.8-mM CaCl2, 2-mM MgCl2, and 5-mM HEPES (pH 7.4), supplemented with 50 mg/l gentamycin sulfate. Two-electrode voltage-clamp recordings were performed at room temperature (18e22 C) using a Geneclamp 500 amplifier (Molecular Devices, Downingtown, PA) controlled by a pClamp data acquisition system (Axon Instruments, Union City, CA). Whole cell currents from oocytes were recorded 1e4 days after injection. Bath solution composition was 96-mM NaCl, 2-mM KCl, 1.8-mM CaCl2,
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2-mM MgCl2, and 5-mM HEPES (pH 7.4). Toxin were applied directly to the bath. Resistances of both electrodes were kept between 0.8 and 1.5 MU. The elicited currents were filtered at 1 kHz and sampled at 20 kHz using a four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol. Sodium current traces were evoked by 100-ms depolarization to the voltage corresponding to maximal sodium current in control conditions. All data were analyzed using pClampClampfit 10.0 (Molecular Devices) and Origin 7.5 software (Originlab, Northampton, MA).
the voltage sensor domain (VS) was included in this study. AaHIV and the receptor sequences were aligned with needle software from EMBOSS package [20] respectively to AaHII and Nav1.7 voltage sensor from the structure of the template [21]. For each complex, we constructed 10 solutions using MODELLER 9.20 software [22] from which we selected the one with the lowest DOPE score. The complex structure was subsequently refined using HADDOCCK protein-protein docking software [23]. The binding energy of each complex was scored with PRODIGY [24].
2.9. Homology search and sequence analysis
3. Results and discussion
Target protein underwent a sequence analysis by establishing its alignment with different other homologous proteins. We started by collecting the sequences using FASTA homology search method [18]. Different peptides were selected to cover a wide range of percentage identity to the query sequence. The collected primary structures were then aligned using T-coffee [19].
3.1. Identification of the most active peptide on DU145 cell viability from negatively charged fractions of Aa venom
2.10. Molecular modeling and docking of AaHIV We used the structure of the complex between Nav1.7-NavPas chimera and AaHII scorpion peptide to establish a homologybased molecular docking solution between AaHIV and two sodium channel subtypes: Nav1.2 (Q99250), Nav1.7 (Q15858). Only
In the present study, we aimed to search for peptides with the lowest toxicity and capable to inhibit DU145 cells viability. Previously, we showed that most negative charged scorpion peptides lack of toxic activity [25]. For this reason, we screened non-retained fractions F1, F2 and F3 by the Resource-S FPLC column from toxic fraction AahG50 (Fig. 1A). We have previously identified KAaH1 and KAaH2, two peptides that target Kv1.3 and Kv1.1 potassium channels, respectively [15] and inhibited cell proliferation, migration and adhesion of U87 (glioblastoma) and MDA-MB-231 (breast cancer) cell lines [5]. Therefore, we assumed that these fractions
Fig. 1. Identification of AaHIV toxin. (A) The toxic fraction of the venom was submitted to FPLC on a Mono S column pre-equilibrated with 0.05 M ammonium acetate buffer. Proteins were eluted with a 40 min linear gradient from 0.05 to 0.5 M ammonium acetate (pH 6.6) at a flow rate of 0.8 ml/min; absorbance was monitored at 280 nm. (B) Two fractions were purified from the non-retained fraction F2 by RP-HPLC on a C18 column as described in the experimental section. (C) The most active molecule, 2F2 was obtained from fraction F2 and was eluted at 44 min. (D) Amino acid sequence determination by Maldi-Isd of the fraction 2F2 showing c ions matching the sequence of AaHIV.
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might contain other pharmacologically active peptides that could inhibit DU145 prostate cancer cell proliferation. These fractions were purified by HPLC. Six major peaks were obtained from F1 (data not shown), three major fractions from F2 (Fig. 1B) and 2 major peaks were obtained from F3 (Data not shown). A total of 11 fractions were screened (at 100 mg/ml) on the viability of DU145 cells (Data not shown). The best activity was found for 2F2 (51.5%) and 1F2 (36%) of F2 fraction (Fig. 1C). When analyzed by MALDI-ISD mass spectrometry, the result confirmed that the fraction 2F2 contains one chemical entity corresponding to different ionization states in the mass spectrum profile (Data not shown). Thus, 2F2 contains one peptide with a molecular mass of 6882 Da. 2F2 was further analyzed by In Source Decay (ISD) MALDI mass spectrometry. We were able to identify a fragment of 55 amino acids (Fig. 1D). The annotation shows 100% of local identity with sequence of the peptide AaHIV (Uniprot database accession number: P45658), a putative voltage-gated sodium channel alpha toxin. Theoretical mono-isotopic mass of this sequence corresponds to 6891.81 Da calculated using ProtParam server. Therefore, the peptide 2F2 corresponds to AaHIV toxin in its oxidized form. AaHIV was previously purified by Martin and Rochat, using a different protocol [13]. It has high sequence identity with alpha scorpion toxins [14]. However, its effect, neither on sodium channels nor on cancer cells, has never been explored. Some sodium channel scorpion toxins, such as the two analgesic peptides BmKAGAP-SUPU2 and r-BmKAGAP from Buthus martensii Karsch scorpion venom have been reported to exhibit antitumor activities against S-180 fibrosarcoma cells, SHG-44 malignant glioma cells, HepG2 hepatic cells, SW480 colon cancer cells, MCF-7 and MDA-MB-231 breast cancer cells [7,26,27]. Cn2, from the Centruroïdes noxius scorpion venom, was also capable to induce apoptosis and inhibits the proliferation of neuroblastoma cells [28]. However, AaHIV is the first scorpion peptide inhibiting DU-145 prostate cancer cells proliferation.
C57bL/6 mice of 20 g by i.c.v root which is concordant with result previously reported by Martin and Rochat (LD50 ¼ 18 ng\20 g mouse) [13]. When compared to AaHII, the most toxic scorpion peptide (0.5 ng/20 g mouse) [13], we found that AaHIV is 30 times less toxic, showing thus that it has low toxicity on animals. 3.3. AaHIV has the best activity on DU145 proliferation compared to other AaH scorpion toxins and veratridine AaHIV exhibited dose-dependent inhibition on the proliferation of DU145 cells with an IC50 of 15 mM (Fig. 2A). Compared to AaHI and AaHII toxins from the same venom, we found that at 15 mM, the two scorpion peptides inhibited cell viability by only 26% and 28%, respectively (Fig. 2B). Comparative adhesion and migration assays were also performed at 15 mM using fibronectin as extracellular matrix (ECM). After 2 h of incubation, we noted that AaHIV and AaHII partially reduced DU145 cells adhesion by only 11% and 16%, respectively, whereas AaHI had no effect (Figure SD). The three scorpion peptides, had no significant effect on DU145 cell migration (Data not shown), suggesting that sodium channels targeted by these peptides are not implicated in the migration of these prostate cancer cells. Functional inference from sequence analysis, suggests that AaHIV target’s is a voltage-dependent sodium channel. For such reason, we tested the peptides alongside with veratridine, an activator for some Nav channels, notably, Nav1.1 and Nav1.7 [29] which are expressed in DU145 cells. Our results show that veratridine had a dose-dependent effect on the viability of DU145 cells with IC50 value of 250 mM (Fig. 2C). Veratridine decreased slightly (25% inhibition at 200 mM) their adhesion (Figure SD) and migration to ECM (Data not shown). 3.4. AaHIV is active on Nav1.6 channel
3.2. In vivo toxicity of AaHIV We found that AaHIV has a LD50 of 15 ng, when injected to
Electrophysiological recordings at a concentration of 5 mM of AaHIV were investigated for its activity against 6 different voltage-
Fig. 2. Effect on DU145 proliferation. (A) Dose-effect of AaHIV on DU145 cell proliferation after 72 h (B) Effect of AaHIV, AaHI and AaHII at 15 mM (C) Dose-effect of veratridine. All data represent at least 3 independent experiments (n 3) and are presented as mean ± standard error.
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Fig. 3. Electrophysiological characterization of AaHIV on Nav channels expressed in X. laevis oocytes. Representative whole-cell current traces in control and peptide conditions are shown. The dotted line indicates the zero-current level. Asterisks mark steady-state current traces after application of 5 mM AaHIV. Traces shown are representative of at least three independent experiments (n 3).
gated sodium channels, shown to be expressed in DU145 cells [30] (Fig. 3). We found that the peptide acted by slowing down the inactivation process of the Nav1.6 channel (I30 ms/Ip ¼ 0.16 ± 0.02) as can be seen by the not fully inactivating current in the presence of the toxin (Fig. 3). However, no activity was seen on Nav1.4, Nav1.5, Nav1.7, Nav1.8 nor Nav1.2. The later is reported to be highly expressed in DU145 cells [30]. Our results show that Nav1.2 might not be involved in the proliferation of DU145 cells. However, Nav1.6 could be involved in the proliferation but not the adhesion or migration of these cells.
3.5. Computational study of AaHIV structure and interaction AaHIV share a sequence identity of 42% with AaHII indicating that they present a similar structure-activity relationship. To get more insight about the interaction of the peptide and sodium channel subtypes, we generated the structure model of AaHIV, as well as its complex with Nav1.2, Nav1.6 and Nav1.7, based on the recently published structure of AaHII and a chimera construction between NavPas channel and Nav1.7. We found that AaHIV interacts with residues 1601e1613 and 1541e1548 of the two extracellular loops formed between the paddle and helices S1eS2 of the
Fig. 4. Docking of AaHIV to Nav1.7 and Nav1.2 voltage sensor domain. (A) Adjustment of AaHIV peptide to the extracellular side of the voltage sensor domain of Nav1.7 showing Helices S1, S2, S3, and S4. (B) Docking complexes of AaHIV/Nav1.7 and AaHIV/Nav1.2 showing the interactions of K83 of AaHIV with residues of the connecting loop between S1 and S2.
Please cite this article as: R. BenAissa et al., AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.115
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channel with inter-helices space being the major interacting pocket in Nav1.6 (Fig. 4). Values of the binding free energy for Nav1.6 is the most favorable evaluated at 6.5 kcal/mol. AaHIV is able to form a salt bridge bond only with Nav1.6 between K75 and D1604. The orientation of K75 side chain seems to be dictated by the presence of K1548 which repulse the former toward a close distance to D1604. In Nav1.7, the peptide relay only on hydrogen bonds which might be stable while in Nav1.2 there could be a strong electrostatic repulsion between the C-termini of AaHIV and the loop between S1eS2. 4. Conclusion In this work, we identified AaHIV as the most active peptide on DU145 cell proliferation purified from natural animal products. However, it showed a weak activity on DU145 cell adhesion and no effect on their migration. The two processes are independent from cell proliferation, relying majorly on signaling pathways involving membrane proteins interacting with the extracellular matrix. Nevertheless, several reports suggest the involvement of Nav channels in such cancerous cell processes [31]. In prostate cancer, Nav1.5 and Nav1.7 seem to be involved more than any other Nav subtypes [31e33]. However, being not active on these two channels, AaHIV highlights the importance of Nav1.6 in the proliferation of DU145 cells. We can also conclude that the Nav1.2 channel, even though highly expressed in DU145 cell, is not involved in their proliferation. Declaration of competing interest The authors declare no conflict of interest. Acknowledgments We would like to thank, Dr. Ines ElBini as well as Thouraya Chagour and AmeneAllah Ellafi (LVMT, Pasteur Institute of Tunis) for their help. Alessandra Pagano Aurrand Lions and Françoise Aix-Marseille, are also knowledged Garrouste, from INP, Universite for their help. This work was supported by the Tunisian Ministry of Higher Education and Scientific Research (LR11IPT08) and the research grant PHC-Utique [code 17G 0811]. JT was funded by GOC2319 N and GOA4919 N (F.W.O. Vlaanderen) and CELSA/17/047 (BOF, KU Leuven). SP is supported by KU Leuven funding (PDM/19/164). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.115. References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, CA Cancer J Clin 64 (2014) (2014) 9e29, https://doi.org/10.3322/caac.21208. [2] P. Colanzi, A. Santinelli, R. Mazzucchelli, R. Pomante, R. Montironi, Changes in the normal-looking epithelium in prostates with PIN or cancer, AdvClin Path 3 (1999) 129e134. [3] B.A. Hellerstedt, K.J. Pienta, The current state of hormonal therapy for prostate cancer, CA Cancer J Clin 52 (2002) 154e179. [4] S.P. Fraser, V. Salvador, E.A. Manning, J. Mizal, S. Altun, M. Raza, R.J. Berridge, M.B.A. Djamgoz, Contribution of functional voltage-gated Naþ channel expression to cell behaviors involved in the metastatic cascade in rat prostate cancer: I. Lateral motility, J. Cell. Physiol. 195 (2003) 479e487, https://doi.org/ 10.1002/jcp.10312. [5] D. Aissaoui, S. Mlayah-Bellalouna, J. Jebali, Z. Abdelkafi-Koubaa, S. Souid, W. Moslah, H. Othman, J. Luis, M. ElAyeb, N. Marrakchi, K. Essafi-Benkhadir, N. Srairi-Abid, Functional role of Kv1.1 and Kv1.3 channels in the neoplastic progression steps of three cancer cell lines, elucidated by scorpion peptides, Int. J. Biol. Macromol. 111 (2018) 1146e1155, https://doi.org/10.1016/
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Please cite this article as: R. BenAissa et al., AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.115