Role of protease and protease inhibitors in cancer pathogenesis andtreatment ScienceDirect

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Review

Role of protease and protease inhibitors in cancer pathogenesis and treatment

Ali Eatemadi

^a,b,

*, Hammed T. Aiyelabegan

^a,c

, Babak Negahdari

^a

,

Mohammad Ali Mazlomi

^a

, Hadis Daraee

^b,d

, Nasim Daraee

^b,d

, Razieh Eatemadi

^e

, Esmaeil Sadroddiny

^a,

*

aDepartmentofMedicalBiotechnology,SchoolofAdvancedTechnologiesinMedicine,TehranUniversityofMedicalSciences,Tehran,Iran

bDepartmentofMedicalBiotechnology,SchoolofMedicine,LorestanUniversityofMedicalSciences,Lorestan,Iran

cTehranUniversityofMedicalSciencesInternationalCampus(TUMS-IC),Tehran,Iran

dDepartmentofMedicalBiotechnology,FacultyofAdvancedMedicalSciences,TabrizUniversityofMedicalSciences,Tabriz,Iran

eUniversityofIlam,Ilam,Iran

ARTICLE INFO Articlehistory:

Received25August2016

Receivedinrevisedform26November2016 Accepted5December2016

Keywords:

Protease Proteaseinhibitor cancer

ABSTRACT

Canceristhesecondcauseofdeathin2015,andithasbeenestimatedtosurpassheartdiseasesasthe leadingcauseofdeathinthenextfewyears.Severalmechanismsareinvolvedincancerpathogenesis.

Studieshaveindicatedthatproteasesarealsoimplicatedintumorgrowthandprogressionwhichis highly dependenton nutrientand oxygensupply.Ontheotherhand,protease inhibitorscould be consideredasapotentstrategyincancertherapy.Onthebasisofthetypeofthekeyaminoacidinthe activesiteoftheproteaseandthemechanismofpeptidebondcleavage,proteasescanbeclassiﬁedinto six groups: cysteine, serine, threonine, glutamic acid, aspartate proteases, as well as matrix metalloproteases.In thisreview,we focusontheroleof differenttypesofproteases andprotease inhibitorsincancerpathogenesis.

Contents

1. Proteases... 2

2. Theroleofproteaseincancerdevelopment ... 2

2.1. Cysteineproteases ... 3

2.2. Serineproteases ... 4

2.3. Aspartateproteases ... 4

2.4. Threonineproteases ... 4

2.5. Matrixmetalloproteases ... 4

3. Roleofproteaseinhibitorsincancertreatment ... 4

3.1. Cysteinecancerproteaseinhibitors ... 5

3.2. Serinecancerproteaseinhibitors ... 5

3.3. Aspartatecancerproteaseinhibitors ... 5

3.4. Glutamicacidcancerproteaseinhibitors ... 7

3.5. Threoninecancerproteaseinhibitors ... 7

3.6. Matrixcancerproteaseinhibitors ... 8

4. Conclusion ... 8

Conﬂictofinterest ... 8

Acknowledgement ... 8

References ... 9

* Correspondingauthorsat:DepartmentofMedicalBiotechnology,SchoolofAdvancedTechnologiesinMedicine,TehranUniversityofMedicalSciences,Tehran,Iran.

E-mailaddresses:[email protected](A.Eatemadi),[email protected](E.Sadroddiny).

http://dx.doi.org/10.1016/j.biopha.2016.12.021

Availableonlineat

ScienceDirect

www.sciencedirect.com

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1.Proteases

One of the most important biological catalytic reactions is proteolysisand this is knownasproteolytic activity,which has beenattributedtoaclassofenzymescalledproteases.Proteolysis isthehydrolysisofpeptidebondbyattackingthecarbonylgroupof the peptide. Proteases are of broad enzymes distribution. In human,thereareabout990knownproteasegenes.Inaddition, about1605knownproteaseinhibitorgeneshavebeenreportedin human[1].

Onthebasisofthenatureofthekeyaminoacidintheactivesite of the protease and the mechanism of peptidebond cleavage, proteases can be classiﬁed into six groups: cysteine, serine, threonine, glutamic acid,aspartate proteases, as well as matrix metalloproteases[2–4].

The cleaving mechanism of a peptide bond witha protease usually occurin the presence of water molecule(in aspartate, metallo-and glutamic acid proteases) or a cysteine, serine, or threonineresidue(typicallyahistidineresidueactivation)asthe nucleophileintheactivesite[5](Fig.1).

Theassociationbetweenstromal and tumorcellsmodulates twoproteasesystemsthatareinvolveinproteolysisoutsidethe cell, these are the MMPs and urokinase plasminogenactivator (uPA)/uPAreceptor(uPAR)/plasminogennetwork.StromalMMP-2 and uPA are synthesized as inactive precursors and then stimulated onthe tumorcells surface (Fig.1), thereby causing malignant cells to rupture the basement membranes. These enzymes also promotes blood vessels sprouting to feed the growing cancer. Antitumor therapies targeting these stromal contributionstometastasis,invasion,andangiogenesis,attacka geneticallyconstantcellpopulation, sotheymaynotattackthe resistance related to the use of traditional chemotherapeutic drugs.

2.Theroleofproteaseincancerdevelopment

Proteases in normal cells are very essential in carrying out imperative biological processes,and canregulate a diversityof differentcellularprocessessuchasgeneexpression,differentia- tion,andcelldeath[6].However,recentstudieshaveindicatedthat

Fig.1.Generalcascadeofproteaseinhibitormechanismofactionontumorcells.

Fig.2. Epithelialtomesenchymaltransitioninmetastasis.

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proteasesarealsoimplicatedintumorgrowthandprogression, bothatprimaryandmetastaticsites[3].

Ithasbeenshown,thattumorcellsstimulatetheexpressionof proteolyticenzymesinnon-neoplasticneighboringcells,hijacking theiractivitytofavortumorexpansion[7,8].

Metastases and tumor progression are highly dependenton nutrient and oxygen supply, which are motivated by various proteasesinthetumorand/orsurroundingtissuesandorgans[3].

Severaltumorshavebeenindicatedtohaveincreasedthelevelsof proteasesatanearlystageandtheseproteasesarenowindicated tobeinvolvedin manyaspects ofcancer,suchasproliferation, immune responses,inﬂammatorycell recruitment, tumorinva- sion,angiogenesis,metastasis,apoptosis,epithelialtomesenchy- maltransition (EMT) as depictedin Fig. 2, the mobilizationof normalcellsfromtheirtissuecompartmentstoassistinmetastasis (Fig.3),aswellasresponsetotherapysuchaschemoresistanceto drugs [9,10]. In thesubsequentsectionof this review, wehave gatheredevidenceemphasizingontheroleofeachproteaseclasses ondifferenttypesofcancers.Table1showsdifferentproteasesand typesofcancercellwheretheirfunctioniselucidated.

2.1.Cysteineproteases

Cysteineproteases(calpains)arelocalizedinthecytosol,orthe lysosome(cathepsinsB,L,HandS)andaresecretedinsomecell

typesunderpathologicalconditions.Thisclassofproteasemediate generalfunctionssuchascatabolismofintracellularproteinand specializedfunctionssuchasselectiveactivationofextracellular protein degradation, macrophage function, bone resorption or signalingmolecules(e.g.interleukin,proteinkinaseC,encephalin) [8].Thefamilyofcathepsincysteineproteasescandegradeboth intracellular and extracellular matrix (ECM) proteins [11,12].

Cathepsins havebeen showntofunction extracellularlyaswell asintracellularly.Cathepsinsarepredictedtobeapotentialtargets for anti-cancer therapy [12,13], as the extracellular activity of cathepsins promotescancercells progression tonearbytissues, blood and lymph vessels and metastasize to outlying tissues [14,15].CathepsinBhasbeenactuallyimplicatedintheremodeling anddissolutionofbasementmembraneandconnectivetissuein the processes of tumor growth, invasion, and metastasis [16], whichmayresultsinpodosome-mediatedECMdegradationand invasionviasecretedlysosomes[17].Cathepsinsarealsoconsid- eredasusefulprognosismarkersforrecognizingpatientswhoare sufferingfromcolorectalcancer[18],breastcancer[19],pancreatic cancer[20]andtonguecarcinoma[21].Increasedlevelsofother lysosomalproteases,suchascathepsinsD,H,orL,havealsobeen reportedinseveral cancertypes[22].CathepsinL2 (CTSL2)has beenshowntobeupregulatedinadifferentmalignancieslikelung, breast, gastric, colon, head and neck carcinomas, gliomas, melanomas[23]aswellasendometrialcancer[24].

Fig.3. Principalstagesintheformationofmetastasis.

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2.2.Serineproteases

Normal regulation of serine proteasesactivity is critical for physiological activities of the cell and tissue. But abnormal regulation of these proteases activity can lead to pathological conditionssuchascancer[25].Justaboutonethirdofallproteases canbe classiﬁedas serine proteases, which are knownfor the existenceof thenucleophilicSerresidue attheactivesite[26].

Serineproteaseswasinitiallywell-knownbytheexistenceofthe Asp-His-Ser“chargerelay” system or “catalytic triad” [27].The Asp-His-Sertriadcanbeestablishinatleastfourdiversestructural contexts;subtilisin, chymotrypsin, carboxypeptidase Y,and Clp protease (MEROPS) nomenclature [28]; signifying that this catalytic machinery have developed on at least four separate occasions[27].

There are another serine proteases with different catalytic triadsanddyads,comprisingSer-His-Glu,Ser-Lys/His,His-Ser-His, andN-terminalSer[27].

One of the well characterized serine proteases is trypsin.

These proteases play a critical roles in a wide spectrum of pathological processes like, inﬂammation, atherosclerosis and cancer[25].

2.3.Aspartateproteases

Cathepsin-D (Cath-D) is an aspartic endo-protease that is universallyfoundinlysosomes[29].Cath-Dhadtwomainroles:

precursor’s activation of biologically active proteins in pre- lysosomal compartments of specialized cells, and as a major protein-degradingenzymeinlysosomesandphagosomes[30].The asparticprotease Cath-D is overexpressedand secreted at high levelsbyhumanepithelialbreastcancercells[31–34],thus,itisa markerofpoorprognosisinbreastcancer[35].Cath-Dmotivates ﬁbroblastoutgrowth,cancercellproliferation,angiogenesis and

metastasis[36–38].Cath-D,likeotheraspartateproteasessuchas renin,pepsinogen,chymosin,hasabilobedstructure[39].

2.4.Threonineproteases

Threonine proteases or proteasomes partake in removing cellular proteins, which is tagged for degradation through a complexmodiﬁcationknownaspoly-ubiquitination;aprocedure of adding a series of ubiquitin molecules to another protein, targeted for degradation [40]. The expression of the majority proteins is controlled bythe proximalactivityof theubiquitin proteasomesystem throughtheactivityofspeciﬁcproteinsand enzymecomplexes,includingthe76-amino-acidproteinupstream ubiquitin, the E1, E2, and E3 ubiquitin ligase machinery, and deubiquitinatingenzymes[41].Aberrantproteasome-dependent proteolysisappearstobeassociatedwiththepathophysiologyof somemalignanciessuchascancer[42].

2.5.Matrixmetalloproteases

Matrix metalloproteases (MMPs) are class of nine or more highlyhomologousZn²⁺endopeptidasethattogethercleavesmost oftheconstituentsoftheECM.IncreasedlevelofMMPsexpression has been reportedin multiple tumor types [43,44]. MMPs are responsiblefortheremodelingandturnoverofECMproteins[7]

andtheincreasedexpressionassociatedwiththepathophysiology ofcancer

3.Roleofproteaseinhibitorsincancertreatment

Whileproteaseareinvolveinpathogenesisofcancer,protease inhibitors have been noted for their role in cancer therapy.

However,proteaseinhibitortherapydesigniscomplicatedsince Table1

Allclassesofproteaseandtypesofcancerwheretheyarelocalize.

Family Protease Location Cancer Ref.

Cysteine Cathepsins

General Intracellular,lysosome Most [1,2]

Cathepsin K

Extracellular,bone Breast [3]

Cathepsin B

Extracellularandpericellularunder pathologicalconditions

Breast,cervix,colon,colorectal,gastric,headandneck,liver,lung,melanoma, ovarian,pancreatic,prostate,thyroid

[4–6]

Cathepsin L

Breast,colorectal [7]

Aspartatic Cathepsins

Cathepsin E

Endosomalstructures,ER,Golgi Cervical,gastric,lung,pancreasadenocarcinomas [8]

Cathepsin D

Lysosome Breast,colorectal,ovarian [8]

Kallikreins(hK) General Intracellular,secreted Most [9,10]

Hk1 Tissue Lungsadenocarcinoma [11]

PSA(Hk3) Prostate,ovarian [2]

Hk10 Colon,ovarian,pancreatic,headandneck [12]

Hk15 Ovarian,prostate [13]

Serine Proteases

Upa,uPAR Membrane,pericellular Cervical,colorectal,gastric,prostate [14]

Caspases Intracellular

MMPs General Extracellular Most [15]

MMP-1, 8, 13

Breast [15]

MMP-2, 9 Breast,Colorectal,lung,malignantgliomas,ovarian [16,17]

MMP 14 Membrane Breast [18]

ADAM Extracellular

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differenttypesofcancersusedifferentproteasesattheﬂuctuating stagesofcancerdevelopmentandnosingleinhibitorcanbeused onallclassesofproteases[45,46].

3.1.Cysteinecancerproteaseinhibitors

Cathepsins(lysosomalcysteineproteases)canbecontrolledby theendogenouscysteineproteaseinhibitor,knownascystatin,in normaltissuesandcells.Thecystatinsuperfamilyareagroupof reversible, tight-binding competitive inhibitors for cysteine peptidases such as cathepsins B, H, and L that inhibits the proteolyticactivityofcysteineproteases[46].

Itwasproposedthatcysteinecancerproteaseinhibitorsplays animportantroleincancer,whicharerelatedwithalterationsof theproteolyticsystem[47].Recentstudieshaveshowedcystatins toblockmetastasisorinvasionofdiversecancersinexperimental systems[48].

Theprimeroleofcystatinsasproteaseinhibitorsistolimitextra cysteineproteaseactivityreleasedfromlysosomesorgenerated duringinﬂammation.Ascysteineproteasesplayimportantrolesin tumorgrowth,development,andmetastasis,thecystatinsshould mediatethecontroltheseprocesses.

Thereare3typesofcystatin;typeIandIIcystatinsbeingthe most studied in cancer. Main species of type I cystatins are cystatinsAandB,whicharecommonlyreferredtoasSteﬁnsAand B.Type2includescystatinsC,D,E/M,F,G(CRES),S,SNandSA [48,49].

DecreasedlevelofStefinAexpressionhavebeenfoundinsome epithelial-type cancers which correlates with lower patient survival [50]. Furthermore,evidence showsthat StefinA levels couldbereducedattheproteinandtranscriptionallevelsduring tumorprogression [51]. Studies havealso showa decreasesin StefinsAandBinbreastcancercelllinesinincreasinginvasiveness [52].ImmunostainingStefinAexistinginbenignbutnotmalignant meningiomashavebeenshown[53,54].

In glioblastoma, the invasive capability of tumors can be confirmedbycystatinmarkers[55].ImmunologicstainingofStefin A was also markedly decreased in pituitary adenomas while cathepsinlevelswereamplified[54].Poorpatientsurvivalinhead andneckcancercanbecorrelatedwithlowStefinAlevels[56].

Altogether,lowerexpressionofstefinsAandBhasbeenshown in aggressivetumortypesandincreaseinprimarytumorStefin levels appear to correlate with a more favorable prognosis [48,49,57]. Patients withsmall cell lung cancer (SCLC) demon- stratedahigherlevelsofStefinAandBintumorthaninnormal tissue [58]. Some of cysteine cancer protease inhibitors are summarizedinTable2.Fig.4depictthe3-Dstructureofseveral proteaseinhibitorsdiscussedinTables2and3.

3.2.Serinecancerproteaseinhibitors

AtypeIItransmembraneserineproteaseknownasmatriptase, is involvedinthedegradationofECM, angiogenesis,and inthe developmentofsomeepithelialcancers[68].Butinnormalcells andtissues,itisrepressedbyhepatocytegrowthfactoractivator inhibitor-1(HAI-1).For theperiodofthedevelopmentofhuman prostate cancer (CaP), there is loss of HAI-1and expression of matriptase. It has been proposed that the ratio of these two proteinsmayserveasafavorablebiomarkerforhumanprostate cancer progression and a probablemarker for establishing the efﬁcacyofchemopreventiveandtherapeuticinterventions[69].

Serpins(serineproteaseinhibitors)areagroupofproteinswith similarstructuresthatwereﬁrstrecognizedasasetofproteins abletoinhibitserineproteases[70,71].Importantserpinsincancer aresummarizedinTable3.

3.3.Aspartatecancerproteaseinhibitors

Therehasbeenseveralstudiesintheimprovementofaspartic proteinase inhibitorsastherapeuticagents forthetreatmentof Table2

Selectedcysteinecancerproteaseinhibitors.

Inhibitors Cancertype Description Ref(s)

SteﬁnA breastcancer Inhibitsdistantmetastasis [59]

SteﬁnA humanprostate

cancer

RatioofcathepsinBtosteﬁnAidentiﬁesheterogeneitywithinGleasonhistologicscores [60]

SteﬁnA inlaryngealcancer ExpressionandclinicalsigniﬁcanceofcathepsinB [61]

SteﬁnA breastcancer Co-expressionofcathepsinBanditsinhibitorSteﬁnAinbreastcancermetastasistolungandbone [62]

StefinB breastcancermodel StefinBdeficiencyreducestumorgrowthviasensitizationoftumorcellstooxidativestress [57]

SteﬁnB Meningiomas Lowerproteinandmessagelevels [63]

SteﬁnB Melanoma down-regulatedgene [64]

SteﬁnB inhumanesophageal carcinoma

downregulatedandthischangewasrelatedto lymphnode-metastasis

[65]

CystatinC gliomatumor levelsweredecreasedinhighgradegliomatumormasses [14]

Cathepsin- cystatinC

colorectalcancers decreaseincathepsin-cystatinCcomplexeswithan increaseinstageofcolorectalcancers

[40]

CystatinFor leukocystatin orCMAP

certainlymphoidcell types

showsaratherrestrictedexpressionpattern, beingconﬁnedtocertainlymphoidcelltypes

[43]

severalmurinecancer types

Thisgeneencodesaglycosylatedcysteineproteaseinhibitorwithaputativeroleinimmuneregulationthrough inhibitionofauniquetargetinthehematopoieticsystem

[44]

theprimarytumor LittleornoexpressionofcystatinFwas foundintheprimarytumor

[45]

CystatinE/M breast cancers

expressedinnormaltissues,butexpressionislostinmostlatestage/metastaticbreastcancers [46]

CystatinM endothelialcells increasedcystatinMexpressionwasshowntodecreasetumorcellproliferation,invasion,andadhesiontoendothelial cells

[47]

CystatinD humancoloncancer cells

TumorsuppressorgeneinducedbyvitaminD [66]

CystatinSN colorectalcancer Tumormarker [67]

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AIDS,hypertension,gastricdiseases,parasiticinfections,amyloid diseases,andcancer.ThecathepsinD(anasparticproteinase)is hyper secreted and overexpressed by cells of epithelial breast cancer[39].Augmentedlevelsoftheasparticproteinasecathepsin

D were ﬁrst reportedin several humanneoplastic, and cancer tissuesinthemid-1980s[94].Theseresultsgeneratedfocusona promisingroleforcathepsinDincancerandneoplasticprocesses.

The ﬁndings showed a strong risk factor for measurement of Fig.4.Collectionsofseveralproteaseinhibitors,showingtheir3-Dstructure(AdaptedfromPDB).

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cathepsinDconcentrationsinbreastcanceraswellasmanyother tumortypes.LevelofcathepsinDiscorrelatedwiththefrequency ofclinicalmetastasisandisthereforeapromisingmarkerofpoor prognosisin breast cancerand othercancers. CathepsinD is a functionalfactorinavarietiesoftissuesduringtheirregressionor remodeling,andoverexpressionoftheenzymepromotesmetas- tasisandtumorigenesis.Theenzymehasalsobeenimplicatedin apoptosis and aids tumor angiogenesis [39]. In the mammary gland,cathepsinDfunctionappearstoberelatedtotheprocessing oftheprolactin(apeptidehormone).ThecathepsinDsynthesisis regulatedbysteroidhormones.Asanexample,inbreastcancercell lines, expression of cathepsin D is controlled by estrogen hormones.Studieshasshown theampliﬁedlevelsof cathepsin D (both at the mRNA and protein levels) in several human neoplastic tissues [95]. The design of antagonists to block the interactionoftheproteinwithitsreceptormaybeapromisingtool inthetreatmentofbreast cancerandotherneoplasticdiseases [39].

Incontrast toothertissue proteases (e.g.metalloproteinases and serine proteases), noendogeneous cath-D tissue proteases inhibitor is known in mammals. Pepstatin, a natural aspartic proteases inhibitor has been isolated from various species of actinomycetes[96],andoftenusednotonlytostudyitsfunctionin some in vitro systems, but also in afﬁnity chromatography puriﬁcationofcath-D.

3.4.Glutamicacidcancerproteaseinhibitors

This is a new class of proteases derived from pepstatin- insensitivecarboxylprotease.Glutamicacidproteaseshavebeen isolated from Aspergillus niger var. macrosporus and Stylidium lignicola.Themechanismofactionisbasedonthetwoenzymes fromthescytalidoglutamicandaspergilloglutamicproteases.The activesiteofdiad glutamicacidandglutamine isimplicatedin catalysisandsubstratebinding.Theseaminoacidsassociationwith watermoleculesbehavesasanucleophilestopossessanacid-base mechanism different from that of the aspartic proteases. The glutamicacidbehavesasageneralacidinthecatalysisﬁrstphase, donatinga protontothecarbonyloxygenofthescissilepeptide bondofthesubstrate.Concurrently,anOH isdonatedbywater

in theactive site of theenzyme tothecarbonyl oxygenof the peptidebondsubstrate.Insomecases,twowatermoleculesplaysa roleinthereaction.Thesubstratetransitionstateisthoughttobe maintainedbyhydrogenbondingwiththetwocatalyticresidues.

Thereafter,glutamicaciddonatesaprotontotheamidenitrogen atomofthescissilepeptidebondstimulatingthebreakdownofthe tetrahedral intermediate, and thus leading to peptide bond cleavage. The glutamine residue is then charged for reversing theoriginalstateoftheglutamicacidresidue[19].

3.5.Threoninecancerproteaseinhibitors

Proteasome, a threonine protease unlike other proteases containsanactivesiteN-terminalthreonineresidue,residingin theMB1subunitsofitsb-ringsthatcanbeaimedbypharmaco- phoreslinkedtoshortpeptides[97,98].ProteasomeInhibitor(PSI) is aninhibitorof chymotrypsin-likeactivityof20Sproteasome.

Recently,PSIhasbeendemonstratedbyOyajobiandMundy[99], tobecapableofreducingthetumoraccumulationinmiceinjected withmurine5TGM1plasmacytomacellsandinhibitsthegrowthof osteolyticlesions.However,thegoodpharmacodynamicsofPSIare outweighedbyitspoorpharmaceuticalproperties.

LactacystinbeingthefirstofsuchspecificPSIisaStreptomyces metabolitethatinhibitthe20Ssubunit,anditspeptidaseenzyme activities include trypsin, chymotrypsin and peptidylglutamyl- peptide-likehydrolyzingactivities,thefirsttwobindirreversibly, buttheyallbindatdifferentratestolactacystin[98].Inspiteofthe selectiveeffectsoflactacystinintumorcells,itstandsasatoolfor proteasomalprocessstudiesonlyininvitrosystems,duetoitspoor metabolicstabilityandbindingirreversiblytoproteasomalsubunit [100].

Severalpharmaceuticalinhibitorshavebeendevelopedtothis end; Epoxomicin is an actinomycetes-derived PSI which binds irreversibly and possesses in vivo and in vitro activities [101].

Epoxomicin likelactacystin binds solely totheproteasome20S catalyticsubunitsleadingtotheinhibitionofthechymotrypsin- like function. It acts more faster with greaterspeciﬁcity when comparedtolactacystin,unlikeotherreversibleinhibitors[101].

Nevertheless,ituseinhumanisrestrictedforsimilarreasonraised by lactacystin. Thus, PSI which binds irreversibly doesn’t have Table3

Selectedserinecancerproteaseinhibitors.

Serpin types

Commonlyname Cancertype Description Ref(s)

SERPINA1 Alpha1antitrypsin Lungcancer,colorectalcancer Serumbiomarkersforthediagnosisoflungcancerandcolorectal cancer

[72,73]

SERPINA3 Alpha1antichymotrypsin prostatecancer Asanindicatorofprostatecancer,predictingbonemetastasesof prostatecancer,

[74,75]

SERPINA4 Kallistatin Lungcancer Inhibitsangiogenesisandtumorgrowth, [76]

SERPINA5 ProteinCinhibitor prostatecancer Asabiomarkerforprostatecancer [77]

SERPINA8 Angiotensinogen LungCancer PolymorphismsinLungCancer [78]

SERPINA9 Centerin(Gcet1) B-celllymphomas Extracellular;inhibitory,maintenanceofnaiveBcells [79,80]

SERPINA12 Vaspin endometrialcancer Lowvaspinareassociatedwithanincreasedriskofdeveloping endometrialcancer

[81,82]

SERPINB2 Plasminogenactivatorinhibitor-2 headandneckcancer Down-regulationcontributestochemoresistanceinheadand neckcancer

[83]

SERPINB3 Squamouscellcarcinomaantigen- 1(SCCA-1)

breastcancer,cervicalcancertreatment, tonguecancer,

Prognostictool [84–

87]

SERPINB4 Squamouscellcarcinomaantigen- 2(SCCA-2)

cervicalcancer Prognostictool [87]

SERPINB5 Maspin Humanmammaryepithelialcells,lung

cancer

Proposedtofunctionasatumorsuppressor,Biomarkerinlung cancer

[88,89]

SERPINI2 Pancpin pancreaticcancer inhibitionofpancreaticcancermetastasishasbeensuggested [93]

SERPINB13 Hurpin/Headpin Brainandovariancancer Singlenucleotidepolymorphismsandtreatmentofcancer [90–

92]

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desirable pharmacological characteristics and are therefore presentlynolongerinvestigatedforcancerdrugdevelopment.

Dipeptide boronicacid(BA)analogues, compared topeptide aldehyde agents, are selective, potent and are PSI which bind reversibly[101–103].BAblocktheactivityofthreonineproteaseby inhibiting chymotryptic activity of proteasome leading to the weakeningofthedegradationofcellcycleregulatoryproteins,e.g., p53orp27,I-jBandcyclinE[102,104,105].Whileaffectingmultiple proteins,BAtreatmentleadstotheinhibitionofcancercellgrowth andinitiationofapoptosis[102].Bortezomib(Velcadee)beingthe ﬁrstintheBAgroupisgenerallyactiveintumorcells,possessing littletoxiceffects onnormalcells anddemonstratedasasingle activeagentinpreclinicalhumancancerxenograftsmodelsand alsoinprimarycultures ofhematologicalandsolid tumortypes like prostate, neck and head cancers, multiple myeloma and lymphoma[40,104].

Ritonavir;anotherPSI hasbeenshowntoreversiblyinteract withtheactivesiteofthesubunitresponsibleforchymotrypsin- likeactivityinisolated20Sproteasomes[106].Gaedickeetal.[107]

showedthatritonavir,likeotherPSIs’hasantitumoractivities,it stronglyreducethedegreeofproliferationofvarioustumorcell lines and induced their apoptosis in vitro. Chymotrypsin-like function of isolated 26S proteasomes is activated by ritonavir compared to its action on isolated 20S proteasomes. The ﬁnal outcome of low micromolar concentrations of ritonavir onthe chymotrypsin-like activityin cells and cell lysateswas a weak inhibition,acid-solubleproteolyticpeptidelevelsmarginalalter- ations, consistent marginal alterations of polyubiquitinated proteinsand a littleaccumulation ofcancersuppressorprotein p53incellsthataretreatedwithritonavir.Accumulationofp21in the presence of this PSI is associated with the hindrance of proteolytic degradation and this shows selective proteasomes inhibitioninlinewithabnormaldegradationofp21,whichdoesn’t need to be ubiquitinated. This discovery proposed that the selective perturbation of proteasomal protein degradation can partakeinritonavirantitumoractivity.

3.6.Matrixcancerproteaseinhibitors

Zinc dependentMMPsarethemostabundantgroupofnon- serine proteases existing in invasive and metastatic cancer, it breakdownstructuralproteinscomprisingtheECM,likeelastin, collagen,ﬁbronectin, laminin, ﬁbrinogen and vitronectin as an important step in the complicated process of hematogenous metastasis. MMPs have long been related with tumor-cell metastasisandinvasion,andthiscontributedtotherationaleof MMPs inhibitors for clinical trials [6,108,109]. The center of attractionofcancerresearchismutationsincancercells,andthis resultineitherloss-of-functionintumor-suppressorgenesorgain- of-functioninoncogenes[110].

Tissue inhibitors of metalloproteases (TIMPs) are groups of smallextracellularproteinsinhibitingMMPs.Directproofforthe role of MMPs in tumor progression comes from xenograft experiments using cancer cells with decreased and increased expressionlevels of TIMPs or MMPs, so alsoas carcinogenesis experimentsusingmicelackingaspecificTIMP-1orMMPorthose havingorgan-specificTIMP-1 orMMPoverexpression.Thereare fourmembersoftheTIMPfamily,TIMP-1, 2, 3and 4,each inhibitingtheactivitiesofdifferentMMPswithvaryingefficiency [6,111].TIMPsarecrucialindeterminingtheinfluenceoftheECM of cell adhesion molecules, chemokines, several cytokines and growthfactorsonthephenotypeofcellsinvariouspathological andphysiologicalconditions[112].

MMPs pharmaceutical inhibitorssuch as batimastat and its chemicalanalog,marimastat,havebeensynthesizedandadmin- isteredtodecreasethecancermetastasisinpatient,mimickingthe

cleavagesitesofMMPsubstrates,but,theclinicaltrialsoutcome withthesedrugsfailed[113].Krügeretal.[108]mentionedthat batimastattreatmenthavebeensaidtogiverisetolivermetastasis ofhumanbreastcarcinomacellsinnudemiceandalsoarisein livermetastasis ofmurine T-cell lymphoma insyngeneic mice, overexpressionofliverspeciﬁcMMP2,MMP9,mRNAangiogenesis up-regulationandcaspase1evenincancerfreeanimalshavebeen observed. Theymentioned that in improving this drug further, induction of organ-speciﬁc side effect should be controlled.

Batimastatcan’tbeadministeredorallyandit’snomore tested fortumortreatmentinhuman[113].

MarimastathavegonethroughvariousPhaseIIIclinicaltrials [113], advanced pancreaticcancer trialwas aimedat detecting differences in survival comparingpatients treated withseveral dosesofthisdrugandconventionalchemotherapy.Althoughitwas unsuccessfultoidentifyincreasedsurvivalforgroupstreatedwith marimastat, however, the highest dose of marimastat was as effectiveastheconventional therapy[114].Bramhallet al.[98]

administered marimastat to some patient in other to prolong survivalinpatientssufferingfromgastro-oesophagealadenocar- cinoma,thisexperimentisoneoftheﬁrsttoshowthetherapeutic use of MMP inhibitor in tumor patients, it was observed that patientwhohavepreviouslyreceivedachemotherapytreatment beforethis drughasa longerspan thanthose who receivethe placebo after chemotherapy. Certainly, an achievementin both academia and pharmaceuticalindustry previouslyaimedat the inhibitionofmainclassofproteasesintumor.ThefailureofMMP inhibitors(MMPIs)duringitsclinicaltrialinthelate1990sledto the abortion of several drug programs and miserably had a negativeeffectonotherproteaseinhibitorclassdevelopment.In analyzingwhyMMPIsreachedphase IIIclinicaltrialsonlytobe unsuccessful,variouspointsthatmightbeimportantforconsider- ation of targeting other classes of proteases have been raised [111,115,116].

4.Conclusion

In this review, we provided an insight into the role of the protease and protease inhibitors in cancer. Proteases, are functionally involved in many processes of cancer progression, frombenign to malignancy [4]. In cancerconditions, proteases wereinitiallyconsideredtostimulatecancercellescapethrough tissuebarriers.However,processofproteolysisiscomplicatedin manyaspectsofcancerinvolvinginﬂammatorycellrecruitment, immune responses,proliferation, andapoptosis[10].Activityof proteasesisregulatedbyinteractionswithendogenousinhibitors oftheprotease(TIMPs)targetingmetalloproteases,serpinsbeing effective against serine proteases, and cystatinspredominantly inhibitingcysteineproteases.Thestudiesoftheroleofprotease and proteaseinhibitorsincancerprogressionextendthethera- peuticwindowforcancertreatment.Collectively,furtherpercep- tiveoftherolesofproteasesintumorprogression,metastasisand cancer development will guide the development of novel therapeuticstrategiesagainstcancer.

Conﬂictofinterest

Theauthorsdeclarenoconﬂictofinterest.

Acknowledgement

The authors thank Department of Medical Biotechnology, SchoolofAdvancedTechnologiesinMedicine,TehranUniversity ofMedicalSciences,Tehran,Iran.