ReviewCationic amphiphilic peptides with cancer-selective toxicityFrank Schweizer ⁎Department of Chemistry,University of Manitoba,Winnipeg,MB,Canada R3T 2N2Department of Medical Microbiology,University of Manitoba,Winnipeg,MB,Canada R3T 2N2a b s t r a c ta r t i c l e i n f o Article history:Received 24April 2009Received in revised form 23July 2009Accepted 3August 2009Available online 14October 2009Keywords:Cationic amphiphilic peptide Cell-penetrating peptide Anticancer peptide Drug delivery PeptidomimeticDuring the last two decades cationic amphiphilic peptides and peptide sequences (CAPs)with cancer-selective toxicity have appeared.Based on their spectrum of anticancer activity CAPs can be divided into two major classes.The first class includes peptides that are highly potent against both bacteria and cancer cells,but not against mammalian cells.The second class includes peptides that are toxic to bacteria,and both mammalian cancer and non-cancer cells.Most antimicrobial and anticancer CAPs share a common membranolytic mode of action that results either in the selective disruption of the cancer cell membrane or permeation and swelling of mitochondria.The electrostatic attraction between the negatively charged membrane components of bacterial and cancer cells and CAPs is believed to play a crucial role in the disruption of bacterial and cancer cell membranes.This mode of action appears to bypass established resistance mechanisms.However,it is currently unclear as to why some CAPs kill cancer cells when others do not.In addition,non-membranolytic mode of actions of CAPs is increasingly recognized to contribute signi ficantly to the anticancer activity of certain CAPs.The development of CAP-based chemotherapeutics is complicated due to the traditionally poor pharmacokinetic properties and high manufacturing costs of peptides and the low intrinsic selectivity for cancer cells.Peptidomimetic approaches combined with novel selective delivery devices show promise in overcoming some of these obstacles.Furthermore,the ability of CAPs to bypass established resistance mechanisms provides an attractive strategy to develop novel lead structures for cancer treatment.©2009Elsevier B.V.All rights reserved.Contents1.Introduction ..............................................................1902.Membrane differences of cancer cells implicated in contributing to the selective permeability and toxicity of CAPs ................1912.1.Membranolytic mode of actions of CAPs against cancer cells ....................................1912.2.Non-membranolytic mode of actions of CAPs ...........................................1923.Chemical modi fications on CAPs designed to improve pharmacokinetics and reduce toxicity ..........................1924.Synergistic effects of CAPs with other anticancer drugs and occurrence of multidrug resistance .......................1935.Conclusions and outlook ........................................................193Acknowledgement..............................................................193References .. (193)1.IntroductionMost of the anticancer agents currently used are based on alkylating agents,antimetabolites and natural products that display little or no selectivity against normal mammalian cells,which results in severe side effects (Smith et al.,2007).These agents interact with intracellular targets and need to penetrate through membranes.Oncethey have reached the cytosol many of these alkylating agents are deactivated by resistance mechanisms that transport these com-pounds out of the cell before interacting with intracellular targets (Pérez-Tomás,2006).Clearly,in order to battle drug resistance and reduce normal cell cytotoxicity,the development of novel drugs and delivery systems with novel mode of actions and high cancer cell selectivity are crucial.In the search for new cancer agents and delivery systems innate immunity peptides and synthetic innate immunity peptidomimetics have recently attracted attention due to their novel mode of actions,decreased likelihood of resistance development,andEuropean Journal of Pharmacology 625(2009)190–194⁎Tel.:+12044747012;fax:+12044747608.E-mail address:schweize@cc.umanitoba.ca.0014-2999/$–see front matter ©2009Elsevier B.V.All rights reserved.doi:10.1016/j.ejphar.2009.08.043Contents lists available at ScienceDirectEuropean Journal of Pharmacologyj o u r n a l h o me p a g e :w w w.e l sev i e r.c om /l oc a te /e j p h a rlow intrinsic cytotoxicity (Hoskin and Ramamoorthy 2008;Papo and Shai,2005;Leuschner and Hansel,2004).Innate immunity peptides are produced by many organisms as a first line of defense to fight antimicrobial invaders (Zasloff,2002).In addition,many of these peptides modulate the immune response in mammals through a variety of mechanisms (Bowdish et al.,2005).Innate immunity peptides show great structural diversity,but have some common features,including:their relatively small size (usually less than 50amino acid residues),their cationic nature (due to the presence of mostly multiple arginine and lysine but also histidine residues),and a substantial portion of hydrophobic amino acids (around 50%)(Zasloff 2002;Hancock and Sahl,2006).For the remainder of this paper we will refer to these peptides as cationic amphiphilic peptides (CAPs).The exact mode of action of CAPs is not completely understood.However there is a consensus that most of the CAPs selectively disrupt cell membranes,possibly by transient pore formation or disruption of lipid packing (Giuliani et al.,2008).In addition,intracellular targets have also been suggested for some CAPs (Park et al.,2000).Many of these peptides are unstructured in solution.However,upon binding to bacterial membranes most of the longer peptides will adopt a well-de fined amphipathic structure that is crucial to enhance membrane permeability (Zasloff,2002;Hancock and Sahl,2006;Giuliani et al.,2008).It should be noted that a well-de fined amphipathic structure is not required.For instance,ultrashort di-,tri-and tetrapeptide sequences (Haug et al.,2008;Svensen et al.,2008;Strom et al.,2003)and ultrashort lipopeptides that contain a polar peptide-based cationic head group (consisting of positively charged arginine and lysine residues and a hydrophobic tail composed of a lipophilic acid or hydro-phobic amino acid(s))are also able to induce a strong antibacterial in vitro activity (Makovitski et al.,2008;Makovitzki and Shai,2005;Malina and Shai 2005).Some of these ultrashort antimicrobial peptides display little hemolytic activity,while others show strong hemolytic activity.It is believed that the selective bacterial cytotoxicity of CAPs is caused by the af finity of the net negative charge found on bacterial cell membranes in contrast to eukaryotic lipid bilayers,which are typically made up of zwitterionic phospholipids (Hancock and Sahl,2006).The overall positive charge of CAPs ensures accumulation at polyanionic microbial cell surfaces that contain acidic polymers such as lipopoly-sacharides and cell wall associated teichoic acids,in Gram-negative and Gram-positive bacteria,respectively.Subsequently,CAPs transit the outer membrane of Gram-negative bacteria via self-promoted uptake (Hancock and Lehrer,1998),insert themselves into the cytoplasmic membrane,and act by either disrupting the physical integrity of the bilayer via membrane thinning,transient poration and/or disruption of the barrier function,or translocating across the membrane and acting on internal targets (Hancock and Sahl,2006).Interestingly,many CAPs also have the ability to kill cancer cells.Based on their spectrum of anticancer activity CAPs can be divided into two major classes (Papo and Shai,2005).The first class includes peptides that are highly potent against both bacteria and cancer cells,but not against mammalian cells.This class of peptides consists ofinsect cecropins and magainins isolated from the skin of frogs and others.The second class includes peptides that are toxic to bacteria,and both mammalian cancer and non-cancer cells.Members of this class include the bee venom melittin,tachyplesin II isolated from the horseshoe crab,human neutrophil defensins,insect defensins and the human LL-37.However,it must be pointed out that many innate immunity-derived CAPs do not possess antitumor activity (Papo et al.,2003;Cruciani et al.,1991;Papo and Shai,2003;Shin et al.,2000;Srisailam et al.,2000).This raises the question,what are the molecular and structural factors of CAPs responsible for inducing cancer toxicity (Table 1)?2.Membrane differences of cancer cells implicated in contributing to the selective permeability and toxicity of CAPsA variety of fundamental differences exists between the cell membranes of cancer cells and normal cells that may explain the cancer-selective toxicity of certain CAPs.Cancer cell membranes typically carry a net negative charge due to elevated expression of anionic molecules such as phosphatidylserine (Utsugi et al.,1991;Dobrzynska et al.,2005),O-glycosylated mucins (high molecular weight glycoproteins consisting of O-glycosidic linkages between serine and threonine that are rich in negatively charged saccharides comprised of sialic acids and sulfates)(Yoon et al.,1997;Burdick et al.,1997),sialilated gangliosides (Lee et al.,2008)and heparan sulfates (Kleeff et al.,1998).In contrast,the surface of normal mammalian cell membranes is mainly composed of neutral zwitterionic phospholipids and sterols (Hoskin and Ramamoorthy,2008).It has been suggested that cholesterol,which is a major component of eukaryotic cell membranes (Simons and Ikonen,2000)may protect eukaryotic cells from the cytolytic effects of antimicrobial CAPs by altering membrane fluidity and thereby interfering with the membrane insertion of lytic peptides.This hypothesis is supported by the observation that membrane insertion of the CAP cecropin,is reduced when the cholesterol content of the membrane is increased (Steiner et al.,1988;Silvestro et al.,1997).Moreover,some breast and prostate cancer cell lines have recently been shown to possess elevated levels of cholesterol-rich lipid rafts (Li et al.,2006),which make these cell lines less susceptible to lysis by CAPs.Another difference between the membrane of cancer and non-cancer cells is based on the relatively higher number of microvilli on tumorigenic cells compared to normal cells (Zwaal and Schroit,1997).This increases the surface area of the tumor cell which may allow the cancer cell to interact with an increased number of CAPs (Chan et al.,1998).2.1.Membranolytic mode of actions of CAPs against cancer cells Similar to the mode of action of antimicrobial peptides that disrupt the bacterial cell wall,the anticancer properties of CAPs may involve selective lysis of the cancer cell membrane.Initial evidence of such a membranolytic mechanism was discovered in a study of magaininTable 1Selected naturally occurring CAPs with anticancer activities.Peptide SequenceAnticancer activityMelittin GIGAVLKVLTTGLPALISWIKRKRQQ Membranolytic Hristova et al.(2001);phospholipase A 2activator Sharma (1992,1993);phospholipase D activator Saini et al.(1999)Tachyplesin KWC 1FRVC 2YRGIC2YRRC 1RActivation of complement C1q Chen et al.(2005);induction of cancer cell differentiation Ouyang et al.(2002)LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Membranolytic Henzler-Wildmann et al.(2003)Cecropin B KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL Membranolytic Hung et al.(1999)Magainin 2GIGKFLHSAKKFGKAFVGEIMNS Membranolytic Cruciani et al.(1991)Buforin IIb RAGLQFPVGRLLRRLLRRLLR Mitochondria-dependent apoptosis,caspase 9activation Lee et al.(2008))Alloferon-1HGVSGHGOHGVHG Immunomodulatory Chernysh et al.(2002)Alloferon-2GVSGHGQHGVHGImmunomodulatory Chernysh et al.(2002)191F.Schweizer /European Journal of Pharmacology 625(2009)190–194and its synthetic analogues against hematopoietic and solid tumors (Cruciani et al.,1991).A crucialfinding of this study was the discovery that the negative charge gradient across the plasma membrane was crucial for membrane disruption and cytotoxicity.Furthermore, replacement of the L-amino acids in magainin by D-amino acids retained anticancer activity,thereby ruling out a receptor-mediated pathway.Similar results were obtained with other antimicrobial CAPs including melittin,cecropin,roctonin and others(Wade et al.,1990; Bessalle et al.,1990;Merrifield et al.,1995;Hetru et al.,2000). Subsequent studies using antimicrobial peptides containing both D-and L-amino acids against various cancer cell types revealed that the cells died after perturbation of the plasma membrane.Two poten-tial mechanisms can account for the membranolytic properties of CAPs;these are referred to as‘carpet’and‘barrel-stave’mechanisms. According to the carpet mechanism(Oren and Shai,1998),CAPs align themselves parallel to the negatively charged membrane surface in a carpet-like fashion until a critical threshold concentration is reached, after which the CAP permeates the membrane.Alternatively,lytic CAPs self aggregate via hydrophobic interactions in the membrane and form transmembrane channels or pores via the‘barrel-stave’mechanism(Shai,1999).The membranolyic mode of action of CAPs is not limited to the cell membrane,and can also be extended to the permeation and swel-ling of mitochondria,which results in a release of cytochrome c and induces apoptosis(Mai et al.,2001;Li et al.,2000).The release of cytochrome c from damaged mitochondria also induces Apaf-1 oligomerization,caspase9activation and conversion of pro-caspase3 to caspase3(Cory and Adams,1998).This mitochondrial pathway of apoptosis takes place with the CAPs granulysin(Pardo et al.,2001)and (KLAKLAKKLAKLAK)fused to a CNGRC homing domain(Ellerby et al., 1999).Recently,it has been shown that the CAP RAGLQFPVG[RLLR]3 (buforin IIb),a histone H2A-derived peptide,displayed cancer-selective toxicity against60human tumor cell lines(Lee et al.,2008).Buforin IIb traversed cancer cell membranes without damaging them and induced mitochondria-dependent apoptosis,as confirmed by caspase9activa-tion and cytochrome c release to the cytosol.However,the exact mechanism for buforin IIb-mediated apoptosis is not clear(Lee et al., 2008).Moreover,the mitochondrial pathway of apoptosis can also be associated with the death receptor pathway(Kaufmann and Earnshaw, 2000).For instance,the CAP tachyplesin conjugated to the integrin RGD homing domain,induces apoptosis via both mechanisms.It rup-tures mitochondrial membranes while at the same time up-regulating members of the death receptor pathway,including Fas ligand,FADD and caspase8(Chen et al.,2001;Park et al.,1992).2.2.Non-membranolytic mode of actions of CAPsBesides the CAP-induced disruption of mitochondrial or plasma membranes in cancer cells there is growing evidence that alternative mechanisms exist.For instance,melittin(GIGAVLKVLTTGLPALIS-WIKRKRQQ)is a26amino acid channel-forming CAP,which specifically counter-selects cells with the overexpressed ras oncogene.Melittin acts through hyperactivation of phospholipase A2(PLA2)in the ras oncogene-transformed cells,resulting in their selective destruction (Sharma,1992).In another study it was shown that the CAPs melittin and cecropin reduce human immunodeficiency virus1(HIV-1)rep-lication and gene expression in acutely infected cells at subtoxic concentrations as either intact peptides,or when expressed in cells using retroviral expression plasmids.Analysis of the effect of melittin on cell-associated virus production revealed decreased levels of Gag antigen and HIV-1mRNAs.Transient transfection assays with HIV long terminal repeat(LTR)-driven reporter gene plasmids indicated that melittin has a direct suppressive effect on the activity of the HIV LTR. This study concluded that both melittin and cecropin are capable of inhibiting cell-associated production of HIV-1by suppressing HIV-1 gene expression(Wachinger et al.,1998).Moreover,a recent study has revealed the cytokine-like modulating properties of insect-derived, histidine-rich antitumor CAPs,termed alloferons.In vitro studies using synthetic versions of HGVSGHGOHGVHG(alloferon1)and GVSGH-GQHGVHG(alloferon-2)revealed that these peptide sequences have stimulatory activities on natural killer lymphocytes,whereas in vivo trials indicate induction of interferon(IFN)production in mice.These results suggest that alloferons have immunomodulatory properties which mayfind future use in the development of novel anticancer and antiviral chemotherapeutic approaches(Chernysh et al.,2002).3.Chemical modifications on CAPs designed to improve pharmacokinetics and reduce toxicityThe development of CAP-based antitumor drugs is hampered by the poor pharmacodynamic properties of peptide-based drugs(Hancock and Sahl,2006).CAPs based on naturally occurring L-amino acids are prone to proteolytic cleavage that limits the half time of these drugs in serum and reduces their oral bioavailability.Increased resistance to proteolytic cleavage can be achieved by partial or full replacement of L-amino acids by D-amino acids or incorporation of unnatural amino acids containing unusual side chains without affecting their antitumor effects. For instance,mice bearing several types of tumors were intraperitone-ally-injected with the CAP magainin2and its all-D amino acid ana-logue MSI-238;both were found to be active against P388leukemia, S180ascites and spontaneous ovarian tumor.While MSI-238showed a10-fold higher in vitro activity when compared to magainin,it was only2-fold more active in vivo against murine tumors(Baker et al., 1993).Serum components such as negatively charged albumins and high-and low-density lipoproteins can also affect the antitumor potency of CAPs.For example,the antitumor activity of human defensins is abolished by low levels of serum due to inactivation by low-density lipoprotein(Lichtenstein et al.,1986).Similar observations have been made with antibacterial CAPs.In this case non-specific binding of CAPs to albumin and lipoproteins significantly decreases their antibacterial activity in vitro(Peck et al.,1993).Another approach to overcome the poor pharmacokinetics and potential toxicity of CAPs involves the vector-mediated delivery of genes encoding membranolytic or apoptotic CAPs into tumor cells. For example,expression of cecropin in tumor cells resulted in either a complete loss or reduced tumorgenicity(Winder et al.,1998).Another strategy to reduce the toxicity of cell-penetrating CAPs can be achieved by conjugation to a homing peptide.Homing peptides are suitable carriers for therapeutic and diagnostic agents to tumors due to their selectivity for a specific site in the body(Ruoslahti et al.,2005).Previous studies have shown that several vasculature homing peptides conju-gated to anticancer drugs improve the anti-angiogenic efficacy by inhibiting tumor angiogenesis,while reducing drug effects on other organs(Arap et al.,1998;Sacchi et al.,2006;Sacchi et al.,2004). However,tumor homing peptides without cell-penetrating properties significantly reduce their applicability,as a result of their inability to carry drug molecules inside cancer cells.In order to overcome this drawback chimeric peptide conjugates containing a cell-penetrating CAP sequence linked to homing peptide sequences such as protein transduction domains(Wadia and Dowdy,2002),integrin receptor ligands(Chen et al.,2001)and the linearfive amino acid long peptide CREKA that selectively recognizes clotted plasma proteins on tumor blood vessels and stroma(Simberg et al.,2007;Mäe et al.,2009)have been studied.Many other tumor homing peptide sequences are known(Shadidi and Sioud,2003).A recent study describes the use of the cell-penetrating cationic amphiphilic peptide p Vec(LLIILRRRI-RKQAHAHSK)that is the conjugated homing peptide CREKA and the DNA alkylating agent Cbl(chorambucil).In vitro studies of the corre-sponding chimeric peptide-chlorambucil conjugate Cbl-CREKA-p Vec reduced the percentage of proliferating MDA-MB-231cells from 100%to approximately40%.At the same time Cbl,CREKA or pVec192 F.Schweizer/European Journal of Pharmacology625(2009)190–194alone did not show any toxicity,confirming the Cbl-mediated effect on cancer cells.This study demonstrated that CREKA-pVec is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent into tumor cells(Mäe et al.,2009).4.Synergistic effects of CAPs with other anticancer drugs and occurrence of multidrug resistanceThe membranolytic mode of action of antitumor CAPs can result in additive or synergistic effects when combined with conventional chemotherapeutics.For instance,the CAP cecropin which displays anticancer activity against ovarian,carcinoma,breast carcinoma and leukemia cells(Chen et al.,1997)synergizes the anticancer drugs S-fluorouracil and cytarabine against acute lymphoblastic leukemia cells(Hui et al.,2002).Similarly,the CAPs OLP-1(Ac-ILKKWPWW-PWRRK)and a diastereomeric version containing two D-amino acids such as D-isoleucine(i)and D-lysine(k)OLP-4(Ac-iLKKWP-WWPWRRk)enhance the in vitro cytotoxic activity of doxorubicin against tumor cells(Johnstone et al.,2000).Previously,magainin analogues in combination with chemotherapeutic agents such as cisplatin,doxorubicin and etoposide were found to have an additive effect against lung cancer cells(Ohsaki et al.,1992).Interestingly,the potency of these anticancer peptides was not affected by the presence of the multidrug resistance1(mdr1)gene.Similar results were obtained with the magainin2against human BRO melanoma cells transfected with the mdr1gene(Lincke et al.,1990).Based on these results,it appears that membranolytic CPAs may be able to bypass established resistance mechanisms thus providing new lead structures to delay or reduce the risk of resistance occurrence.5.Conclusions and outlookCurrent cancer treatments have many harmful side effects and give rise to multidrug resistance.Clearly,novel therapeutic ap-proaches with reduced risk of resistance development and higher cancer-selectivity are urgently required.Over the last two decades several CAPs with high cancer-selective toxicity have appeared.Most of these CAPs also kill microbial pathogens.However,many CAPs that kill pathogens do not have anticancer activity.In addition,antimi-crobial CAPs with cancer-selective toxicity may or may not be hemolytic.The reasons for this selectivity are poorly understood.It is anticipated that biophysical studies dealing with the structure, dynamics,topology,mechanisms of membrane disruption and the role of individual components of membranes will provide new insight and rationalizations to explain the observed selectivity differences.While a variety of CAPs displays cancer-selective toxicity it is currently difficult to develop peptide-based drugs due to poor pharmacokinetics,potential systemic toxicity and the high cost of manufacturing(Hancock and Sahl,2006).Ultrashort antimicrobial CAP sequences 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