Mauriporin, a Novel Cationic a-Helical Peptide with SelectiveCytotoxic Activity Against Prostate Cancer Cell Linesfrom the Venom of the Scorpion Androctonus mauritanicus

Ammar Almaaytah • Shadi Tarazi •

Nizar Mhaidat • Qosay Al-Balas • Tareq L. Mukattash

Accepted: 10 May 2013

� Springer Science+Business Media New York 2013

Abstract Prostate cancer is the second most common

cancer in men and the second leading cause of cancer-related

deaths among men in the western world. Finding a cure for

prostate cancer is urgently needed. Scorpion venoms are rich

sources of biologically active peptides, among which the

non-disulfide bridged peptides constitute an important group

displaying multifunctional activities. The non-disulfide

bridged scorpion venom peptides are rarely identified and

poorly characterized so far. In this work, we report the

molecular cloning and functional characterization of a novel

non-disulfide bridged peptide from the venomous gland

cDNA library of the Moroccan scorpion Androctonus mau-

ritanicus. Named Mauriporin, the peptide was found to be

composed of 48 residues and circular dichroism analysis

revealed the peptide to display a well defined a-helical

structure in membrane mimicking environments. A synthetic

replicate of Mauriporin was found to exert potent selective

cytotoxic and antiproliferative activity against prostate can-

cer cell lines (IC50 4.4–7.8 lM) when compared with non-

tumorigenic cells. In this concentration range, Mauriporin

produced also negligible degrees of hemolytic activities

against mammalian erythrocytes. Apoptotic studies dis-

played that Mauriporin is not causing cell death through an

apoptotic-mediated pathway but possibly through a necrotic

mode of cell death. In conclusion Mauriporin may offer a

novel therapeutic strategy in the treatment of prostate cancer

considering its significant cytotoxic potency against prostate

cancer cells and low toxicity to non-tumorigenic cells.

Keywords Peptide � Scorpion � Venom �Molecular cloning � Prostate cancer

Introduction

Prostate cancer is considered to be the most commonly

diagnosed malignancy in elderly males and the second

leading cancer killer in the Western world. About 241,740

new prostate cancer cases are expected to be diagnosed in

2012 and 28,170 men were expected to die as a result of

this disease (Siegel et al. 2012). Prostate cancer has also

become a major malignancy and health burden facing the

developing countries as well (Jemal et al. 2011). The cur-

rent treatment regimens for patients with localized prostate

cancer include prostatectomy and a combined treatment

approach employing radiation therapy and androgen sup-

pression therapy (Leibowitz and Tucker 2001). However, a

significant proportion of these patients (30–50 %) will

develop recurrent disease in less than 10 years (Wilt et al.

2008; Klotz et al. 2010; Zelefsky et al. 2007). Treatment

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10989-013-9350-3) contains supplementarymaterial, which is available to authorized users.

A. Almaaytah (&)

Department of Pharmaceutical Technology,

Faculty of Pharmacy, Jordan University of Science

and Technology, Irbid, Jordan

e-mail: amalmaaytah@just.edu.jo

S. Tarazi

Department of Applied Biological Sciences,

Faculty of Science and Arts, Jordan University of Science

and Technology, Irbid, Jordan

N. Mhaidat � T. L. Mukattash

Department of Clinical Pharmacy, Faculty of Pharmacy,

Jordan University of Science and Technology, Irbid, Jordan

Q. Al-Balas

Department of Medicinal Chemistry and Pharmacognosy,

Faculty of Pharmacy, Jordan University of Science and

Technology, Irbid, Jordan

123

Int J Pept Res Ther

DOI 10.1007/s10989-013-9350-3

options for metastatic prostate cancer are limited to cas-

tration surgery, LH-releasing hormone agonists and che-

motherapy with no curative therapies available to patients

experiencing advanced or metastatic prostate cancer

(Antonarakis and Eisenberger 2011). Therefore novel

therapeutic options for the treatment of metastatic prostate

carcinomas are highly needed.

Scorpion venoms contain a diverse mixture of bioactive

peptides that supply scorpions with a formidable defence

mechanism against predators and also play a major role in

aiding the scorpion in capturing prey (Rodriguez de la Vega

and Possani 2005; Almaaytah et al. 2012; Goudet et al.

2002; Kozminsky-Atias et al. 2008). The scorpion venom

polypeptides are classified into two major groups, the

disulfide bridged peptides (DBPs) which usually target

membrane bound ion channels (Chen et al. 2003; Chen et al.

2005; Schwartz et al. 2007; Possani et al. 2000) and the non-

disulfide bridged peptides (NDBPs) which represent a

smaller group within the scorpion polypeptide arsenal but

display a diverse set of functions such as antimicrobial and

bradykinin potentiating activities with some peptides

belonging to this group with no characterized activities

identified so far (Zeng et al. 2000; Zeng et al. 2005). Some

scorpion venom peptides belonging to the (DBPs) have been

reported to display potent anticancer activities, Chlorotoxin

from the venom of scorpion Leiurus quinquestriatus have

been found to specifically inhibit the proliferation of glioma

cells by inhibiting chloride channels (Deshane et al. 2003).

Bengalin a 72 kDa peptide isolated from the venom of the

Indian black scorpion H. bengalensis was found to inhibit

the proliferation of leukemic cells through an apoptotic-

mediated pathway (Gupta et al. 2010). To the best of our

knowledge no (NDBPs) displaying anticancer activities

from scorpion venoms have been identified so far.

In this study we report the molecular and functional

characterization of a novel linear cationic a-helical peptide

with no disulfide bridges displaying cytotoxic activity

against prostate cancer cell lines. This peptide named

Mauriporin was identified from the venomous gland cDNA

library of the scorpion Androctonus mauritanicus. Mau-

riporin was found to be composed of 48 residues and

bioinformatic analysis in addition to circular dichroism

studies revealed the peptide to display both a cationic

charge and an a-helical structure. The synthetic replicate of

Mauriporin displayed potent selective cytotoxic activity

against prostate cancer cell lines when compared to its

activity against non-tumorigenic cells. Apoptotic studies

revealed that Mauriporin is not inducing cell death through

an apoptotic pathway but possibly through a necrotic mode

of cell death. Mauriporin also didn’t exhibit any significant

hemolytic activity against blood erythrocytes. These results

clearly indicate that Mauriporin is a novel tumour selective

cytotoxic peptide that has been identified from the venom

of the scorpion A. Mauritanicus and a potential lead agent

that could be exploited to offer new therapeutic options for

the treatment of metastatic prostate cancer.

Materials and Methods

Acquisition of Androctonus mauritanicus Venom

Ten milligrams of lyophilized venom from the Androctonus

mauritanicus scorpion were obtained from Latoxan, France.

The scorpions were collected by experts in the field and

relocated to France. Electric stimulation was used to extract

the venom from the scorpions without causing damage to the

animals. The venom was prepared as a lyophilized powder.

‘‘Shotgun’’ Cloning of A. mauritanicus Mauriporin

cDNA

5 mg of the lyophilized scorpion venom was used for the

construction of the cDNA library. Initially Poly (A)? RNA

(mRNA) was isolated from the venom using the Dynabeads

mRNA� DIRECTTM kit (Invitrogen, USA). The cDNA

library was constructed by using SMARTer RACE cDNA

Amplification Kit (Clontech, USA) as described by the man-

ufacturer. First-strand cDNA was synthesized by PowerScript

reverse transcriptase using 30-RACE CDS Primer and 50-RACE CDS Primer. Full-length Mauriporin precursor-

encoding nucleic acid was obtained by subjecting the resultant

cDNA library to 30-rapid amplification of cDNA ends

(RACE). The 30-RACE reactions were conducted using a

sense primer (S1; 50 GGTTAAACAACTCTGCAAAATG-30)and a universal primer that was supplied by the manufacturer.

The sense primer was designed based on a highly conserved

domain of the 50-untranslated region of an antimicrobial pep-

tide precursor cDNAs that was identified from the Chinese

scorpion Buthus martensii. PCR products were gel-purified

and cloned using a pGEM-T vector system (Promega, USA)

and sequenced (Macrogen, Korea). The sequence data

obtained from the 30-RACE product was used to design an

antisense primer (AS: 50-TTTTGTCGAAAGTTGTTCTTTT

ATC-30) based on the sequence of the 30-non-translated region

of the novel Mauriporin transcripts. 50-RACE was carried out

using these primers with the universal primer and the resultant

products were cloned and purified as mentioned previously

and sequenced.

Bioinformatic Analysis

Sequence similarity searches were performed using the

NCBI BLAST routine (http://blast.ncbi.nlm.nih.gov/

Blast.cgi). Multiple sequence alignments were aligned by

a ClustalW2 software (http://www.ebi.ac.uk/Tools/msa/

Int J Pept Res Ther

123

clustalw2/). The putative signal peptide was identified

using SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/)

software. Helical wheel projections of Mauriporin were

obtained using the Heliquest software (http://heliquest.ipmc.

cnrs.fr/cgi-bin/ComputParamsV2.py).

Reverse Phase HPLC Fractionation of A. Mauritanicus

Scorpion Venom

The lyophilised venom of the scorpion A. mauritanicus

(5 mg) was dissolved in 0.05/99.5 (v/v) trifluoroacetic acid

(TFA)/water (1 mL) before being cleared of microparticles

and other debris by centrifugation. The supernatant was

then injected onto a reverse phased HPLC system fitted

with an semi-perparative column (C18; 250 9 4.6 mm,

ACE, UK) using a gradient formed from 0.05/99.95 (v/v)

TFA/water to 0.05/29.95/70 (v/v/v) TFA/water/acetonitrile

over 240 min at a flow rate of 1 mL/min. Absorbance was

constantly monitored at k 214 nm and all fractions (1 mL)

were collected. Aliquots of each fraction (100 lL) were

lyophilised and redissolved in phosphate saline buffer

(PBS) to determine their antiproliferative activity against

prostate cancer cell lines using the MTT assay.

Identification and Structural Characterization of Mature

Mauriporin in the Scorpion Venom

Reverse phase HPLC fractions # 93, 94, 95 and 96 were

found to possess antiproliferative activity against the PC-3

cell line using the MTT assay. These fractions were then

analyzed to determine the molecular mass of their com-

ponents using matrix-assisted laser desorption/ionization,

time-of-flight mass spectrometry (MALDI-TOF MS).

Briefly the samples were spotted on a MALDI plate and

analyzed by a MALDI-TOF 5800 Proteomics Analyzer

(AB Sciex, MA, USA). MS spectra were collected in mass

range 1,000–8,000 Da. The mass spectrometer was cali-

brated on a six member calibration mixture in the mass

range of 900–3,600 Da.

Peptide Synthesis and Purification

The synthetic replicate of Mauriporin was synthesized by

GL Biochem Ltd. (China). The identity and purity of the

synthetic peptide was confirmed by high performance

liquid chromatography and ESI-MS mass spectrometry

(supplementary material).

Circular Dichroism Analysis

Circular dichroism (CD) experiments were performed to

determine the secondary structure of Mauriporin in aque-

ous phosphate buffer solution (10 mM PBS) and in the

presence of a membrane-mimetic solvent (50 % trifluoro-

ethanol/water) CD spectral analysis was conducted using a

Jasco 810 spectrometer (Jasco, Victoria, British Columbia,

Canada) at 20 �C, using a 1 mm path length cuvette and at

a scan speed of 100 nm/min. The spectra were recorded

between 190 and 260 nm and five replica scans were col-

lected and the average recorded. Final spectra represent

buffer subtracted data. Percentage of a-helical structure

was calculated using the delta-epsilon calculation method

using K2D2 circular dichroism spectra deconvolution

software (http://www.ogic.ca/projects/k2d2/).

Cell Culture

The prostate cancer cell lines PC-3, LNCaP, DU145 and

Vero (African green monkey kidney epithelial cell lines)

were maintained using RPMI-1640 culture medium (PAA

Laboratories GmbH, Austria). This medium was supple-

mented with 10 % (v/v) foetal bovine serum (FBS) (PAA

Laboratories GmbH, Austria) and 1 % (w/v) penicillin/

streptomycin (PAA Laboratories GmbH, Austria). HUVECs

(Human umbilical vein endothelial cells) were maintained in

M199 medium supplemented with 20 % FBS, 60 lg/mL

endothelial cell growth supplement, 2 mM 1-glutamine, and

50 lg/mL heparin and 1 % (w/v) penicillin/streptomycin

(PAA Laboratories GmbH, Austria).

Confluent HUVECs (passages 3–6) were used in the

experiment. All cells were seeded into 75 or 150 cm2

culture flasks (Jet Biofill, China) and were digested with

0.025 % trypsin when grown into confluence, stained

with 0.04 % trypan blue, and counted manually using a

haemocytometer. Cells were cultured as monolayers in

a humidified environment of 5 % CO2 95 % air at

37 �C.

Cell Proliferation Assay

The antiproliferative activity of Mauriporin was deter-

mined by using the MTT assay. Each cell line used in this

experiment (PC-3, LNCaP, DU 145, Vero and HUVEC)

was seeded at a density of 5 9 103 cells per well into a

96-well microtitre plate 24 h before peptide treatment.

Cells were incubated with various concentrations of Mau-

riporin. After 24 h of incubation, 20 lL of 5 mg/mL of

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-

mide (MTT) was added to each well and incubated for 4 h.

Conversion of MTT into purple formazan by metabolically

active cells indicates the extent of cell viability. The

medium was replaced by 200 lL of Dimethyl sulfoxide

(DMSO) and mixed to dissolve the formazan crystals that

had developed. Absorbance was measured using an ELISA

Microplate Reader at 550 nm. The GraphPad prism soft-

ware was used for statistical analyses.

Int J Pept Res Ther

123

Lactate Dehydrogenase (LDH) Release Assay

LDH activity present in the cell culture medium can be

measured using a coupled two-step reaction. In the first

step NAD? is reduced to NADH and H? by oxidizing

lactate to pyruvate through the catalyzing effect of LDH. In

the second step, the newly formed NADH and H? use

diaphorase to catalyze the reduction of a tetrazolium salt

(INT) to highly colored formazan. This assay was per-

formed according to the manufacturer’s instructions (LDH

Cytotoxicity Assay Kit, Cayman, USA). Briefly, each cell

line used in this experiment (PC-3, LNCaP, DU 145, Vero

and HUVEC) was seeded on a 96-well plate at a density of

5,000 cells/well for 24 h at 37 �C in the absence or in the

presence of Mauriporin at different concentrations. Fol-

lowing the incubation period the plates were centrifuged at

4009g for 5 min and 100 uL each of the supernatants were

transferred into a new 96-well plate and mixed with 100 uL

of the supplied reaction solution. The plates were incubated

with a gentle shaking on an orbital incubator for 30 min

and absorbance values were recorded at 492 nm with a

96-well microplate reader. As a control for maximum LDH

release, cells were treated with 2 % triton-X100 (Santa

Cruz, USA) in RPMI medium for 10 min before running

the assay. LDH enzyme activity was expressed as the

percentage of cellular LDH release to the total LDH release

of the cells treated with 2 % triton-X100.

Time Course for Cell Killing by Mauriporin

To test the efficacy of Mauriporin over time, PC-3 cells

were treated with Mauriporin at a standard concentration of

50 lM and the effects were observed and recorded over a

24 h period. Briefly 5 9 103 of PC-3 cells were seeded into

each well of a 96-well culture plate and incubated over-

night. The following day the cells were treated in triplicates

with 50 lM of Mauriporin over a 24 h period at different

time points (0.5, 1, 3, 6, 12, and 24 h). The induction of cell

death by Mauriporin was analysed by the MTT assay as

described previously.

Hemolysis Assay

The hemolytic activity of Mauriporin was tested against

defibrinated sheep blood. A suspension of red blood cells

(2 %) (v/v) was prepared by centrifuging 2 mL fresh blood

at 9309g for 5 min. The supernatant was discarded and

cell pellet was washed three times with phosphate-buffered

saline (PBS) followed by resuspension of the cell pellet in

50 mL of PBS. Mauriporin in different concentrations was

prepared in saline, followed by mixing 2 mL of each

peptide solution with equal amounts of erythrocyte sus-

pension. Zero hemolysis and 100 % hemolysis consisted of

RBC suspended in PBS and 0.2 % Triton X-100 (Santa

Cruz, USA), respectively. Following incubation of the

erythrocytes with the peptide for 60 min, 1 mL of each

sample was centrifuged at 9309g for 5 min. Following

centrifugation 200 lL of each sample was loaded on a 96-

well plate. Plates were placed in n ELISA reader and their

absorbance measured at k = 550 nm. The following

equation was used to calculate percentage hemolysis:

% Hemolysis ¼ A� AOð Þ= AX � AOð Þ � 100

where A = absorbance of test solution, AO = absorbance of

negative control and AX = absorbance of positive control.

DNA Laddering

To assess DNA ladder formation, PC-3 cells (2 9 106) were

seeded into 50 cm2 culture flasks (Jet Biofill, China) and

incubated with Mauriporin for 12 and 24 h and later were

lysed with TE lysis buffer, scraped, and harvested in treat-

ment medium to ensure that all apoptotic cells detached

from the plate were included in the analysis. An apoptotic

DNA ladder isolation kit (Abcam, Cambridge, UK) was

used to extract low molecular weight DNA and the protocol

was performed according to the manufacturer’s instructions.

A total of 20 lL of the product was loaded onto a 1 %

agarose gel containing 0.5 lg/mL ethidium bromide. The

gel was visualized by Compact Digimage System (Major

Science, USA) and documented using UN-SCAN-IT gel

(Silk Scientific, USA) Image Analysis software.

Caspase-3 Colorimetric Assay

The analysis of caspase-3 activation was analyzed using a

commercially available caspase-3 assay kit from (Abcam,

Cambridge, UK). Briefly, 4 9 106 PC-3 cells were seeded

into 50 cm2 culture flasks (Jet Biofill, China) and incubated

with Mauriporin for 6, 12 and 24 h, centrifugation was used

to collect the cells and the cells were resuspended in 50 lL

cell lysis buffer. The resultant lysates were then centrifuged

at 9,0009g for 5 min. The caspase-3 assay was performed in

96-well microtiterplate by incubating 50 lg of total protein

with the caspase-3 substrate DEVD-pNA for 1 h at 37 �C.

Following the incubation period, the absorbance was mea-

sured with a microplate reader at 405 nm.

Results

Cloning and Sequence Analysis of Mauriporin

From the cDNA library prepared from A. Mauritanicus

venom mRNA, we identified a novel full length cDNA

clone which encoded an open reading frame consisting of

Int J Pept Res Ther

123

73 amino acids that encoded a single copy of Mauriporin

precursor. The complete cDNA sequence and deduced

amino acid sequence are shown in (Fig. 1). The cDNA

sequence had been deposited into EMBL Nucleotide

Sequence Database (accession number: HF545613). The

Mauriporin precursor has a putative signal peptide of 22

residues constituting the N-terminal domain of the open

reading frame that was identified with the signalIP 4.0

server (http://www.cbs.dtu.dk/services/SignalP/). The pre-

cursor is flanked C-terminally with an extra Arg–Arg–Arg

tail which is cleaved at the processing step and is consistent

with the sequence of other homologous peptides identified

from other scorpion species (Zeng et al. 2000; Zeng et al.

2012). A protein sequence similarity search using protein

BLAST revealed that Mauriporin is homologous to Tx297

(78 %) from the scorpion Buthus occitanus israelis,

Bmkbpp (74 %) from the scorpion Mesobuthus martensii

(Zeng et al. 2000), NDBP6 (76 %) from the scorpion

Lychas mucronatus (Ruiming et al. 2010), Venom AMP

(64 %) and Meucin (64 %) from the scorpion Mesobuthus

eupeus, TdBPP (64 %) from the scorpion Tityus discrepans

(D’Suze et al. 2009), and Parabutoporin (62 %) from the

scorpion Parabuthus schlechteri (Moerman et al. 2002)

(Fig. 2a). Additionally the main organizational sequence of

these peptides including the signal peptide, the mature

peptide and the C-terminal processing site seem to be

highly conserved which indicates that Mauriporin belongs

to a novel class of scorpion non-disulfide bridged peptides

(Fig. 2b). The peptide was found to display an isoelectric

point of 10.56 at pH 7 with a net positive charge of 6 that

confirms the cationic nature of the peptide. Helical wheel

projections of Mauriporin revealed the peptide to exhibit an

amphipathic nature (Fig. 2c).

Detection and Structural Characterization

of Mauriporin in HPLC Fractions of

A. Mauritanicus Venom

A broad region of antiproliferative activity was detected

in fractions 93–96 of the scorpion venom, the maximal

activity was detected in fraction 95 (Fig. 3a). Molecular

mass analysis of the contents of the HPLC fraction

exhibiting maximal antiproliferative activity was achieved

by the use of a matrix-assisted laser desorption ionization

time-of-flight (MALDI-TOF) mass spectrometer (AB

Sciex, MA, USA). Mauriporin cDNA clone revealed the

peptide to possess a computed molecular mass of

5397.36 Da and a peptide corresponding to this mass was

identified in fraction number 95. Fraction 95 was found to

contain a single charged peak at approximately 5398.1 Da

and a doubly charged peptide ion form (M?2H)2? of

2699.0 Da (Fig. 3b). This data confirms the peptide is

being actively secreted within the scorpion venom. Mau-

riporin was successfully synthesized and obtained with

a high degree of purity and the molecular mass of

the purified product was confirmed to be identical to that

of the natural peptide by use of MALDI-TOF mass

spectrometry.

Fig. 1 Nucleotide sequence of

a full length cloned A.mauritanicus venom derived

cDNA encoding Mauriporin

precursor. The predicted protein

sequence is given above the

nucleotide sequence. Putative

signal peptide is double-underlined, mature peptides are

single underlined and stop

codons are indicated by

asterisks

Int J Pept Res Ther

123

CD Analysis

To investigate the secondary structure of the peptide, the

CD spectrum of Mauriporin was obtained in 10 mM

sodium phosphate buffer solution and in a membrane-

mimicking environment (50 % TFE/H2O). In the aqueous

solution, the CD spectrum of Mauriporin displayed a

negative band around 200 nm (Fig. 4a), which is typical

for peptides in a random coil conformation. In 50 % TFE,

the CD spectra of Mauriporin exhibited double-negative

bands at 208 and 222 nm, suggesting that Mauriporin is

adopting an a-helical structure when transformed into a

membrane mimicking environment (Fig. 4b). This con-

formational change from a random-coil structure in aque-

ous buffer to an a-helical structure in membrane-mimetic

environments is common for many membrane-binding

peptides. The measure of helical content using the K2D2

deconvolution software revealed that the helical content of

Mauriporin is 73.65 % in 50 % TFE which is showing that

Mauriporin is adopting a well defined a-helical structure

within membrane-mimicking environments.

Antiproliferative Activity of Mauriporin

The impact of Mauriporin on prostate tumor cell viability

and proliferation was assessed by the MTT assay. The MTT

assay is considered as an indicator of mitochondrial activity

and has been usually applied for determination of cell via-

bility. Mauriporin was found to inhibit the proliferation of

prostate cancer cells in a potent and dose-dependent fashion

(A)

(B)

(C)

Fig. 2 Bioinformatic analysis of Mauriporin. a Peptide sequence

alignment of Mauriporin with Tx297, Bmkbpp, NDBP6, Venom

AMP, Meucin 49, TdBPP and Parabutoporin. Asterisks indicate fully

conserved amino acid residues. b Multiple sequence alignment and

comparison of domain organization of the precursors of Mauriporin,

Tx297, Bmkbpp and NDBP6. (1) The signal peptides (2) Mature

peptides (3) C-terminal Cleavage site. c Helical wheel representations

of Mauriporin displaying the amphipathic nature of the peptide.

Nonpolar and hydrophobic residues are highlighted in yellow color;

basic residues are in blue color; small neutral residues are in graycolor; small polar residues are in purple color; acidic residues are in

red color (Color figure online)

Int J Pept Res Ther

123

(Fig. 5a). Mauriporin displayed potent activity with an

average IC50 of 6.63 lM (range 4.4–7.8 lM) against all

three prostate cancer cell lines tested. The maximal anti-

proliferative activity of Mauriporin was against the DU-145

cell lines with an IC50 of 4.4 lM followed by PC3 with an

IC50 of 7.7 lM and the weakest activity was against the

LNCaP cell line with an IC50 of 7.8 lM (Table 1). The

effects of Mauriporin on non-tumorigenic cells were also

assessed using the mammalian non-tumorigenic Vero and

HUVEC cell lines. As shown in Fig. 5b, Mauriporin man-

aged to inhibit the proliferation of the Vero cell lines with an

IC50 value of 59.7 lM while the IC50 value for Mauriporin

against the HUVEC cell lines was 62.5 lM. The IC50 values

of Mauriporin against non-tumorigenic cell lines clearly

displays the apparent selectivity of the peptide against

prostate cancer cell lines as the IC50 value was approxi-

mately ninefolds higher with the non-tumorigenic mamma-

lian cells than those values which were obtained when

Mauriporin was administered against prostate cancer cell

lines.

Membrane Disrupting Activity of Mauriporin

LDH is an intracellular enzyme that is retained by viable

cells. Cells with damaged membranes release LDH into the

extracellular environment. Thus the LDH cytotoxicity

assay is a good indicator of Mauriporin lytic ability and its

ability to cause membrane damage.

Mauriporin increased LDH release from prostate cancer

cell lines in a dose dependent fashion displaying a corre-

lation with the decrease in the viability of prostate cancer

cell lines (Fig. 6). The maximal membrane damage and

lytic activity of Mauriporin was observed against the DU-

145 cell line with an IC50 of (4.8 lM) while the lowest

activity was against the PC3 cell line with an IC50 of (6.9

lM) (Table 1). The LDH assay results correspond with the

behaviour of Mauriporin against prostate cancer cell lines

in terms of cell viability. Mauriporin’s ability to disrupt

non-tumorigenic cellular membrane integrity was also

assessed using the LDH cytotoxicity assay, Mauriporin

increased LDH release from the Vero and HUVEC cell

Fig. 3 a Region of reverse-phase HPLC chromatogram of the

scorpion venom A. mauritanicus. The elution position/retention time

of the anticancer peptide Mauriporin is indicated (arrow). b MALDI-

TOF mass spectrum of Mauriporin as present in fraction 95 indicating

a singly protonated molecular mass (M?H)? of 5398.1 Da and a

doubly charged peptide ion form (M?2H)2? of 2699.0 Da

Int J Pept Res Ther

123

lines in a dose dependent fashion showing a correlation

with the behaviour of the peptide in the previous MTT

assays. However the increase in LDH release from the non-

tumorigenic cells was achieved at significantly higher

concentrations when compared to the tumorigenic cells as

the IC50 value for the Vero and HUVEC cell lines were

57.4 and 67.8 lM respectively. The results from the LDH

assay experiments confirm the selective activity of Mau-

riporin against tumorigenic prostate cancer cell lines.

Time Course for Mauriporin Induced Tumour Cell

Death

Figure 7 summarizes the time course for PC3 cell killing

induced by Mauriporin.

The percentage cell viability value for each time point

using the MTT assay was plotted as a function of time. As

shown in the figure, Mauriporin managed to inhibit the

proliferation of PC-3 cells within 30 min of treatment as

the decrease in cell viability was *30 %. The cell death

increased over the remainder of the time course reaching

50 % within 1.3 h and appears to reach a maximum at

about 6 h. The behaviour of Mauriporin is showing that the

inhibition of PC-3 proliferation seems to start instantly and

is it is activated as soon as the peptide comes in contact

with the tumorigenic cells.

Hemolytic Activity

To examine the potential toxicity of Mauriporin against

mammalian cells, we tested the peptide at several con-

centrations against sheep erythrocytes. At concentrations

equal to the IC50 value needed to inhibit the proliferation of

prostate cancer cell lines, Mauriporin caused hemolysis of

(0–1.7 %) when incubated with erythrocytes for 60 min.

When the concentration was raised to 40 lM, percentage

hemolysis value determined for Mauriporin was 3.3 %

(Table 2). At a concentration of 80 lM which is 10 times

Fig. 4 CD spectra of Mauriporin in a 10 mM sodium phosphate

buffer, pH 7.0. b 50:50 TFE:dH2O, pH 7.2, plotted as mean residue

ellipticity (deg cm2/dmol) versus wavelength (nm). Five replica scans

were collected and the average recorded. Final spectra represent

buffer subtracted data

(A)

[Mauriporin], µM

[Mauriporin], µM

% C

ell V

iab

ility

0 5 10 15 20 25 30 35 40 45 50 55 600

20

40

60

80

100

DU-145

LNCAP

PC3

(B)

% C

ell V

iab

ility

30 40 50 60 70 80 90 100 1100

20

40

60

80

100

Vero

HUVEC

Fig. 5 Cell survival curves as measured by MTT assays for

Mauriporin against a three tumorigenic prostate cancer cell lines

(PC-3, DU-145, and LNCAP), and b two non tumorigenic cell lines

(Vero and HUVEC). Cells were incubated with various concentra-

tions of Mauriporin for 24 h at 37 �C. The results are shown as

mean ± SD of three independent experiments

Int J Pept Res Ther

123

higher than the IC50 concentration of Mauriporin against

PC-3 cell lines, only 4.8 % hemolysis was observed which

clearly indicates that Mauriporin has a weak hemolytic

activity against mammalian cells a behaviour consistent

with other homologous peptides identified from scorpion

species such as Parabutoporin and BmKbpp (Zeng et al.

2000; Moerman et al. 2002).

Analysis of Mauriporin’s Effect on DNA

Fragmentation and Caspase-3 Activity

DNA fragmentation is considered to be one of the bio-

chemical hallmarks of apoptosis. Apoptotic DNA frag-

mentation results in the generation of regular DNA

fragments of 180 bp that can be visualized by agarose gel

electrophoresis. PC-3 cells treated with 20 uM of Mau-

riporin were incubated for 12 and 24 h to examine Mau-

riporin’s ability to induce apoptotic DNA fragmentation.

No evidence of apoptotic DNA fragmentation pattern with

regular DNA fragments was observed with PC-3 cells

treated with Mauriporin but on the contrary a random DNA

fragmentation pattern (smearing) was observed after 24 h

which indicates a mode of cell death that is not related to

apoptosis but possibly through a necrotic pathway

(Fig. 8a). To further characterize the cellular death

Table 1 Effect of Mauriporin on cell proliferation and cytotoxicity in

three prostate cancer cell lines PC3, DU-145, LNCAP and the non-

tumorigenic Vero and HUVEC cell lines

Cell line Cell proliferation

MTT assay

(IC50 ± SEM in lM)

Cytotoxicity

LDH assay

(IC50 ± SEM in lM)

PC3 7.7 ± 0.24 6.9 ± 0.83

DU-145 4.4 ± 0.45 4.8 ± 0.95

LNCAP 7.8 ± 0.27 6.3 ± 0.10

Vero 59.7 ± 0.13 72.4 ± 0.36

HUVEC 62.5 ± 0.19 67.8 ± 0.55

The values present mean ± SEM of three dependent experiments

VERO/24 H

Peptide Concentration (µM)

% L

DH

Rel

ease

40 50 60 70 80 90 1000

102030405060708090

100HUVEC/24 H

Peptide Concentration (µM)

% L

DH

Rel

ease

40 50 60 70 80 90 1000

102030405060708090

100

PC3/ 24 H

1 2.5 5 10 20 30 400

102030405060708090

100

Peptide concentration (µM)

LD

H R

elea

se %

Fig. 6 Effect of Mauriporin on cytotoxicity in three prostate cancer

cell lines (PC-3, DU-145, and LNCAP), in addition to two non-

tumorigenic cell lines (Vero and HUVEC). Cells were incubated with

various peptide concentrations of Mauriporin for 24 h at 37 �C. As a

control for maximum lactic dehydrogenase (LDH) release, cells were

treated with 2 % triton X-100 in serum-free RPMI medium for 2 h

before running the assay. The results are shown as mean ± SD of

three independent experiments

Time (hours)

0.5 1 3 6 12 240

10

20

30

40

50

60

70

80

90

100

% C

ell V

iab

ility

PC3

Fig. 7 PC-3 cell growth as a function of time in the presence of

50 lM Mauriporin, measured by MTT assay. PC-3 cells were treated

with Mauriporin for different times (0.5, 1, 3, 6, 12, 24 h). Data

represents the mean ± SD of three independent experiments

Int J Pept Res Ther

123

mechanism induced by Mauriporin, we performed an

in vitro assay based on the spectrophotometric detection of

the chromophore p-nitroanilide (pNA) cleavage by the

enzyme caspase-3 from the labeled substrate DEVD-pNA

which can be measured spectrophotometrically. PC-3 cells

were treated with 20 lM Mauriporin and incubated for 6,

12 and 24 h. No significant increase in caspase-3 activities

was observed with Mauriporin treatment (Fig. 8b), which

further indicates that Mauriporin is probably causing cell

death through a necrotic rather than an apoptotic mode of

cell death.

Discussion

In spite of considerable progress in the treatment of cancer

recently, Cancer is still considered one of the leading

causes of death among humans and a major burden of a

disease worldwide. Most cancer treatment regimens focus

primarily on chemotherapy as the treatment of choice for

advanced or metastatic disease. Current chemotherapeutic

drugs suffer from high toxicity and induce a wide range of

side effects that decrease the overall quality of life in

patients undergoing chemotherapy (Coughlin 2008).

Additionally cancer cells display a high tendency to

develop resistance against chemotherapeutic compounds

by expressing high efflux glycoprotein pumps that identify

and expel a wide range of chemotherapeutic agents

regardless of their structural identity (Fletcher et al. 2010).

The cationic a-helical peptides lack the toxicities and the

intracellular based mechanism of action that chemothera-

peutic drugs possess and may prove to be promising can-

didates for development as novel anticancer agents for the

treatment of metastatic disease.

In this study, we have identified and functionally char-

acterized a novel scorpion peptide named Mauriporin from

the venom constructed cDNA library of the Moroccan

scorpion A. mauritanicus. Mauriporin belongs to the group

of scorpion non-disulfide bridged peptides (NDBPs). This

group represents a diverse assembly of polypeptides that

seem to display numerous biological activities including

antimicrobial, haemolytic, bradykinin potentiating, and

immune-modulatory activities. Scorpion (NDBPs) possess

highly diverse primary and secondary structures which are

thought to be responsible for this complex biological

Table 2 Hemolytic activity of Mauriporin

Peptide concentration (lM) Hemolysis (%)

1.0 0

10 1.7

20 3

40 3.3

60 4

80 4.8

100 5.2

(A)

(B)

Time (hours)

Cas

pas

e-3

acti

vity

(Ab

sorb

ance

at

OD

405

)

6 12 24

0.00

0.02

0.04

0.06

0.08Control

Mauriporin

Fig. 8 a Lack of apoptotic DNA fragmentation in Mauriporin treated

PC-3 cell lines. Cells were cultured in medium with or without

Mauriporin (20 lM) for 12 and 24 h. DNA was isolated, subjected to

agarose gel electrophoresis, and visualized by ethidium bromide

staining. M molecular weight marker, Control control PC-3 DNA,

lane 1 PC-3 cells treated with Mauriporin for 12 h, lane 2 PC-3 cells

treated with Mauriporin for 24 h. b Effects of Mauriporin on caspase-

3 activity in cultured PC-3 cell lines. Proteolytic activity of caspase-3

was evaluated as a function of time in cellular extracts from control

PC-3 cells and from PC-3 cells exposed to Mauriporin for 6, 12 and

24 h by measuring the cleavage of the caspase-3 colorimetric

substrate peptide, DEVD–pNA. Data were obtained from three

different experiments using separate culture preparations, and are

expressed as optical density units at 405 nm (±SD)

Int J Pept Res Ther

123

diversity. Protein sequence similarity analysis of Mau-

riporin revealed the peptide to share a high degree of

sequence homology with Parabutoporin and Bmkbpp

which were identified from the venom of the scorpion

Parabuthus schlechteri and Mesobuthus martensii,

respectively (Fig. 2). This significant degree of homology

and sequence conservation indicates that these peptides

seem to constitute a new class of scorpion (NDBPs) that

probably have evolved from a common ancestral protein.

Both Parabutoporin and Bmkbpp are multifunctional pep-

tides exhibiting antimicrobial, immune-regulatory and

bradykinin potentiating activities. In addition to the activ-

ities displayed by both peptides, Mauriporin was found to

exhibit potent anticancer activities confirming the multi-

functional nature of this class of peptides and additionally

indicating the probability that Mauriporin could display a

wider range of activities in which we intend to explore in

future studies.

In terms of physicochemical properties Mauriporin was

found to adapt an amphipathic a-helical conformation in a

membrane mimetic environment, a feature that is essential

for allowing this group of peptides to exert their biological

activity (Wang et al. 2008). CD studies revealed that

Mauriporin is present in an unordered conformation under

benign conditions such as aqueous phosphate buffer solu-

tion and only when the peptide is shifted to a solution

composed of 50 % TFE that the peptide adopts an a-helical

structure. This behaviour is consistent with many mem-

brane-binding a-helical peptides studied in previous studies

(Wang et al. 2012; Ilic et al. 2013; Huang et al. 2012). The

majority of these peptides lack well defined secondary

structures in water and conformational transition induced

by TFE reflects the potential ability of Mauriporin and

structurally similar peptides to interact with anionic

membranes. TFE has the ability to act as a membrane

mimetic as it is responsible for stabilizing the hydrogen

bonds within the peptide and its surrounding solutes and

has the ability to induce an a-helical structure only in

peptides that have the potential to adopt such a confor-

mation (Corbier et al. 2001).

The results of the anti-proliferative studies showed that

Mauriporin significantly inhibited the proliferation of the

three prostate cancer cell lines tested in a dose dependent

fashion using the MTT assay. IC50 values obtained for

Mauriporin ranged between 4.4 and 7.8 lM. Additionally

high LDH, released from damaged cells, was observed

when the cells were treated with Mauriporin in a similar

manner to the behaviour of the peptide in the anti-prolif-

erative studies. Mauriporin also displayed selective anti-

proliferative and membrane lytic activities against cancer

cells when compared to non-tumorigenic cells as the

human HUVEC and monkey Vero cell lines were resistant

to Mauriporin at the IC50 values that were obtained with

the prostate cancer cell lines. This data indicates that

Mauriporin is relatively nontoxic to cells unassociated with

tumors and displays cell selectivity.

To confirm Mauriporin’s ability to selectively kill can-

cer cell lines rather than non-tumorigenic cells, we assessed

the hemolytic activity of Mauriporin against mammalian

erythrocytes. Mauriporin showed diminished hemolytic

activity against the erythrocytes in the concentration range

needed to kill the cancer cells. Even at a concentration of

80 lM, which is *10 times higher than the IC50 values

obtained for Mauriporin against the prostate cancer cells,

no significant hemolytic activity was observed and the

percentage of hemolysis never exceeded 4.8 %. This data

confirms the selective nature of the Mauriporin towards

tumorigenic cells. This behaviour could be attributed to

structural differences between tumorigenic cells and their

non-tumorigenic counterparts. Tumorigenic cells exhibit a

higher net surface negative charge due over-expression of

anionic molecules on the surface of their membranes such

as O-glycosylated mucins and Phosphatidylserines

(Dobrzynska et al. 2005; Yoon et al. 1996). This significant

difference in net negative charge could be the major

driving force of attracting a higher number of cationic

peptides towards the surface of the cells, a force that is

mainly driven by electrostatic interactions. In addition to

that, transformed cancer cells have been characterized by a

significant increase in membrane fluidity and higher sur-

face area than normal non-tumorigenic cells (Matsuzaki

et al. 1995; Mason et al. 2007), a feature which could

accumulate large amounts of cationic peptides on the sur-

face of transformed cells and increase their lytic activity

due to increased membrane fluidity.

The basis of selective killing of tumor cells by Mau-

riporin has not been completely explained. However, in this

study, we investigated Mauriporin’s ability to induce an

apoptotic mode of cell death and consequently act upon an

intracellular target within the cell. DNA laddering assays

showed that Mauriporin is not causing DNA fragmentation,

a major biochemical hallmark indicative of apoptosis. In

addition to that no increase in caspase-3 activity was

observed when PC-3 cell lines were treated with Mauriporin

at different time intervals. As caspase-3 is one of the major

enzymes activated during the cascade of events associated

with apoptosis (Saraste and Pulkki 2000), an increase in its

activity is an indicator of an apoptotic mode of cell death.

These results indicate that Mauriporin is responsible for

selective killing of tumor cells through a mechanism other

than apoptosis and possibly exerts its cytotoxic activity

through a necrotic mode of cell death which is consistent

with the mechanism of action of other cationic a-helical

peptides identified from venomous animals (Lehmann et al.

2006; Leuschner and Hansel 2004). The mode of action

suggested for these peptides indicate a lytic mode of cell

Int J Pept Res Ther

123

death caused by the ability of the peptides to inflict damage

on the membranes of cancer cells which eventually leads to

cell burst and lysis (Hoskin and Ramamoorthy 2008;

Schweizer 2009). This proposed mechanism of action

allows the peptides to evade the multi-drug resistance phe-

nomenon of cancer cells that is often associated with che-

motherapeutic drug treatment, as these peptides do not seem

to act upon an intracellular target.

In conclusion we report the identification and functional

characterization of a novel tumor selective cytotoxic pep-

tide from the venom of the Moroccan scorpion A. mauri-

tanicus. To our knowledge this is the first peptide

belonging to the NDBPs group of scorpion peptides to

display potent cytotoxic activities against cancer cells.

Several studies are needed to assess the safety and effec-

tiveness of Mauriporin within biological models as several

obstacles hinder the development of anticancer peptides as

effective therapeutics, but the initial data generated from

this study suggest that Mauriporin could be exploited to be

successfully developed as a novel antitumor agent that

could avoid the multi-drug resistance problem associated

with chemotherapy and prove to be a good candidate for

drug development for the purpose of combating metastatic

prostate cancer.

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