Icariin and Its Derivatives

18 Pages·2016·3.62 MB·English

REVIEW
published:29June2016
doi:10.3389/fphar.2016.00191
Anti-Cancer Properties of the
Naturally Occurring Aphrodisiacs:
Icariin and Its Derivatives
Hui-LiTan1,2,Kok-GanChan3,PriyiaPusparajah2,SurasakSaokaew1,4,5,
AcharapornDuangjai4,6,Learn-HanLee1,4*andBey-HingGoh1,4*
1NovelBacteriaandDrugDiscoveryResearchGroup,SchoolofPharmacy,MonashUniversityMalaysia,BandarSunway,
Malaysia,2BiomedicalResearchLaboratory,JeffreyCheahSchoolofMedicineandHealthSciences,MonashUniversity
Malaysia,BandarSunway,Malaysia,3DivisionofGeneticandMolecularBiology,FacultyofScience,InstituteofBiological
Sciences,UniversityofMalaya,KualaLumpur,Malaysia,4CenterofHealthOutcomesResearchandTherapeuticSafety
(Cohorts),SchoolofPharmaceuticalSciences,UniversityofPhayao,Phayao,Thailand,5PharmaceuticalOutcomes
ResearchCenter,FacultyofPharmaceuticalSciences,NaresuanUniversity,Phitsanulok,Thailand,6DivisionofPhysiology,
SchoolofMedicalSciences,UniversityofPhayao,Phayao,Thailand
Epimedium (family Berberidaceae), commonly known as Horny Goat Weed or Yin
Yang Huo, is commonly used as a tonic, aphrodisiac, anti-rheumatic and anti-cancer
agent in traditional herbal formulations in Asian countries such as China, Japan, and
Korea. The major bioactive compounds present within this plant include icariin, icaritin
and icariside II. Although it is best known for its aphrodisiac properties, scientific
Editedby: and pharmacological studies suggest it possesses broad therapeutic capabilities,
LyndyJoyMcGaw,
especially for enhancing reproductive function and osteoprotective, neuroprotective,
UniversityofPretoria,SouthAfrica
cardioprotective,anti-inflammatoryandimmunoprotectiveeffects.Inrecentyears,there
Reviewedby:
Fang-RongChang, hasbeengreatinterestinscientificinvestigationofthepurportedanti-cancerproperties
KaohsiungMedicalUniversity,Taiwan of icariin and its derivatives. Data from in vitro and in vivo studies suggests these
AmitKumarTyagi,
compounds demonstrate anti-cancer activity against a wide range of cancer cells
TheUniversityofTexasMDAnderson
CancerCenter,USA which occurs through various mechanisms such as apoptosis, cell cycle modulation,
*Correspondence: anti-angiogenesis,anti-metastasisandimmunomodulation.Ofnote,theyareefficientat
Learn-HanLee
targeting cancer stem cells and drug-resistant cancer cells. These are highly desirable
[email protected];
Bey-HingGoh propertiestobeemulatedinthedevelopmentofnovelanti-cancerdrugsincombatting
[email protected] theemergenceofdrugresistanceandovercomingthelimitedefficacyofcurrentstandard
treatment.Thisreviewaimstosummarizetheanti-cancermechanismsoficariinandits
Specialtysection:
Thisarticlewassubmittedto derivatives with reference to the published literature. The currently utilized applications
Ethnopharmacology, of icariin and its derivatives in cancer treatment are explored with reference to existing
asectionofthejournal
patents. Based on the data compiled, icariin and its derivatives are shown to be
FrontiersinPharmacology
compoundswithtremendouspotentialforthedevelopmentofnewanti-cancerdrugs.
Received:01March2016
Accepted:16June2016
Keywords:icariin,icaritin,icarisideII,YinYangHuo,anti-cancer,ethnopharmacology
Published:29June2016
Citation:
TanH-L,ChanK-G,PusparajahP, INTRODUCTION
SaokaewS,DuangjaiA,LeeL-Hand
GohB-H(2016)Anti-Cancer
Cancerisagrowinghealthproblem,representingoneoftheleadingcausesofmortalityworldwide
PropertiesoftheNaturallyOccurring
(Goh and Kadir, 2011). The current standard of care includes conventional medicine, surgery,
Aphrodisiacs:IcariinandIts
Derivatives.Front.Pharmacol.7:191. radiotherapy,andchemotherapy(HsiaoandLiu,2010)howeverthelimitationsofexistingcareare
doi:10.3389/fphar.2016.00191 clearasevenaftercompletionofthestandardtreatmentregimens,therearehighratesofmetastasis
FrontiersinPharmacology|www.frontiersin.org 1 June2016|Volume7|Article191
Tanetal. AnticancerMechanismsofIcariinandItsDerivatives
andrelapse(Liangetal.,2010).Inaddition,therearesignificant tosupporttheefficacyofHerbaEpimedii-basedtraditionalherbal
problems associated with the standard treatments such as formulations for the treatment of cancer. Overall, the existing
significanttoxicitylevelsandtheemergenceofresistancetoward literaturehighlightstheremarkablepotentialofthesecompounds
anti-cancer drugs, making treatment of cancer even more forthedevelopmentofnoveldrugsforcancertreatment.
challenging (Crespo-Ortiz and Wei, 2012). Consequently, the
annualdeathratesduetocancercannotbesignificantlylowered
PLANT DESCRIPTION AND TRADITIONAL
(Chanetal.,2015)unlessnewtreatmentstrategiescanbedevised.
USES
Scientific exploration of medicinal plants and herbs is now
of increasing interest in the hope that they will yield novel
Epimedium is a low-growing, deciduous plant with leathery
compounds with properties that can overcome the limitations
leaves that spreads by underground stems. The flowers of
ofthedrugscurrentlyavailable(Lauetal.,2015;Supriadyetal.,
Epimedium vary in color and they have eight sepals. A tough
2015). Plant based compounds are also of interest due to their
andlong-livedperennialspecies,Epimediumisfoundmainlyon
multitargeting properties, low cost, relative absence of toxicity
cliffsinmoistforests,nearstreamsandwetlandsataltitudesof
andhighavailability(Guptaetal.,2011;Tanetal.,2015;Sayyad
between200and3700m(Maetal.,2011).Epimediumiswidely
etal.,inpress).
distributed from Japan to Algeria but it is mostly found in the
Epimedium (family Berberidaceae), (Latin name Herba
EastAsianandMediterraneanregion(Ariefetal.,2015).
Epimedii) commonly known as Horny Goat Weed or Yin Yang
Epimedium species have a long history of use in traditional
HuoinChinese,isagenusconsistingofapproximately52species
medicine as they have been used in botanical supplements for
ofherbaceousfloweringplants(Maetal.,2011).Inrecentyears,
more than 2000 years. The extracts of Epimedium plants are
therehasbeenasignificantvolumeofresearchontheanti-cancer
includedintraditionalherbalformulationsforthetreatmentof
effectofthemainactivecompoundsfromHerbaEpimediisuch
infertility, rheumatism and cancer in Asian countries such as
as icariin, icaritin, and icariside II. Icariin and its derivatives,
China, Japan, and Korea. In China, Epimedium is taken as a
icaritin, and icariside II seem to be promising compounds for
supplementforpreventionofchronicdiseasesandtostrengthen
cancer treatment, with studies having shown that they exhibit
the body (Cassileth et al., 2010; Ma et al., 2011). Today, Herba
anti-cancer activity against a wide range of cancer cell types
Epimedii is still popular in the treatment of cancers; it has
such as osteosarcoma (Geng et al., 2014), prostate (Lee et al.,
been commonly used as one of the main ingredients together
2009), lung (Zheng et al., 2014), gastric (Wang et al., 2010),
with other herbs, for the preparation of traditional Chinese
and kidney cancer cells (Li et al., 2013b). These compounds
formulations to treat various cancers such as digestive system
exert their anticancer action via a multitude of cellular targets
cancers, hepatocarcinomas, lung cancers, breast cancers and
andthroughavarietyofpathwaysincludingapoptosisinducing
cervical cancers (Zhang, 1991; Qi and Qi, 2002; Wang, 2003;
effect, cell-cycle modulation, anti-angiogenesis, anti-metastasis,
Teng, 2010). With reference to formal research, the extracts of
and immunomodulation. Of particular interest, they effectively
Herba Epimedii were reported to display anti-cancer activity
targetcancerstemcellsanddrugresistantcancercells.Research
in cancer cell lines such as colon cancer cells, hepatoma and
also suggests they are able to potentiate the current standard
leukemiacells(Linetal.,1999;GuonandChung,2014).Given
cancer treatments. The purpose of this review is to provide
the apparent efficacy of Herba Epimedii in treating cancers,
an up-to-date of the anti-cancer mechanisms of icariin and its
phytochemical analyses have also been carried out to identify
derivatives;andtoprovidescientificevidencethatthereisabasis
the bioactive components responsible for its pharmacological
activities.
Abbreviations: ADM, adrenomedullin; AIF, Apoptosis inducing factor; Apaf-
1, Apoptotic protease activating factor 1; ATF, activating transcription factor;
Bad, Bcl-2-associated agonist of cell death; Bax, Bcl-2-associated X protein;
ICARIIN AND ITS DERIVATIVES
Bcl, B-cell lymphoma 2; CAM, Chick chorioallantoic membrane; CDK, cyclin-
dependentkinases;CHOP,C/EBPhomologousprotein;COX-2,cyclooxygenase-
2;c-src,Proto-oncogenetyrosine-proteinkinaseSrc;CXCR4,chemokine(C-X- More than 260 moieties can be detected in Herba Epimedii,
C Motif) receptor 4; EBV, Epstein-Barr virus; eIF2α, α -subunit of eukaryotic including flavonoids, lignins, ionones, phenol glycosides,
translational initiation factor 2; EGFR, Epidermal growth factor receptor;
phenylethanoid glycosides, sesquiterpenes, and other types of
EMMPRIN,extracellularmatrixmetalloproteinase;EMT,epithelial-mesenchymal
compounds(Maetal.,2011).Amongthecompoundsidentified,
transition;ERK,extracellularsignal-regulatedkinases;ERS,endoplasmicreticulum
stress;FADD,Fas-associatedproteinwithdeathdomain;GRP,glucoseregulated icariin is thought to be the principal bioactive compound in
protein; HIF-1α, hypoxia-inducible factor 1-alpha; HUVECs, human umbilical Epimedium extracts. Aside from icariin, derivatives such as
vein endothelial cells; iNOS, Inducible nitric oxide synthase; IR, Ionizing icaritin, icariside I, icariside II, and desmethylicaritin can also
radiation; JNK, Jun N-terminal kinases; LMP1, Latent membrane protein 1;
be found in Herba Epimedii (Ma et al., 2011). Metabolic and
MAPK, Mitogen-activated protein kinases; MMP, matrix metalloproteinase;
pharmacokinetic studies have shown that these derivatives can
mTOR,mechanistictargetofrapamycin;NF-κB,nuclearfactor-kappabeta;PARP,
poly ADP-ribose polymerase; PGE2, prostaglandin E2; PI3K, phosphoinositide alsobeobtainedthroughthemetabolismoficariinbyintestinal
3-kinase; Piwil4, piwi-like protein 4; pRb, retinoblastoma protein; PTEN, flora,byconvertingicariintoicaritin,icarisideI,icarisideII,and
phosphatase and tensin homolog; PUMA, p53 upregulated modulator of desmethylicaritin (Liu et al., 2005; Xu et al., 2007). As shown
apoptosis;ROS,reactiveoxygenspecies;STAT3,signaltransducerandactivator
in Figure1, icariin (1) is a prenylated flavonol glycoside with
of transcription 3; TLR4, toll-like receptor 4; uPAR, urokinase plasminogen
rhamnosyl, glucosyl, and methoxy groups. Deglycosylation or
activatorreceptor;VASP,Vasodilator-stimulatedphosphoprotein;VEGF,vascular
endothelialgrowthfactor. demethylation of icariin will form different metabolites. For
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Tanetal. AnticancerMechanismsofIcariinandItsDerivatives
FIGURE1|ChemicalstructuresoficariinanditsderivativesisolatedfromHerbaEpimedii.Icariin(1),icaritin(2),andicarisideII(3).
instance, icariside I will be formed when the rhamnose residue compounds hold promise for cancer treatment because they
is removed whereas icariside II (3) will be formed when the are effective against a wide range of cancer cell types including
glucose residue is removed. Deglycosylation of icariin will osteosarcoma (Geng et al., 2014), prostate (Lee et al., 2009),
result in icaritin (2) and demethylation of icaritin will result in lung (Zheng et al., 2014), gastric (Wang et al., 2010), kidney
desmethylicaritin(Shenetal.,2009;Khanetal.,2015). (Lietal.,2013b).Furthermore,thesecompoundsseemtoexert
Scientific studies have provided evidence of the broad their anticancer actions through different cellular targets and
therapeutic capabilities of icariin, especially with reference pathways.Itiscrucialtoattainamorecompleteunderstanding
to its effects on reproductive function (Chen et al., 2014), of the underlying mechanisms of the anticancer properties of
osteoprotective (Qin et al., 2008), neuroprotective (Nie et al., icariinanditsderivativestoprovidedirectionforfutureresearch
2010), cardioprotective (Ding et al., 2007), anti-inflammatory incancerdrugdiscovery.
(Xuetal.,2010),andimmunoprotectiveeffects(Lietal.,2011).
Icariin’s derivatives have also been shown to possess significant ANTICANCER PROPERTIES
biologicalactivities.Forexample,icaritinwasshowntopossess
osteopromotive(Yaoetal.,2012),neuroprotective(Wangetal., Apoptotic Activity
2007)andcardioprotectiveeffect(Leietal.,2015).Thenumber Overthepastdecade,triggeringapoptosisintumorcellshasbeen
ofstudiesonicarisideIarelimitedbutitwasreportedtoalleviate utilized as an effective approach for treatment of cancer (Han
sexualdysfunction(Lenobleetal.,2002).IcarisideII,isreported et al., 2015). Apoptosis occurs through two different pathways:
topossessanti-hepatotoxic(Choetal.,1995),anti-osteoporosis the extrinsic or death receptor pathway and the intrinsic or
effects (Liu et al., 2014) and the ability to ameliorate diabetic mitochondrial pathway (Goh et al., 2014). Both pathways of
neuropathy (Tian et al., 2015). Icariin, icaritin and icariside II, apoptosis involve the activation of initiator caspases, followed
have also been reported to possess anticancer activity. Results by activation of effector caspases (caspase-3, -6, and -7) which
of both in vitro and in vivo experiments have suggested these the major executioners of apoptosis (Mongiat et al., 2007;
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Tanetal. AnticancerMechanismsofIcariinandItsDerivatives
Ahmed and Othman, 2013). With reference to the intrinsic Aside from mitochondria, organelles such as endoplasmic
pathway,mitochondrialoutermembranepermeabilizationisthe reticulumandlysosomesalsoplayanimportantroleinapoptosis
crucialeventforinitiatorcaspaseactivation(Chanetal.,2012). of cancer cells (Geng et al., 2015). The rapid rate of glucose
Herba Epimedii was shown to induce apoptosis of cancer cells; metabolism and growth in cancer cells causes endoplasmic
70% ethyl alcohol extracts of Epimedium koreanum Nakai has reticulum stress (ERS) and activation of the unfolded protein
causedactivationofmitochondria-dependentapoptoticpathway response(UPR).AstheUPRisoneoftheresistancemechanisms
in colon cancer cells (Guon and Chung, 2014). This led to againstcancertherapy,itcanoftenenablecancercellstoadaptto
furtherstudiesontheapoptoticactivityinducedbythebioactive ERSandevadeapoptoticpathways(Yadavetal.,2014).However,
components isolated from this herb. Generally, the apoptotic some studies have shown that therapeutic induction of ERS-
effect of icariin and its derivatives has been associated with induced apoptosis can be useful in eradicating cancer cells if
the activation of the intrinsic pathway of apoptosis. Treatment there is forced activation of ERS (Di et al., 2015; Fan et al.,
with icariin and icaritin has resulted in increase in Bax/Bcl2 2016). Recently, the role of ERS signaling in the anticancer
ratio, release of cytochrome c, cleavage of poly (ADP-ribose) activity of icariin has been studied on human adenocarcinoma
polymerase and activation of caspases in a wide range of andesophagealcancercells.IcariintreatmentupregulatedERS-
cancer cells (He et al., 2010; Li et al., 2010, 2013a, 2014b; relatedmoleculessuchasp-PERK,ATF,GRP78,p-eIF2α,CHOP
Tong et al., 2011; Wang et al., 2011a; Zhu et al., 2011; Sun as well as apoptosis-related protein PUMA. At the same time,
et al., 2015b; Wu et al., 2015b); these can be regulated by therewasdownregulationofanti-apoptosis-relatedproteinBcl2.
various signaling pathways, as shown in Table1. Similarly, Thisdemonstratedthattheapoptoticactivityoficariinincancer
icariside II has been shown to increase the ratio of Bax/Bcl2, cellsmightalsobeduetoactivationofERSsignaling(Dietal.,
causingtranspositionofcytochromecandactivationofcaspase- 2015;Fanetal.,2016).
3 and -9 in cancer cells including lung cancer cells and acute Lysosomesareacidicorganellescontainingvarioushydrolytic
myeloid leukemia cells. It also causes degradation of poly enzymes for recycling of intracellular proteins. When the
ADP-ribose polymerase (PARP) and reactive oxygen species alteration in lysosomal membrane permeabilization occurred,
(ROS) over-production (Kang et al., 2012; Song et al., 2012). there will be release of its hydrolytic enzymes, leading to a
Additionally, apoptosis of esophageal squamous cell carcinoma change in mitochondrial membrane potential and subsequent
treated with icariside II has been reported to be associated pro-apoptotic events of cells (Geng et al., 2015). In a
with lowered expression of β-catenin and downregulation of recent study, it was shown that treatment with icariside
expression of survivin and cyclin D1, suggesting that icariside II induced both mitochondrial and lysosomal dysfunction
II is capable of simultaneously inducing apoptosis while also through mitochondrial membrane and lysosomal membrane
inhibiting proliferation of cancer cells (Wang et al., 2011b). permeabilization in human hepatoblastoma cells, leading to
Other mechanisms involved include inhibition of epidermal caspase activation and cell apoptosis (Geng et al., 2015). This
growth factor receptor (EGFR) pathway activation (Wu et al., showsthaticarisideIIisabletoinducecancercellapoptosisby
2013b) which then prevents the downstream activation of the causingbothmitochondrialandlysosomaldamage.
JAK/STAT3 pathway (Kim et al., 2011; Wu et al., 2013a). This Therefore,icariinanditsderivativesappeartobeeffectivein
showstheabilityoficarisideIItoovercomethesurvivalsignals inducingapoptosisinawiderangeofcancercelltypesthrougha
of tumor cells because STAT3 is one of the STAT proteins that varietyofcellsignalingmechanisms(Figure2).
is constitutively active in cancer cells where it promotes the
expressionofantiapoptoticproteins(Wuetal.,2013a).Icariside Cell-Cycle Modulation
II also helps to overcome the survival signals of tumor cells by ThecellcycleconsistsofphasesknownasG ,G ,S,andG M.
0 1 2
inhibitingcyclooxygenase-2(COX-2)/PGE pathway(Leeetal., Cyclin-dependent kinases (CDK) are the protein translating
2
2009);expressionofCOX-2hasbeenassociatedwithtumor-cell signalsthatpushthecellsthroughthecellcycleandthegrowthof
resistancetoapoptosis(Greenhoughetal.,2009). cellsisdependentontheircapabilitytotranslatethesesignalsfor
The extrinsic pathway, on the other hand, is activated effective replication and division. When the normal regulation
by specific receptors of the tumor necrosis factor receptor ofcell-cycleprogressionanddivisionisdisrupted,uncontrolled
superfamily, such as Fas, DR4 (TRAIL-R1), and DR5 (TRAIL- proliferationcanoccurwhichmayresultinthedevelopmentof
R2). The binding of their specific ligands will result in the cancer(Crespo-OrtizandWei,2012;Dinicolaetal.,2014).
production ofdeath-inducingsignalingcomplexandactivation Icariinanditsderivativesinhibitthegrowthofcancercellsby
ofcaspase-8andcaspase-10whichwillthenactivatetheeffector causingcellcyclearrest.IcariininducescellcyclearrestatG /G
0 1
caspases (Mongiat et al., 2007). In some studies, both intrinsic and G /M phases in gallbladder carcinoma cells and colorectal
2
and extrinsic pathways seem to be involved in the induction cancercellsrespectively,throughtheinhibitionofNF-κBactivity
of apoptosis by icariin and its derivatives. For example, the (Zhang et al., 2013, 2014). Icaritin inhibits growth of human
treatment of human hepatocellular carcinoma with icaritin not prostate carcinoma cells through G cell cycle arrest which is
1
only triggered mitochondrial/caspase apoptotic pathway but attributed to induction of p27Kip1, p16Ink4a, and pRb, as well
alsoinducedapoptosisthroughFas-mediatedcaspase-dependent as the down-regulation of expression of phosphorylated pRb,
pathway as evidenced by increased levels of Fas and cleaved cyclinD1andCDK4(Huangetal.,2007).Inotherwork,icaritin
caspase-8 (Sun et al., 2015b). Similar results were obtained in caused cell cyclearrestattheG /Mphasein extranodal NK/T-
2
breastcancercellstreatedwithIcarisideII(Huangetal.,2012). cell lymphoma cells (Wu et al., 2015b). Similarly, treatment
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TABLE1|Apoptoticactivityoficariinanditsderivatives.
Compound CellLine StudyType Mechanism References
Icariin Humanlungadenocarcinomacells(A459) Invitroand ActivationofERSsignaling,increasedexpressionofERS-related Dietal.,2015
Invivo molecules(p-ERK,ATF6,GRP78,p-eIF2α,andCHOP,
downregulationofBcl2expressionandupregulationofPUMA
Humanesophagealcancercells(EC109and Invitroand ActivationofERSsignaling,increasedexpressionofERS-related Fanetal.,2016
TE1) Invivo molecules(p-ERK,ATF4,GRP78,p-eIF2α,andCHOP,
downregulationofBcl2expressionandupregulationofPUMA
Humanhepatomacells(SMMC-7721, Invitroand ActivationofROS/JNK-dependentmitochondrialpathwaythat Lietal.,2010
Bel-7402,andHepG2) Invivo involvedthegenerationofROSandJNKactivation,resultedin
enhancedBax-to-Bcl-2ratio,lossofmitochondrialmembrane
potential,cytochromecrelease,andcaspasecascade
MousetumorLeydigcells(MLTC-1) Invitro Activationofcaspase-9,-3,enhancedBax/Bcl-2ratio,and Wangetal.,2011a
releaseofcytochromec,aswellasdown-regulationofthe
expressionofpiwil4
Icaritin HepatocellularCarcinoma(HepG2) Invitro EnhancedBax/Bcl-2ratio,activationofcaspase-3,activationof Heetal.,2010
JNK1signaling
Leukemiacells(Primaryacutemyeloid Invitro Activationofcaspase-9,-3,-7,cleavageofPARP,downregulation Lietal.,2013a
leukemiacells,NB4,HL60,andU937) ofc-myc,inhibitionofMAPK/ERK,andPI3K/AKTsignals.
HumanBurkittlymphomacells(Rajiand Invitro Activationofcaspase-8,-9,cleavageofPARP,downregulationof Lietal.,2014b
P3HR-1) c-mycandenhancedBax/Bcl-2ratio
Humanlivercells(L02)andHuman Invitro Activationofcaspase-3,-8,enhancedBax/Bcl-2ratio,increased Sunetal.,2015b
hepatocarcinomacells(SMMC-7721) expressionlevelsofFas
Humanendometrialcancercells(Hec1A) Invitro Activationofcaspase-3andcaspase-9,cleavageofPARP,release Tongetal.,2011
ofcytochromec,enhancedBax/Bcl-2ratio,andsustained
activationERK1/2signaling
ExtranodalNK/T-celllymphomacells(SNK-10 Invitro Activationofcaspase-3andcaspase-9,enhancedBax/Bcl-2ratio, Wuetal.,2015b
andSNT-8) downregulationofp-Bad,inhibitionofJak/Stat3,andPI3K/Akt
signalingpathwaymediatedbyreducedexpressionofLMP1
Leukemiacells(Primarychronicmyeloid Invitroand Activationofcaspase-3,-9,releaseofcytochromec,enhanced Zhuetal.,2011
leukemiacellsandK562) Invivo Bax/Bcl-2ratio,downregulatedexpressionofApaf-1,inhibitionof
MAPK/ERK/JNKsignalsanddown-regulatedkinaseactivityof
Jak-2/STAT3/Aktsignalnetwork
IcarisideII Humanhepatoblastomacells(HepG2),Mouse Invitroand Mitochondrionmembraneandlysomalmembrane Gengetal.,2015
livercarcinomacells(H22) Invivo permeabilization,activationofcaspase-3,-7,-8,-9,cleavageof
PARP,enhancedBax/Bcl-2ratio.
Humanbreastcancercells(MCF-7) Invitro Activationofcaspase-8,-9,decreasedmitochondrialpotential, Huangetal.,2012
releaseofcytochromeCandAIF,increasedexpressionofFasand
FADDaswellasincreasedexpressionofBaxandBimL
Humanacutemyeloidleukemiacells(U937) Invitro Activationofcaspase-3,cleavageofPARP,anddecreasedbcl-xL Kangetal.,2012
andsurvivin,inactivationofSTAT3-relatedsignalingpathway.
Multiplemyelomacells(U266) Invitro Down-regulationofexpressionofBcl-2,Bcl-xL,survivin,Cyclin Kimetal.,2011
D1,COX-2,andVEGF,enhancedPARPcleavageandcaspase-3
activation,inhibitionofSTAT3activationandenhancedexpression
ofSHP-1andPTENthroughinhibitingJAK2andc-Src
Humanprostatecancercells(PC-3) Invitro Activationofcaspase-8,-9,-3,cleavageofPARP,decreased Leeetal.,2009
mitochondrialpotential,COX-2,iNOS,andVEGFexpression,
releaseofcytochromeC,inhibitionofCOX-2/PGE2pathway
Humannon-smallcelllungcancercells(A549) Invitro Activationofcaspase-3,-9,cleavageofPARP,releaseof Songetal.,2012
cytochromec,enhancedBax/Bcl-2ratio,Activationof
ROS/MAPKpathwaythatinvolvedactivationofROSdownstream
effectors,JNKandp38MAPK
Humaneosophagealsquamouscarcinoma Invitroand DownregulationofsurvivinandCyclinD1,inhibitionof Wangetal.,2011b
(Eca109) Invivo β-catenin-dependentsignaling.
Humanepidermoidcarcinomacells(A431) Invitro Activationofcaspase-9,cleavageofPARP,inhibitionof Wuetal.,2013b
JAK/STAT3andMAPK-ERKpathways,activationofPI3K/AKT
pathway,inhibitionofEGF-inducedactivationofEGFRpathway.
Humanmelanomacells(A375andSK-MEL-5) Invitroand Activationofcaspase-3,decreasedexpressionofsurvivin, Wuetal.,2013a
andMousemelanomacells(B16) Invivo inhibitionofJAK/STAT3andMAPKpathwaysaswellasactivation
ofPI3K/AKTpathway
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FIGURE2|Icariin,icaritin,andicarisideIIexertapoptoticeffectsthroughmultiplemechanisms,whichincludetheinhibitionofβ-catenin-dependent
signaling,EGFRsignaling,MAPK/ERKsignaling,PI3K/Aktsignaling,JAK/STAT3signaling,andCOX-2/PGE2signaling.Theactivatedpathwaysaresuch
asROS/JNKdependentmitochondriapathway,FAS-dependentapoptosis,JNK1signaling,ERSsignalingandERK1/2signaling.PI3K/Aktsignalingcanalsobe
activatedtoinactivateJAK/STATpathway.
of breast cancer cells with icaritin resulted in cell cycle arrest mighttargetmTOR,themasterswitchoftumorcellproliferation
at the G /M phase, accompanied by down-regulation of the (Gengetal.,2014).
2
expression of G /M phase regulatory proteins including cyclin Based on these studies, icariin and its derivatives were
2
B, cdc2 as well as cdc25c (Guo et al., 2011). The cell cycle demonstrated to exert cell cycle modulation at different phases
arrestwasassociatedwithsustainedextracellularsignal-regulated resulting in an anti-proliferative effect on cancer cells; mainly
kinases (ERK) activation, consistent with the findings of Tong attributedtothedownregulationofcellcycleregulatoryproteins,
et al. (2011). Aside from these, icaritin has been shown to assummarizedinTable2.
induce cell cycle arrest in the S phase associated with down-
regulated expression levels of S regulatory proteins including Angiogenesis Inhibition
Cyclin A, Cyclin B, and CDK2 in lung cancer and chronic Angiogenesis is the vital physiological process of formation
myeloidleukemiacells(Zhengetal.,2014;Zhuetal.,2015).As of new blood vessels; it is a normal process in growth and
shown in Figure3, the cell cycle modulation of icaritin seems granulationtissueformation.Angiogenesisiscrucialforcancer
to vary from study to study and this might be due to the cell development because blood vessels supply the nutrition and
type species-differences of the cancer cell lines (Zheng et al., transferchannelsateverystageofthegrowthoftumor(Zhang,
2014).IcarisideIIalsoinhibitscancercellproliferationthrough 2014).Therefore,disruptionofangiogenesiscancauseinhibition
cellcyclemodulation(Wangetal.,2011b);itwasabletoinduce of tumor invasion and metastasis (Tan et al., 2016). Currently,
cellcyclearrestatG0/G1 andG2/Mtransitions.Theunderlying anti-angiogenesis treatment represents one of the strategies
mechanismwassuggestedtobeduetothegenerationofreactive in cancer therapy but the currently known effective anti-
oxygen species as well as activation of p38 and p53 (Wu et al., angiogenesisagentsareprohibitivelyexpensive.
2015a). In a study on osteosarcoma, the inhibition of cancer The potential of Herba Epimedii to inhibit angiogenesis was
cellproliferationbyicarisideIIwasassociatedwithitsabilityto investigated in vitro and in vivo, as summarized in Table3.
inactivateepidermalgrowthfactorreceptor(EGFR)/mammalian Anti-angiogenic effects of the ethyl acetate fraction of Herba
targetofrapamycin(mTOR)pathway,showingthaticarisideII Epimediiextractwasdemonstratedinbothexperimentalmodels
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FIGURE3|Cellcyclemodulationbyicariinanditsderivatives.Icariin,icaritin,andicarisideIIinducecellcyclearrestthroughmodulationofexpressionofcell
cycleregulatoryproteinsatdifferentstagesofcellcycle,resultedininhibitionoftumorgrowth.
and might be due to inhibition of ERK signaling pathway studies showed a decrease in VEGF levels in both the icaritin-
(Yu et al., 2013). Preliminary studies on the effect of active andicariside-IItreatedgroups(Choietal.,2008;Lietal.,2013b).
compounds of Herba Epimedii such as icariin and icaritin on BasedonthestudyreportedbyChoietal.(2008),thedecreased
angiogenesis have been carried out by using human umbilical levelofVEGFisprobablyduetothereductionintranscriptional
vein endothelial cells as a cell model of angiogenesis. Both activityofhypoxiainduciblefactor1-alpha,theprimaryregulator
icariin and icaritin were found to inhibit the proliferation, oftheexpressionofVEGF.Takingalloftheaboveintoaccount,
migration as well as tube formation of the cells (Zhang, 2014; inhibition of VEGF is likely to be the target of icariin and its
Ye et al., 2015). In a chick embryo chorioallantoic assay, there derivativeswithrespecttheiranti-angiogeniceffect.
wassignificantreductioninnewly-formedbloodvesselsaswellas
reducedareaandlengthofbloodvesselsthatoccurredinadose- Inhibition of Metastasis
dependent manner following treatment of icariin and icaritin The malignancy of cancers is mainly due to the ability of
(Hongetal.,2013;Yeetal.,2015).Icariinanditsderivativeshave cancer cells to invade their neighboring tissues. This process
also exhibited their anti-angiogenic activity in vivo in different of invasion allows neoplastic cells to enter the blood stream
xenograftmodelsoftumorssuchashepatocellularcarcinomaand and lymphatic system and reach other organs where secondary
renalcellcarcinoma.CD31isapan-endothelialmarkerwhichis tumors may form. The development of metastases is the cause
specificallyexpressedontheendothelialcellssurfaces;intumors oftreatmentfailureanddeathincancerpatients.Thekeysteps
of mice treated with icariin and icaritin, there was a significant for metastases are cell attachment, local proteolysis and cell
decreaseintheareaspositiveforCD31(Yangetal.,2009;Lietal., migration (Harlozinska, 2005). Icariin and its derivatives have
2013b). The anti-angiogenic effect appears to be moderated by also been investigated with reference to their anti-metastatic
down-regulation of vascular endothelial growth factor (VEGF), activity.Astudyonhighlymetastatichumanlungmaincancer
acrucialgrowthfactorthatactsasthefundamentalregulatorof cells demonstrated a reduced ability of these cells to invade
angiogenesis(Figure4).Theresultsofbothinvitroandinvivo and migrate following treatment with icariin (Mao et al.,
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Tanetal. AnticancerMechanismsofIcariinandItsDerivatives
TABLE2|Cellcyclemodulationoficariinanditsderivatives.
Compound CellLine StudyType Mechanism References
Icariin Humangallbladdercarcinomacells(GBC-SD Invitro G0/G1phasecellcyclearrestduetoinhibitionofexpressionof Zhangetal.,2013
andSGC-996) survivin
ColorectalCancerCells(HCT116andHT29) Invitro G2/MphasecellcyclearrestthroughsuppressionofNF-κB Zhangetal.,2014
activationanddownregulationofCyclinD1
Icaritin Breastcancercells(MDA-MB-453andMCF7) Invitro G2/Mphasecellcyclearrestaccompaniedbydownregulationof Guoetal.,2011
theexpressionlevelsoftheG2/Mregulatoryproteinssuchas
CyclinB,cdc2andcdc25C
Humanprostatecarcinomacells(PC-3) Invitro G1cellcyclearrestthroughincreasedexpressionofpRb,p27Kip1, Huangetal.,2007
andp16Ink4aaswellasdecreasedexpressionofphosphorylated
pRb,CyclinD1andCDK4.
Humanendometrialcancercells(Hec1A) Invitro ReductionsofcyclinD1andcdk4proteinexpressionand Tongetal.,2011
inductionsofp21andp27expression.
ExtranodalNK/T-celllymphomacells(SNK-10 Invitro G2/MphasecellcyclearrestthroughinhibitionofJak/Stat3and Wuetal.,2015b
andSNT-8) PI3K/Aktsignalingpathwaymediatedbyreducedexpressionof
LMP1.
Humanlungcancercells(A549) Invitro Sphasecellcyclearrestthroughdownregulationofexpression Zhengetal.,2014
levelsofSregulatoryproteinssuchasCyclinAandCDK2
HumanmultiplemyelomacellsU266(ATCC InvitroandIn SphasecellcyclearrestdownregulationofexpressionlevelsofS Zhuetal.,2015
TIB-196)andHumanprimarymultiplemyeloma vivo regulatoryproteinssuchasCyclinA,CyclinBandCDK2and
cells upregulatedtheexpressionofCyclinE
IcarisideII Humanosteosarcomacells(MG-63and InvitroandIn Inhibitionofepidermalgrowthfactor(EGF)-inducedactivationof Gengetal.,2014
Saos-2) vivo EGFR/mTORsignalingpathway,includingEGFR,
PI3K/AKT/PRAS40,Raf/MEK/ERKaswellasmTOR.
Humaneosophagealsquamouscarcinoma InvitroandIn DownregulationofsurvivinandCyclinD1,inhibitionof Wangetal.,2011b
(Eca109) vivo β-catenin-dependentsignaling.
Humanmelanomacells(A375) Invitro G0/G1andG2/Mphasescellcyclearrestthroughgenerationof Wuetal.,2015a
ROSandactivationofp38andp53,accompaniedbyInhibitedthe
expressionofcell-cyclerelatedproteins,includingCyclinE,CDK2,
CyclinB1,andP-CDK1
TABLE3|Anti-angiogeniceffectoficariinanditsderivatives.
Compound Model/CellLine StudyType Observation/Mechanism References
HerbaEpimediiethyl HUVECSandZebrafish Invitroand InhibitionofHUVECsproliferation,migration,andVEGF-inducedtubeformationas Yuetal.,2013
acetatefraction embryos Invivo wellassignificantinhibitionofbloodvesselformationinzebrafishembryos,dueto
inhibitionofERKsignalingpathways
Icariin HepG2tumor Invivo DecreaseinCD31+vesselsintumor Yangetal.,2009
HUVECSandCAM Invitroand InhibitionofproliferationofHUVECsandinhibitionofangiogenesisinCAM,reduced Yeetal.,2015
Assay Invivo expressionofVEGF
Icaritin CAMAssay Invivo InhibitionofangiogenesisinCAM Hongetal.,2013
Rencatumor Invivo DecreaseinthemeanpositiveareaofCD31inimmunohistochemicalanalysesof Lietal.,2013b
tumor,reducedexpressionofVEGF,duetoinhibitionofactivityofSTAT3
HUVECS Invitro InhibitionofHUVECproliferation,migrationandtubeformation Zhang,2014
IcarisideII HUVECS Invitro Inhibitionofangiogenicfunctionwithreducedwidthsandlengthsoftheendothelial Choietal.,2008
network-likestructuresinHUVECS,reducedexpressionofVEGFinhuman
osteosarcomacells
2000). In another study, icariin suppressed the adhesion of tumor metastasis. VASP function in turn is regulated by Rac, a
lung adenocarcinoma (Di et al., 2015). The possible mode of member of the Rho family proteins (Schlegel et al., 2000). The
actionincludeseffectsonvasodilator-stimulatedphosphoprotein study has also revealed the inhibition of human gastric cancer
(VASP)whichisreportedtobecrucialincellmigrationaswell cell invasion and cell migration by icariin and the underlying
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FIGURE4|Anti-angiogeniceffectoficariinanditsderivatives.Thetreatmentoficariin,icaritin,andicarisideIIhaveresultedinanti-angiogeniceffect.Thiscan
beduetothereductioninexpressionofVEGF,acrucialgrowthfactorthatactsasthefundamentalregulatorofangiogenesis.
mechanismwassuggestedtobethesuppressionofexpressionof tumormetastasisbecauseaninflammatorymicroenvironmentis
cellmotility-relatedgenesRac1andVASP(Wangetal.,2010). favorableforcancerinvasionandmetastasisthroughepithelial-
Icaritin was also tested on glioblastoma cell line. There mesenchymaltransition(EMT).Studieshavebeendoneonthe
was suppression of adhesion, migration and invasion of effect of exposure to icariside II on the migration of non-small
glioblastomacells,duetodownregulationofextracellularmatrix cell lung cancer cells in an inflammatory microenvironment;
metalloproteinase via PTEN/Akt/HIF-1α pathway (Xu et al., theexposureoficarisideIIresultedintheinhibitionofTNF-α-
2015). Icariside II was also found to exert anti-metastatic boosted EMT, migration and invasion of cancer cells, probably
effects as it inhibited the migration of human osteosarcoma throughtheinhibitionofAkt/NF-κBpathway.Thisissupported
cells by suppressing the transcription of hypoxia-inducible by the results of an in vivo study that showed anti-metastastic
genes involved in tumor cell invasion, including urokinase effectoficarisideIIontumorinnudemice(Songetal.,2016).
plasminogen activator receptor, adrenomedullin, and matrix The findings of these studies revealed that icariin and
metalloproteinase 2 (Choi et al., 2008). The CXCR4/CXCL12 its derivatives inhibit the metastasis of cancer cells through
signalingaxisisknowntobeinvolvedinthemetastasisofcancer downregulationofproteinsthatarecrucialforcancermetastasis,
cells and CXCR4 chemokine receptors are highly expressed assummarizedinTable4andFigure5.
in various tumors compared to normal cells (Kim and Park,
2014). In a study, the anti-metastatic activity of icariside II Inhibition of Hormone Dependent Cancers
was shown to be correlated with downregulation of CXCR4 at Breastandprostatecancersarethecommonestinvasivehormone
transcriptionallevelthroughthesuppressionofNF-κBactivation dependentcancersinfemalesandmales,respectively.Although
(Kim and Park, 2014). The chronic inflammation that occurs these two types of cancers have different physiological features
during cancer treatment is an important factor that induces depending on the organs where they arise, they have common
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TABLE4|Anti-metastaticeffectoficariinanditsderivatives.
Compound CellLine StudyType Mechanism References
Icariin Humanlungadenocarcinomacells Invitro Decreasedmigrationandadhesionofcells Dietal.,2015
(A459)
Humanlungcancercells(PG) Invitro Decreasedcelladhesiveratiotolamininsubstrateandreduced Maoetal.,2000
cellabilityofinvasionormigration
Gastriccarcinomacells(BGC-823) Invitro Inhibitionofcellsmigrationthroughdownregulationofcell Wangetal.,2010
motility-relatedgenesRac1andVASP
Icaritin Humanglioblastomacells(U87MG) Invitro DownregulationofEMMPRINviathePTEN/Akt/HIF-1αsignaling Xuetal.,2015
pathway
Humanosteosarcomacells(SaOS2) Invitro Decreasedcellmotility,throughdownregulationofMMP-2and WangandWang,2014
MMP-9expression
IcarisideII Humanosteosarcomacells(HOS) Invitro Inhibitionofthetranscriptionofhypoxia-induciblegenesinvolved Choietal.,2008
ininvasion/migration,suchasuPAR,ADM,andMMP2
Humancervicalcancercells(HeLa) Invitro DownregulationoftheexpressionofCXCR4,throughsuppression KimandPark,2014
andBreastcancercells ofNF-κBactivation
(MDA-MB-231)
Humannon-smallcelllungcancer Invitroand InhibitofTNF-α-inducedmigrationandEMTofcancercellsinvitro Songetal.,2016
cells(A549andH1299) Invivo andinvivo,throughinhibitionofAkt/NF-κBpathway
FIGURE5|Anti-metastaticeffectoficariinanditsderivatives.Icariin,icaritin,andicarisideIIinhibitmetastasisoftumorbyinhibitingcelladhesion,migration,
andinvasionthroughmultiplemechanismsthatresultedindownregulationofVASP,CXCR4,uPAR,ADM,andmatrixmetalloproteinases.
pathological features of “hormonal-dependence” (Belev and receptor alpha (ERα). It has been found that some ERα –
Vrbanec,2013).Theinitiationandprogressionofthesediseases positivetumorscandevelopresistancetowardhormonetherapy.
are based on the modulation of hormone effects through These indicate a role of ERα in initiation and progression of
specificsteroidhormonereceptorsincludingestrogenreceptors, cancer (Ali and Coombes, 2000). Icaritin has been reported to
progesterone receptors and androgen receptors (Rau et al., inhibit the estradiol-stimulated growth of ERα-positive breast
2005). cancer cells by destabilizing ERα protein. Icaritin activates the
Estrogens are known to stimulate the growth of breast aryl hydrocarbon (Ahr)-mediated pathways by acting as an
cancerandmostbreastcancershaveoverexpressionofestrogen Ahr agonist; resulting in Ahr-mediated proteosomal pathways
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