pharmaceuticals
Article
Cryptolepine Suppresses Colorectal Cancer Cell Proliferation,
Stemness, and Metastatic Processes by Inhibiting
WNT/β-Catenin Signaling
Jude Tetteh Quarshie , Kwadwo Fosu , Nicholas Awuku Offei , Augustine Kojo Sobo, Osbourne Quaye
and Anastasia Rosebud Aikins *
West African Centre for Cell Biology of Infectious Pathogens (WACCBIP), Department of Biochemistry Cell
and Molecular Biology, University of Ghana, Accra P.O. Box LG 54, Ghana
* Correspondence: araikins@ug.edu.gh
Abstract: Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause
of cancer-related deaths globally. Evidence shows that over 90% of CRC cases are initiated by a
deregulated Wingless Integrated Type-1 (WNT)/β-catenin signaling pathway. The WNT/β-catenin
pathway also promotes CRC cell proliferation, stemness, and metastasis. Therefore, modulators of the
WNT/β-catenin pathway may serve as promising regimens for CRC. This study investigated the effect
of cryptolepine—a plant-derived compound—on the WNT/β-catenin pathway in CRC. Two CRC
cell lines, COLO205 and DLD1, were treated with cryptolepine or XAV 939 (a WNT inhibitor) in the
presence or absence of WNT3a (a WNT activator). Using a tetrazolium-based assay, cryptolepine
was found to reduce cell viability in a dose- and time-dependent manner and was a more potent
inhibitor of viability than XAV 939. RT-qPCR analyses showed that cryptolepine reverses WNT3a-
induced expression of β-catenin, c-MYC, and WISP1, suggesting that cryptolepine inhibits WNT3a-
mediated activation of WNT/β-catenin signaling. Cryptolepine also repressed WNT3a-induced OCT4
and CD133 expression and suppressed colony formation of the cells, indicating that cryptolepine
inhibits the stemness of CRC cells. Additionally, cryptolepine inhibited WNT3a-induced epithelial-to-
mesenchymal transition by reducing the expression of SNAI1 and TWIST1 genes. In a wound healing
Citation: Quarshie, J.T.; Fosu, K.; assay, cryptolepine was found to suppress cell migration under unstimulated and WNT3a-stimulated
Offei, N.A.; Sobo, A.K.; Quaye, O.;
conditions. Moreover, cryptolepine downregulated WNT3a-induced expression of MMP2 and MMP9
Aikins, A.R. Cryptolepine Suppresses
genes, which are involved in cancer cell invasion. Altogether, cryptolepine suppresses CRC cell
Colorectal Cancer Cell Proliferation,
Stemness, and Metastatic Processes proliferation, stemness, and metastatic properties by inhibiting WNT3a-mediated activation of the
by Inhibiting WNT/β-Catenin WNT/β-catenin signaling pathway. These findings provide a rationale for considering cryptolepine
Signaling. Pharmaceuticals 2023, 16, as a potential WNT inhibitor in CRC.
1026. https://doi.org/10.3390/
ph16071026 Keywords: cryptolepine; WNT/β-catenin signaling; colorectal cancer; proliferation; stemness; metastasis
Academic Editor: Giorgio Cozza
Received: 31 May 2023
Revised: 25 June 2023 1. Introduction
Accepted: 4 July 2023 In the year 2020, colorectal cancer (CRC) accounted for 1.9 million new cancer cases
Published: 19 July 2023 and 935,000 cancer-related deaths [1]. It causes severe morbidity in affected persons, and
the probability of survival depends on the stage at diagnosis [2]. Research is ongoing to
find the etiology of CRC, and although this remains elusive, evidence shows that CRC
Copyright: © 2023 by the authors. is initiated when genetic and epigenetic alterations in oncogenes and tumor suppressor
Licensee MDPI, Basel, Switzerland. genes accumulate in mucosal epithelial cells of the intestines [3]. Predisposing factors
This article is an open access article include gender (males are more susceptible than females), obesity, a sedentary lifestyle,
distributed under the terms and consumption of high amounts of alcohol and red meat, and smoking cigarettes. Protective
conditions of the Creative Commons factors include the consumption of whole grains, dairy products, and fibers [4].
Attribution (CC BY) license (https:// In the healthy human gut, the entire lining of the colonic lumen self-renews weekly to
creativecommons.org/licenses/by/ replace the glandular epithelial cells that are lost to stress from digestion and evacuation.
4.0/). This is made possible by the abundant intestinal stem cells at the bottom of the intestinal
Pharmaceuticals 2023, 16, 1026. https://doi.org/10.3390/ph16071026 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2023, 16, 1026 2 of 16
crypts, which have the capacity for continual proliferation and differentiation. As these
pluripotent cells differentiate into epithelial cells, they migrate from the crypt to the villus
and become fully differentiated epithelial cells such as enterocytes, goblet cells, Paneth
cells, and enteroendocrine cells [5]. In the event of oncogenic insults, the epithelial cells
lose their ability to properly regulate proliferation and transform into a benign adenoma
that may turn cancerous (adenocarcinoma) and metastasize over time [2].
Among the suite of signaling cascades that drive CRC, the canonical Wingless Inte-
grated Type-1 (WNT) pathway is principal. Indeed, over 90% of CRC cases occur due to
driver mutations that result in constitutive activation of the WNT signaling pathway. The
pathway is an evolutionarily conserved pathway that can either be transduced via canon-
ical (WNT/β-catenin) or non-canonical cascades. It is activated when WNT proteins—a
family of secreted cysteine-rich glycoproteins—activate their cell surface receptors, known
as Frizzled (FZD) [6,7]. In the canonical WNT cascade, WNT ligands bind FZD, which
in turn propagates a signal via Dishevelled to cause cytosolic accumulation of β-catenin.
Accumulated β-catenin translocates into the nucleus where it forms heterodimers with T
cell factor/lymphoid enhancer factor (TCF/LEF) to upregulate the transcription of genes
such as Axin2, c-MYC, cyclin D1, WISP1, and OCT4. These genes promote the proliferation,
survival, apoptosis, and stemness of the CRC cells [7,8]. Constitutive activation of WNT
signaling confers a clonal advantage to CRC cells, which increases their fitness through
modification of the tumor microenvironment [9]. Additionally, WNT/β-catenin signaling
promotes stem cell-like properties that are associated with drug resistance and tumor recur-
rence [5]. Considering the significance of WNT/β-catenin signaling in CRC development
and progression, WNT-modulating compounds may serve as promising regimens for CRC.
Due to their chemical diversity, some studies are focusing on naturally occurring
compounds in plants and their structural analogs as sources of pharmaceutically use-
ful small organic molecules for cancer treatment. One such compound is cryptolepine,
the main alkaloid extracted from the roots of an African-indigenous shrub, Cryptolepis
sanguinolenta [10,11]. Cryptolepine is pharmacologically active and a potential antiplas-
modial, antibacterial, antiprotozoal, antifungal, anti-hyperglycemic, antithrombotic, anti-
inflammatory, and anticancer agent [10,12]. The anticancer property of cryptolepine is
particularly of interest because, unlike many anticancer agents, cryptolepine exhibits low
mutagenic and genotoxic effects [13]. Moreover, a study showed that cryptolepine is highly
selective in inhibiting the viability of skin cancer cells versus normal cells [14]. Another
study reported no abnormalities in the hematological, liver, and kidney function indices
within 6 months of cryptolepine exposure in a 49-year-old male [15], suggesting that it may
be a safe alternative for cancer treatment.
Although the molecular basis for its anticancer activity remains largely ambiguous,
mechanistic studies have shown that cryptolepine acts through mechanisms such as topoi-
somerase II and telomerase inhibition, as well as DNA intercalation [16–18]. Using hep-
atocellular carcinoma cell models, Domfeh et al. (2021) demonstrated that cryptolepine
mediates its anticancer activity by upregulating anticancer pathway genes and downreg-
ulating pro-cancer pathway genes. In their study, it was found that cryptolepine inhibits
the transcriptional activity of the TCF/LEF transcription factor [19]. Given the modulatory
activity of cryptolepine on WNT/β-catenin signaling, it is imperative to assess its effect
on CRC models. In this study, we demonstrate that cryptolepine suppresses CRC cell
proliferation, stemness, and metastatic processes through inhibition of the WNT/β-catenin
signaling pathway.
2. Results
2.1. Effects of Cryptolepine, XAV 939, and WNT3a on Cell Viability
To determine the effects of cryptolepine, XAV 939, and WNT3a on cell viability, an
MTT assay was performed. Cryptolepine and XAV 939 reduced cell viability in a dose-
and time-dependent manner. The IC50 values shown in Table 1 indicate that cryptolepine
is more cytotoxic than XAV 939. Furthermore, cryptolepine was more cytotoxic against
Pharmaceuticals 2023, 16, 1026 3 of 16
DLD1 than COLO205 cells, whereas the potency of XAV 939 was identical in both cell lines
(Figure 1A,B). WNT3a had no obvious effect on the viability of both cell lines (Figure 1C).
Pharmaceuticals 2023, 16, x FOR PEERB RaEsVeIEdWo n a literature search [20–22], 100 ng/mL of WNT3a was used to ac3t iovf a1t8e WNT
 signaling in further experiments.
Table 1. IC50 values for treatment conditions.
2. Results 
2.1. Effects of Cryptolepine, XAV 939, and WNT3a on Cell ViabilIitCy5 0 (µM) of Compounds
CelTl Lo idneetsermine the effects of Tcirmypet(ohle) pine, XAV 93C9r, yapntdo lWepNinTe3a on cell viabXiAliVty,9 a3n9 
MTT assay was performed. Crypto2l4epine and XAV 9395 .0re1d±uc0e.4d3 cell viability 3in2 .1a5 d±os3e.-1 9
anCdO tLimOe2-0d5ependent manner. The IC4850 values shown in2 T.4a5bl±e 10 .i5n9dicate that cry2p4t.8o7le±pin2.e4 5
is more cytotoxic than XAV 939. Fu72rthermore, cryptole1p.4i3ne± w0.a1s5 more cytotox1i6c. 5a1ga±in0s.t2 3
DLD1 than COLO205 cells, whereas the potency of XAV 939 was identical in both cell lines 
(Figure 1A,B). WNT3a had no obvio2u4s effect on the viab3i.l5it1y± of0 b.3o1th cell lines (F3ig6.u5r1e± 1C1).0. 4
BaDsLedD 1on a literature search [20–22]47, 
8
2100 ng/mL of WNT
13.16 ± 0.070.8a6 w±as0 .u0s2ed to activate
2 W5.5N9T± 3.2219.19 ± si2g.-70
naling in further experiments. 
 
Figurree 11. .EEfffefectcst sofo cfrcyrpytpolteopleinpei,n XeA, XVA 9V399, 3an9d, a WndNWT3Na oTn3 aceolln vciaebllilvitiya.b CilOitLyO. C20O5L aOnd2 0D5LaDn1d cDellLsD 1 cells
weerree ttrreeaatteedd wwitiht hinicnrceraesainsgin cgonccoenncteranttiroantsi oonf s(Ao)f c(rAy)pctorlyepptinoele opri n(Be) oXrA(VB )9X39A fVor9 2349, 4fo8,r a2n4d, 4728 ,ha, nd 72 h,
oorr ((CC)) WWNNTT3a3 afofro 2r42 h4. hC.elCl veilalbviliiatyb iwlitays dweatesrmdeinteerdm uisninegd auns iMngTTa nassMayT.T Daastas aayr.e pDraetsaenatreed pasr esented
mean ± SEM of three independent experiments performed in triplicate. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 
a0.s00m1,e *a*n**± p ≤S E0.M000o1f vths.r uenetirneadteepd.e nnsd: ennott esixgpneifiricmanetn. ts performed in triplicate. * p ≤ 0.05, ** p ≤ 0.01,
*** p ≤ 0.001, **** p ≤ 0.0001 vs. untreated. ns: not significant.
  
 
Pharmaceuticals 2023, 16, 1026 4 of 16
2.2. Cryptolepine Inhibits WNT/β-Catenin Signaling in CRC Cells
To assess the effect of cryptolepine on WNT/β-catenin signaling, the cells were
treated as described, and mRNA levels of β-catenin, c-MYC, and WISP1 were evalu-
ated [7]. WNT3a induced the expression of β-catenin, c-MYC, and WISP1, while XAV
939 inhibited their expression. Cryptolepine did not affect the basal levels of all three genes
in both cell lines but significantly suppressed WNT3a-induced expression of the genes
Pharmaceuticals 2023, 16, x FOR PEER( R FEiVgIuErWe 2A–C). This suggests that cryptolepine inhibits WNT3a-mediated ac5t iovf a1t8i on of the
WNT/β-catenin pathway.
 
FFiigguurree 22. .CCryryptpotloepleinpein ienhinibhitisb WitsNWT/Nβ-Tca/tβen-icna tseignninalisnigg nina lCinRgCi nceCllsR. CCOceLlOls2.0C5 OanLdO D2L05D1a ncdellDs LD1 cells
wweerree ttrreeaatetedd wwithit hcrycrpytoplteoplienpei nore XoArVX 9A3V9 f9o3r9 48fo hr i4n8 thhe ipnrethseenpcer eosre anbcseenocrea obf sWenNcTe3oaf. TWhNe mTR3aN. AT he mRNA
levels of (A) β-catenin, (B) c-MYC, and (C) WISP1 were determined by RT-qPCR. Data are presented 
alesv melesano f±( ASE)Mβ -ocfa ttehnreine, i(nBd)epc-eMndYeCnt, aexnpder(iCm)eWntIsS pPe1rfworemreedd ient etrrmipliincaetde.b *y* pR ≤T -0q.0P1C, R**.* Dp a≤t a0.0a0r1e, presented
*a*s**m p e≤a 0n.0±00S1E vMs. uonfttrhearetedi.n #d pe p≤ e0n.0d5e, n##t pe x≤p 0e.0ri1m, #e#n#t ps p≤ e0r.0fo01r,m #e##d# ipn ≤t r0i.p0l0i0c1a tves.. *W* Np T≤3a0.. 0C1R,Y**P*: p ≤ 0.001,
*c*r*y*ppto≤lep0i.n0e0;0 X1AvVs:. XuAnVtr e9a3t9e; dm.R#NpA≤: m0.e0s5s,e#n#gepr ≤rib0o.n0u1,cl#e#ic# apci≤d; 0n.s0:0 n1o, t# s#i#g#nipfic≤an0t..0 001 vs. WNT3a. CRYP:
cryptolepine; XAV: XAV 939; mRNA: messenger ribonucleic acid; ns: not significant.
2.3. Cryptolepine Downregulates Stem Cell Markers in CRC 
Aberrant WNT signals promote stemness in CRC, which is linked to poor disease 
outcomes [23]. To this end, the impact of cryptolepine on the expression of two stem cell 
markers—OCT4 and CD133—was examined [7]. WNT3a induced the expression of OCT4 
and CD133 in both cell lines and XAV 939 decreased their expression in unstimulated and 
 
Pharmaceuticals 2023, 16, 1026 5 of 16
2.3. Cryptolepine Downregulates Stem Cell Markers in CRC
Pharmaceuticals 2023, 16, x FOR PEER REVIEAWb errant WNT signals promote stemness in CRC, which is linked to poor d6i soefa s1e8 
 outcomes [23]. To this end, the impact of cryptolepine on the expression of two stem cell
markers—OCT4 and CD133—was examined [7]. WNT3a induced the expression of OCT4
and CD133 in both cell lines and XAV 939 decreased their expression in unstimulated and
WNNTT33aa--ssttiimuullaatteedd cceellllss ((FFiigguurree 33AA,,BB)).. CCrryyppttoolleeppiinnee ddiidd nnoott aaffffeecctt tthhee bbaassaall lleevveell ooff OOCCTT44 
iinn bbootthh cceellllss bbuutt ssuupppprreesssseedd WNTT33aa--iinndduucceedd OCCTT44 eexxpprreessssiioonn ((FFiigguurree 33A)). .IInntteerreessttiinngglyly,, 
ccrryyppttoolleeppiinnee inindduucceeddC DCD131333ex epxrepsrseisosnioinn biont hbocethll scbeulltsr ebpurte srseepdreWssNedT3 aW-iNndTu3cae-dinCdDuc1e3d3 
CexDp1r3e3s seioxnpr(eFsisgiounre (3FBig)u, rseu g3gBe),s tsinugggtehsattincgr ytphtaot lecpryinpetomleapyinpe rmomayo tperoormsoutpe porre sssuCppDr1e3s3s 
CexDp1r3e3ss eioxnprdeesspieond dinegpeonndWinNg oTna cWtiNviTty aicntitvhietyc einll .the cell. 
 
Figure 3.. Cryptollepiine downrregullatteess sttem cellll markerrss iin CRC.. COLO205 and DLD1 cells were 
ttrreeaatteedd wiitthh ccrryyppttoolleeppiinnee oorr XXAV 993399 ffoorr 4488 hh iinn tthhee pprreesseennccee oorr aabbsseennccee ooff WNTT33aa.. TThhee mRRNA 
lleevveellss ooff ((AA)) OOCCTT44 aanndd ((BB)) CCDD113333 wweerree ddeetteerrmmiinneedd bbyy RRTT--qqPPCCRR.. DDaattaa aarree pprreesseenntteedda assm meeaann± ± SSEEMM 
ooff tthhrreeee iinnddeeppeennddeenntt eexxppeerrimimeenntstsp peerfroformrmededi nint rtirpilpicliactaet.e*. **p* p≤ ≤0 0.0.011, ,* *****pp≤ ≤ 00..000011,, ********pp ≤≤ 00.0.0000011 vvss.. 
untreated. ## p ≤≤ 0.01, ### p ≤ 0≤.001, #### p ≤ 0.≤0001 vs. WNT3a. CRYP: cryptolepine; XAV: XAV 939; munRtNreAat:e md.e#ss#epnger0 r.0ib1o, n##u#clpeic a0c.i0d0;1 n,s#:# n#o#t psign0ifi.0c0a0n1t.v s. WNT3a. CRYP: cryptolepine; XAV: XAV
939; mRNA: messenger ribonucleic acid; ns: not significant.
22..44.. CCrryyppttoolleeppiinnee IInnhhiibbiittss CClloonnooggeenniicciittyy iinn CCRRCC 
GGiivveenn tthhee iinnhhiibbiittoorryy eeffffeecctt ooff ccrryyppttoolleeppiinnee oonn tthhee eexxpprreessssiioonn ooff sstteemnneessss maarrkkeerrss,, wee 
aasssseesssseedd iittss iinnflfluueennccee oonnt htheec ocololonnyy-f-oformrminingga baibliitlyityo foCf RCCRCce cllesl.lsT.h Tehaes saasysaays saesssseessstehse thcae- 
cpaapciatcyitoyf oafs ain sgilnegcleel lcteollg troo wgroinwto inatcoo lao cnoyloonf yat olef aastt l5e0ascte l5l0s, cwelhlsic, hwshigicnhifi seigs nstiefimesn setsesmbnaseesds 
boanstehde ornat tihone arlaetitohnaat lfeo ltlhoawt ifnogllotrweaintmg etrneta, tsmteemntc, esltlesmus cuealllsly upseuraslilsyt panerdsirsetg aende rraetgeetnoeirnaittei- 
taot eintuitmiaoter itguemneosriisg[e2n4e].siIsn [t2h4i]s. sItnu dthyi,sW stNudTy3a, WprNomT3oate pdrcoomloonteydf ocromloantiyo nfoirnmbaottihonc eilnl lbinoeths, 
ceolnl filirnmesin, cgotnhfierimnflinuge nthce ionfflWueNnTces iogfn WalNinTg osingnstaelmingn eosns isnteCmRnCescse ilnls .CSRuCrp creisllisn. gSluy,rpXrAisV-
i9n3g9lyin, cXreAaVse d93t9h eincocrloenasyefdo rtmhea ticonloonfyu nfosrtimautiloante dofc eullnssatinmdurleaptereds sceedllsW aNnTd3 ar-eipnrdeussced 
WNT3a-induced clonogenicity in DLD1 cells but not COLO205 cells. On the other hand, 
cryptolepine suppressed clonogenicity in both unstimulated and WNT3a-stimulated 
COLO205 and DLD1 cells, suggesting that cryptolepine inhibits colony formation and 
hence stem cell-like properties of the CRC cells (Figure 4A,B). 
 
 
Pharmaceuticals 2023, 16, 1026 6 of 16
clonogenicity in DLD1 cells but not COLO205 cells. On the other hand, cryptolepine
Pharmaceuticals 2023, 16, x FOR PEER REVIEW suppressed clonogenicity in both unstimulated and WNT3a-stimulated C7O LoOf 2108 5 and
 DLD1 cells, suggesting that cryptolepine inhibits colony formation and hence stem cell-like
properties of the CRC cells (Figure 4A,B).
 
Figure 4. CryptolepineF ignuhrieb4i.tsC crylopntoolegpeineicinithyib iints CclRonCo.g (eAni)c iCtyOinLCOR2C0.5(A a)nCdO (LBO) 2D05LaDn1d c(Be)llDs LwDe1rcee ltlrsewaeteredt reated
with cryptolepine or XwAitVh  c9r3yp9 tionle pthinee porreXsAeVnc9e3 9oirn athbesepnrecsee nocfe WorNabTse3nac,e aonfdW aN cTo3alo, annyd faocromloantyiofnor masastaioyn assay
was performed. Colonwya asrpeearsfo armree dex. pCroelsosneyda raesa sa apreerecxepnretsasgeed oasf ua npterreceantteadg eceolflsu natnreda tpedrecseellnstaendd apsr ebsaern ted as
charts. Data are presenbtaerdc haasr mts.eDanat ±a SarEeMpr eosfe tnhterdeea sinmdeeapne±ndSeEnMt eoxf ptherreieminednetps epnedrefnotremxpeedr iimne tnrtisppliecrafoterm. ed in
*** p ≤ 0.001, **** p ≤ 0.0tr0i0p1li cvaste. .u*n**trpe≤at0e.d00. 1#,#* *p* *≤ p0.≤010,. 0#0#0#1#v ps .≤ u0n.0tr0e0at1e dv.s.# W# pN≤T30.a0.1 C, #R#Y##Pp: c≤ry0p.0t0o0l1epvsin. eW; NT3a.
CRYP: cryptolepine; XAV: XAV 939.
XAV: XAV 939. 
2.5. Cryptolepine Represses Epithelial-to-Mesenchymal Transition (EMT) in CRC 
Metastasis is a multistep process that involves landmark events like EMT, where can-
cer cells become highly motile. Because deregulated WNT/β-catenin signaling drives EMT 
in CRC [25], the expression of two EMT genes—SNAI1 (encodes SNAIL1 protein) and 
TWIST1—were examined. WNT3a induced the expression of SNAI1 and TWIST1 genes 
in both cell lines. XAV 939 induced the expression of SNAI1 in both cell lines, whereas 
cryptolepine did not affect SNAI1 levels in unstimulated cells. In contrast, both XAV 939 
and cryptolepine significantly inhibited WNT3a-induced SNAI1 expression (Figure 5A). 
Furthermore, XAV 939 and cryptolepine did not affect the basal levels of TWIST1 mRNA 
in unstimulated cells but suppressed WNT3a-induced TWIST1 upregulation (Figure 5B). 
The results, therefore, show that cryptolepine represses EMT in CRC cells with hyperac-
tive WNT signaling via downregulation of SNAI1 and TWIST1. 
 
 
Pharmaceuticals 2023, 16, 1026 7 of 16
2.5. Cryptolepine Represses Epithelial-to-Mesenchymal Transition (EMT) in CRC
Metastasis is a multistep process that involves landmark events like EMT, where
cancer cells become highly motile. Because deregulated WNT/β-catenin signaling drives
EMT in CRC [25], the expression of two EMT genes—SNAI1 (encodes SNAIL1 protein) and
TWIST1—were examined. WNT3a induced the expression of SNAI1 and TWIST1 genes
in both cell lines. XAV 939 induced the expression of SNAI1 in both cell lines, whereas
cryptolepine did not affect SNAI1 levels in unstimulated cells. In contrast, both XAV 939
and cryptolepine significantly inhibited WNT3a-induced SNAI1 expression (Figure 5A).
Furthermore, XAV 939 and cryptolepine did not affect the basal levels of TWIST1 mRNA
Pharmaceuticals 2023, 16, x FOR PEERi nREuVnIsEtiWm ulated cells but suppressed WNT3a-induced TWIST1 upregulation (Figure 5B). 8 of 18 
 The results, therefore, show that cryptolepine represses EMT in CRC cells with hyperactive
WNT signaling via downregulation of SNAI1 and TWIST1.
 
Fiigguurere5 .5C. rCyprtyopletpoilneeprienper ersesepsrepsistehse lieapl-itoth-meleisaeln-tcoh-ymeasletrnacnhsiytimona(lE tMraTn) sinitiCoRnC .(ECMOLTO) 2i0n5 aCnRdC. COLO205 
DanLdD 1DcLelDls1w cereelltsr ewatedrew tirtehactreydp twoleitphin ceroyrpXtAoVlep93in9 efo or r4 8XhAiVn  th9e39p rfeosre n4c8e ohr ianb stehnec eporfeWseNnTce3a o. r absence of 
TWheNmTR3Na.A Tlhevee lms oRfN(AA)  SlNevAeIl1sa onfd ((AB)) TSWNISATI11 waenrde  d(Bet)e rTmWinIeSdTb1y wRTe-rqeP CdRe.teDramtainareedp rbeyse nRtTed-qaPsCR. Data are 
mpreeasne±ntSeEdM aos fmtheraeeni n±d SeEpeMnd oefn tthexrpeeer iimndenetpsepnedrfeonrmt eedxpinetrriimpleicnattes. p**e*r*fpor≤m0e.0d0 0i1n vtsr.ipunlitcraeatete. d*.*** p ≤ 0.0001 
#v#s.p u≤n0tr.0e1a,t#e#d#.# #p# ≤p 0≤. 000.0011,v #s.##W#N pT ≤3 a0..0C0R0Y1P v: csr. yWptNoleTp3ian.e ;CXRAYVP: :X cArVyp9t3o9l;empRinNeA; X: mAeVs:s eXnAgeVr 939; mRNA: 
rmibeosnsuecnlegicera criidb;onns:uncolet isci ganciifidc;a nnts.: not significant. 
2.6. Cryptolepine Reduces CRC Cell Migration 
A wound healing assay was performed to evaluate the effect of cryptolepine on cell 
migration. DLD1 cells were treated with IC50 and IC30 of cryptolepine or IC50 of XAV 
939 in the presence or absence of WNT3a. Cell migration was monitored at 0, 24, and 48 
h. Activating or inhibiting WNT/β-catenin signaling promoted or inhibited cell migration, 
respectively. The IC30 and IC50 of cryptolepine remarkably inhibited wound closure in 
both unstimulated and WNT3a-stimulated DLD1 cells (Figure 6A,B). These findings 
demonstrate that cryptolepine is a potent inhibitor of DLD1 cell migration. 
 
 
Pharmaceuticals 2023, 16, 1026 8 of 16
2.6. Cryptolepine Reduces CRC Cell Migration
A wound healing assay was performed to evaluate the effect of cryptolepine on cell
migration. DLD1 cells were treated with IC50 and IC30 of cryptolepine or IC50 of XAV
939 in the presence or absence of WNT3a. Cell migration was monitored at 0, 24, and 48 h.
Activating or inhibiting WNT/β-catenin signaling promoted or inhibited cell migration,
Pharmaceuticals 2023, 16, x FOR PEER REVIEreWsp ectively. The IC30 and IC50 of cryptolepine remarkably inhibited wound closure 9 of 18 
 in both unstimulated and WNT3a-stimulated DLD1 cells (Figure 6A,B). These findings
demonstrate that cryptolepine is a potent inhibitor of DLD1 cell migration.
 
FigureF i6gu. rCer6y.pCtorylepptoilnepei nredreudcuecse sCCRRCC ccell miiggraratitoino.nD. DLDL1Dc1e llcsewllse rwe terreeat etrdewatiethd cwryipttho lcerpyinpetolepine or 
XAV 9o3r9X fAoVr 94389 hfo irn4 8thhei n(Ath) ep(rAe)spenrecsen ocre (oBr )( Ba)basbesnencece ooff WNT33aa. . TThheew wouonudnadre araetae acth etaicmhe time point 
is exppreosinsteids eaxsp are spseedrcaesnatapgeerc eonft athgee o0f hth.e O0rhi.gOinraigl inmalamgnaigfincifiactaiotinon ××101000. .DDaattaa aarreep prerseesnetnedteads as mean ± 
SEM omfe tahnr±eeS EinMdoefptehnredeeinndt eepxepndeernimt eexnptesr impeernftos rpmerefodr mined tirniptrliipclaicteat.e *. ** *pp ≤≤ 00..0011,, ********p ≤p ≤0. 000.001001 vs. un-
treatedvs.. #u#n#tr pea ≤te 0d..0#0#1#,p #≤##0#.0 p0 1≤,  #0#.#0#0p01≤ v0s.0. 0W01NvsT. 3WaN. CT3RaY. CPR: YcrPy: cprtyoplteopleipnien;e ;XXAAVV:: XXAAVV9 3993.9. 
2.7. Cryptolepine Reduces CRC Cell Invasiveness
2.7. CryptTooleepxianme iRneedthueciems pCaRctCof Ccreylpl tIonlvepaisniveeonnecsesl l invasion, the levels of MMP2 and MMP9
Twoe reexdaetmerimnien etdh.eT himesepgaecnt eos fe nccroydpetporloetpeiinnaese sotnh actedlel girnadveasthioenh,i sttohleo gliecavleblasr roiefr MtoMP2 and 
MMPfacilitatea9n wuperreeg 
cdanecteerrcmell invasion [26]. Activating or inhibiting WNulation orindeodw.n Trehgeuslaet iogne,nreessp eenctcivoedlye, opfrMotMeiPn2as
Te/sβ t-chaatetn dinesgigrnaalingand MMP9 in botdhece tllh
leed tolin ehsi.stological 
barrieCrr ytop tfoalecpiliinteathea dcannocoebrv cioeulls ienffveactssioonn M[2M6]P. 2AacntdivMatMinPg9 oerx pirnehssiibointiinngu WnstNimTu/lβat-ecdatenin sig-
naling led to an upregulation or downregulation, respectively, of MMP2 and MMP9 in 
both cell lines. Cryptolepine had no obvious effects on MMP2 and MMP9 expression in 
unstimulated cells but significantly reduced WNT3a-induced expression of these genes, 
confirming the selectivity of cryptolepine for cancer cells with hyperactive WNT signaling 
(Figure 7A,B). 
 
 
Pharmaceuticals 2023, 16, 1026 9 of 16
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 10 of 18 
 cells but significantly reduced WNT3a-induced expression of these genes, confirming the
selectivity of cryptolepine for cancer cells with hyperactive WNT signaling (Figure 7A,B).
 
FFiigurre 7.. Crrypttooleleppinienere rdeudcuesceCsR CCRcCel lceinllv iansivvaesnievsesn. eCsOs.L COO20L5Oa2n0d5D aLnDd1 DcLelDls1w ceerlelst rweaetreed treated with 
cwryitphtcorlyepptionleep oinre XorAXVA 9V3993 9fofor r4488 hh iinn tthheep prerseesnecnecoer aobrs eanbcseenofceW oNfT W3aN. TTh3eam. TRNheA mleRveNlsAo flevels of (A) 
M(AM) MP2M aPn2da n(dB)( BM) MMP9P 9wweerree ddeetteerrmiinneeddb byyR TR-TqP-qCPRC. RD.a tDa aatrae parrees epnrteesdenastemde aans m± eSaEnM ±o Sf EM of three 
itnhdreeepiennddepeennt deexnpteerxipmereinmtesn ptserpfeorrfmormede dinin trtirpipliliccaattee.. ** pp ≤≤ 00..0055,, **** pp ≤≤ 00..0011,, ******** pp≤ ≤ 00..00000011v vs.s. untreated. 
#u#n tpr e≤a t0ed.0.1#,# #p#≤# 0p. 0≤1 ,0#.#0#0p1,≤ ##0.#0#0 1p,  #≤# #0#.0p0≤010 .v00s0. 1WvsN. WT3NaT. 3Ca.RCYRPY:P c: rcyryppttoolleeppiinnee; ;X XAAV:VX:A XVA9V39 ;939; mRNA: 
mmeRsNseAn:gmeers sreibnogenrurcibleoincu accleiidc;a ncisd: ;nnos:t nsoigt nsiigfincifiancatn. t.
3. Discussion
3. DiTshcue simsipoonr tance of WNT/β-catenin signaling in CRC has been established [9], and
reseaTrchei sismtipllorntgaoninceg toof dWevNeloTp//βd-cisactoevneirns msiaglnl maloinlegc uilne iCnhRiCbi tohrasso fbteheenp aetshtwabalyisthoed [9], and 
rteresaetaCrcRhC i.sC srtyipllt oolnepgionienigs  atop ldanevt-edleorpiv/eddisccoomvpeoru snmdathlla mt hoalsebceuelne fionuhnidbittoorinsh oibf itththee pathway to 
ttrreaants cCriRptCio. nCarl yapcttiovilteypoinf eT CisF /aL pElFanptr-odteeirniv(aedtr acnosmcrpipotiuonndal tfhacatto rhians WbeNeTn/ fβo-ucantden tino inhibit the 
signaling) in hepatocellular carcinoma cells [19]. This study, therefore, investigated the
tproatnesnctirailpotfiocnryapl taocletpiviniteya os fa TmCoFd/uLlaEtFor porfoWteNinT /(aβ -tcraatnenscinrispigtinoanlianlg fiancCtoRrC inc eWllsN. T/β-catenin sig-
nalinIgn) tihnis hsetupdayt,occreyllputolalerp cinaerceinxhoimbitae dcemllso r[e19p]o.t eTnhtiscy sttoutodxyic, tehffeercetsfotrhea,n inXvAeVst9ig39a.ted the po-
tCenrytpiatol loefp icnreyptatrogletps iDneN aAs, ato mpooisdoumlaertoasre oIfI ,WanNdTt/eβlo-mcaetreansien,  asnigdnaaflfienctgs ian mCyRrCia dceolfls. 
signaIlnin gthpiast hswtuadyys ,[ 1c6r–y1p9t]o. lXeApVine9 3e9x, honibtihteedo tmheorrhea npdo,teisnat tcayntkoytroaxsiec inehffiebcittosr tthhaatn XAV 939. 
Cspreycpifitocalellpyiinneh itbaitrsgWetNs TDsNigAna, ltiongpo[2i7s]o.mTheerasusep eIrIi,o arncydt otetoloximc eefrfeacsteo, facnrydp atoffleepcitnse am mayyriad of sig-
nthaelrinefgo rpeabtehawttraiybus t[e1d6t–o1i9ts].a bXiAlitVy  t9o3r9e,g uolna ttehme uolttihpeler shigannadli,n igs paa tthawnkayysrsaismeu ilntahnieboiutoslry .that specif-
ically inhibits WNT signaling [27]. The superior cytotoxic effect of cryptolepine may there-
fore be attributed to its ability to regulate multiple signaling pathways simultaneously. 
Surprisingly, stimulating WNT activity in the CRC cells with recombinant WNT3a had no 
obvious effect on viability. In their study, Reischmann et al., (2015) found that, although 
WNT3a enhances HEK293 cell proliferation, this effect can only be detected by measuring 
DNA synthesis (BrdU assay) rather than mitochondrial activity (MTT assay) [28]. While 
this study did not assess the proliferative effects of WNT3a using other assays, the appar-
ent ineffectiveness of WNT3a may be ascribed to limitations in the method of measuring 
cell viability i.e., MTT assay. 
 
Pharmaceuticals 2023, 16, 1026 10 of 16
Surprisingly, stimulating WNT activity in the CRC cells with recombinant WNT3a had no
obvious effect on viability. In their study, Reischmann et al., (2015) found that, although
WNT3a enhances HEK293 cell proliferation, this effect can only be detected by measuring
DNA synthesis (BrdU assay) rather than mitochondrial activity (MTT assay) [28]. While
this study did not assess the proliferative effects of WNT3a using other assays, the apparent
ineffectiveness of WNT3a may be ascribed to limitations in the method of measuring cell
viability i.e., MTT assay.
β-catenin is the main effector in canonical WNT signaling. It interacts with TCF/LEF
and other proteins to induce the transcription of WNT-target genes [29]. The effect of
tankyrase inhibition on the expression of β-catenin shows that XAV 939 does not only
facilitate the degradation of β-catenin protein but also reduces its mRNA level as previously
reported [30]—either by inhibiting transcription of the CTNNB1 gene (encodes for β-
catenin) or by facilitating the degradation of β-catenin mRNA. Although cryptolepine
is reported to suppress the transcriptional activity of TCF/LEF [19], it is not known if
cryptolepine indeed affects the WNT/β-catenin signaling pathway, or how it affects the
pathway. This study revealed that cryptolepine inhibits WNT3a-induced expression of
β-catenin, c-MYC, and WISP1. This may explain the findings by Domfeh et al. (2021) who
showed that cryptolepine suppresses the transcriptional activity of both TCF/LEF and
MYC proteins [19]. Additionally, our findings are consistent with previous reports in which
cryptolepine reduced c-MYC levels in melanoma cells [31]. In CRC, deregulated c-MYC
is indispensable in cell growth and proliferation and is linked to unfavorable prognosis
and poor patient survival [32]. WISP1 also enhances survival by protecting cancer cells
from p53-mediated apoptosis [33]. The inhibitory effect of cryptolepine on the expression
of these genes is therefore favorable for CRC treatment. Moreover, cryptolepine suppressed
WNT/β-catenin signaling in WNT3a-stimulated cells only. We, therefore, hypothesize that
the influence of cryptolepine on the WNT/β-catenin pathway is selective for cells with
hyperactive WNT signals.
Colorectal cancer stem cells (CSCs) express cell surface markers such as OCT4 and
CD133 (AC133 or prominin 1). OCT4 promotes stem cell maintenance and immune evasion
and positively correlates with liver metastasis in CRC [34,35]. CRC cells expressing CD133
are more resistant to radiochemotherapy and are an indication of worse overall survival
and metastatic relapse [36,37]. WNT3a and XAV 939 had opposing effects on the expression
of OCT4 and CD133, supporting evidence on the influence of WNT signaling on the
expression of these genes and hence stemness of CRC cells. Cryptolepine reduced the
mRNA levels of OCT4 in WNT3a-stimulated cells, signifying its effect on WNT-meditated
OCT4 expression. Treatment of the cells with cryptolepine upregulated basal CD133
mRNA levels. We propose two theories to explain this observation. First, cryptolepine
enhances the transcriptional activity of SMAD2/SMAD3/SMAD4 proteins [19], which
drive transforming growth factor β (TGFβ) signaling. TGFβ has been shown to enhance
CD133 expression through the demethylation of CD133 promoter-1 [38]. Thus, cryptolepine
increased the expression of CD133 through the activation of TGFβ signaling. Second,
cryptolepine promoted MAPK/ERK signaling by enhancing the transcriptional activity of
the serum response element (SRE) protein [19]. Hyper-activated MAPK pathway leads to
overexpression of CD133 mRNA and protein in CRC [39]. Therefore, cryptolepine increased
CD133 expression by upregulating MAPK signaling. Experimental evidence is required to
validate these theories, especially because cryptolepine also enhances the transcriptional
activity of the p53 protein [19], which has been shown to suppress the activity of the CD133
promoter and downregulate CD133 expression [40]. This study also found that cryptolepine
reduced WNT3a-induced CD133 expression, suggesting that it may promote or suppress
CD133 expression depending on WNT activity in the cell.
The current study found that WNT3a induced colony formation in both cell lines.
Contrary to previous findings [41,42], tankyrase inhibition increased the colony formation
of the cells. This may be attributed to an inability of XAV 939 to exert a long-term effect
on the CRC cells, allowing them to regenerate after treatment. Conversely, cryptolepine
Pharmaceuticals 2023, 16, 1026 11 of 16
significantly suppressed the colony-forming ability of unstimulated and WNT3a-stimulated
cells. The superior ability of cryptolepine over XAV 939 to inhibit colony formation may be
attributed to the capacity of cryptolepine to exert a long-lasting effect on the cells. Moreover,
the ability of cryptolepine to inhibit telomerase activity and cause DNA damage may have
accounted for this observation, as these events also suppress colony-forming ability [43,44].
One essential step in metastasis is EMT, a phenomenon where cells undergo rearrange-
ment of the cytoskeleton and switch from a non-motile epithelial state to a motile mesenchy-
mal state. EMT facilitates cell migration and generates and maintains CSCs [45]. Several
transcription factors, including SNAIL1 and TWIST1, regulate EMT [25]. In this study,
WNT3a induced SNAI1 expression, corroborating previous reports [46,47]. Tankyrase
inhibition with XAV 939 upregulated basal SNAI1 expression in both cell lines. Contrasting
findings have been reported in this regard. While some research found that XAV 939
decreased SNAIL1 expression [48,49], other studies found that XAV 939 increased SNAIL1
expression [50,51]. Indeed, XAV 939 stabilizes and increases the abundance of Axin proteins,
which in turn activate SNAIL1 expression [47]. This may explain the increase in SNAI1 ex-
pression following tankyrase inhibition. Cryptolepine significantly reduced SNAI1 mRNA
levels in WNT3a-stimulated but not unstimulated cells, demonstrating the ability of cryp-
tolepine to inhibit EMT through WNT signaling. We also found that WNT3a induced
TWIST1 expression, which is consistent with previous studies that identified TWIST1 as a
WNT-inducible transcription factor whose expression can be activated by WNT3a [52,53].
Cryptolepine did not affect basal levels of TWIST1 but reduced WNT3a-induced TWIST1
levels. In cancer, the expression of TWIST1 is associated with lymphatic vessel invasion,
lymph node metastasis, and perineural invasion [25]. Additionally, TIWST1 binds to
β-catenin and enhances the transcriptional activity of the β-catenin/TCF4 complex [54].
Therefore, the ability of cryptolepine to suppress WNT3a-induced TWIST1 expression is a
favorable outcome.
Cancer cell migration is critical for distant metastasis [45]. The WNT/β-catenin path-
way is essential for this phenomenon, hence the impact of cryptolepine on CRC cell migra-
tion was studied. Stimulating WNT signaling promoted cell migration, whereas inhibiting
WNT signaling suppressed migration. This is supported by reports that WNT signaling
supports cell migration, and inhibition of tankyrase activity attenuates WNT3a-induced
cell migration [30,55]. Furthermore, cryptolepine remarkably impeded cell migration, both
under unstimulated and WNT3a-stimulated conditions. These findings, therefore, indicate
that cryptolepine suppresses the migration of CRC cells.
A key event in cancer invasion is the breakdown and remodeling of the extracellular
matrix by matrix metalloproteinases (MMPs). In CRC, MMP2 and MMP9 degrade the his-
tological barrier that would normally prevent invasion. Their overexpression is associated
with the transition from colon adenoma to adenocarcinoma and is correlated with poor
prognosis in CRC [26,56]. In this study, WNT3a induced MMP2 and MMP9 expression.
Conversely, tankyrase inhibition suppressed the expression of these genes. This corrobo-
rates findings that WNT3a upregulates downstream WNT targets MMP2 and MMP9 [53,57],
while XAV 939 suppresses their expression [48,49]. Additionally in this study, cryptolepine
had no effect on MMP2 and MMP9 basal mRNA levels but suppressed WNT3a-induced
expression of these genes, supporting the theory that the effect of cryptolepine on the
WNT/β-catenin pathway is selective for cells with hyperactive WNT signaling.
In conclusion, cryptolepine reduces WNT3a-induced expression of WNT target genes
and suppresses the proliferation, stemness, and metastatic processes of CRC cells (Figure 8).
Our findings suggest that the effect of cryptolepine on the WNT/β-catenin pathway is
specific for CRC cells with hyper-activated WNT signals. Thus, cryptolepine may be best
used against WNT-driven (WNT-mutant) cancers. Moreover, while both cell lines used in
this study have a mutant APC gene, COLO205 has additional β-catenin and BRAF mutations,
whereas DLD1 has an additional KRAS mutation [58]. It is uncertain how the additional
mutations will affect the activity of cryptolepine. However, the ability of cryptolepine to
inhibit the anticancer properties of both cell lines is a favorable outcome as it indicates that
Pharmaceuticals 2023, 16, 1026 12 of 16
in spite of the additional mutations that may cause CRC, cryptolepine may be an effective
Pharmaceuticals 2023, 16, x FOR PEERt hReErVaIpEeWu tic option. Further in vivo studies are required to confirm our findings. Novel 13 of 18 
 evidence may, in the future, confirm the administration of cryptolepine to be an effective
treatment for colorectal cancer patients.
 
Figuurree8 .8P. rPorpoopseodsemde cmheanchisamntihsmrou tghhrowuhgichh wcrhypicthol ecpryinpetionlheipbitnseC iRnChipbriotgs rCesRsiCon pvrioagWreNsTsi/oβn- via WNT/β-
catteenninins isginganlianlgin. Wg. NWTN3aTin3tae riancttesrwacitths iwtsictohg intast ceorgecneapteo rrteocetrpigtgoerr tao sterriigegs eorf  ian tsrearcieellsu olafr ienvternatcsellular events 
tthaattc cuulmlminianteatine tihne tuhper eugpurlaetgiounlaotfiWonN oTft WargNetTg etanregs.eCt rgyepntoelse.p Cinreydpotwolnerpeginulea tdesowthenWreNguTltaatregse tthe WNT tar-
geent egseineCsR iCn cCelRlsC, r ecseulllsti,n rgeisnutlhtienign hinib itthioen ionfhpirboiltiifoerna toiof np,rsotelmifenreastsi,oan,d smteemtansteasssis,. and metastasis.  
4. Materials and Methods
44..1 M. CaeltleLriinaelssa annd dC uMltuertehods 
4.1. CThelel CLOinLeOs a2n05d aCnudltDuLrDe 1 cell lines used in this study were originally obtained from
the AmTherei cCanOTLyOpe2C05u latunrde  CDoLllDec1ti ocnelaln ldinweesr eusaekdin idng tihftifsr osmtuPdryo fwesesroer Roergiginina aAlplyp ioahb-tained from 
Opong of the Department of Clinical Pathology at the Noguchi Memorial Institute for Med-
itchael  RAemseearrcihca, Gnh Taynpa.eT Cheuclteullrsew Ceroellmecaitniotanin aenddin wReorsew ae lkl iPnadrk gMifetm froormial PInrsotfiteustseo(Rr PRMegI)ina Appiah-
1O6p40onsugp polfe mtheen tDedepwairthtm10e%ntf eotfa lCbloivniinceals ePrautmho(FloBgSy)  aantd t1h%e pNenoigcuillcihn-is tMreepmtoomryiaciln I-nstitute for 
gMluetdamicianle R(aelslepaurrcch,a sGehdafrnoam. TGhibec coe-llilfse twecehrneo mlogaiienst,aCinareldsb iand ,RCoAs,wUeSlAl P),aartk3 7M◦Ceminoarial Institute 
h(RuPmMidIifi) e1d64at0m sousphpeleremcoenttaeidni nwgit5h% 1C0O%2 .fetal bovine serum (FBS) and 1% penicillin-strepto-
mycin-glutamine (all purchased from Gibco-life technologies, Carlsbad, CA, USA), at 37 
4.2. Compounds/Drugs
°C in a humidified atmosphere containing 5% CO2. 
Cryptolepine (C7124-10MG), XAV 939 (X3004-5MG), and human WNT3a (H17001-
10UG) were purchased from Sigma-Aldrich, St. Louis, MO, USA. XAV 939 is a specific
i4n.2h.i bCitoomr opfouWnNdsT/Dsigrunaglsi ng, while human WNT3a is an activator of WNT signaling. The
compoCurnydpstwoleerpe ipnrep (aCre7d1a2s4i-n1s0trMucGte)d, bXyAthVe m93a9n u(fXac3t0u0re4r-5i.Me.,Gcr)y,p atonledp ihnuemanadnX WAVN93T93a (H17001-
w10eUreGd)is swoelvreed pinudrcimhaestheydl sfurolfmox iSdiegamnda-WANldTr3icAhw, aSst.r eLcoounsisti,t uMteOd ,w UithSA1×. XphAoVsp 9h3a9te -is a specific 
binuhffiebreitdosra olifn eW(PNBTS) .signaling, while human WNT3a is an activator of WNT signaling. The 
4c.o3m. CpeloluVniadbisli twy eArses apyrepared as instructed by the manufacturer i.e., cryptolepine and XAV 
939 Twheereef fdeicstssoolfvcerdyp itnol edpiimnee,tXhAyVl s9u3l9f,oaxniddeW aNnTd3 aWonNCTO3ALO w20a5s arnedcoDnLsDti1tuvtieabdi lwityith 1× phos-
wpheraeted-ebteurffmeinred usasilningea n(PMBTST). assay. Briefly, the cells were seeded into 96-well plates at
a density of 1 × 104 cells/well and incubated at 37 ◦C for 24 h. They were treated with
c4r.y3p. tColeelpl iVnieaobrilXitAyV A9s3s9ayfo r 24, 48, and 72 h. For WNT3a, treatment was conducted for 24 h.
Then 20 µL of 2.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT)T(hSieg meffa-eAcltds roicfh c, Sryt Lpotouilse,pMinOe,,U XSAAV),  w9a3s9a, dadnedd Wto eNacTh3wa eollna CndOinLcOub2a0t5e daantd3 7D◦LCD1 viability 
fworer4eh d. Aetfeterrmwianrded, 1 u00siµnLgo afna cMidiTfiTed aisssoapyro. pBarnieofll wy,a tshaed dceedllst oweearceh wseeeldl aendd iinntcou b9a6t-ewd ell plates at 
at d37en◦Csitfoyr o30f m1 i×n .1A0b4 scoerlblasn/wceewll aasnreda dinactu5b70atnemd watit h37a V°Car ifooskr a2n4™ hL. UTXhemyu lwtimeroed etreated with 
cryptolepine or XAV 939 for 24, 48, and 72 h. For WNT3a, treatment was conducted for 24 
h. Then 20 μL of 2.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 
(MTT) (Sigma-Aldrich, St Louis, MO, USA), was added to each well and incubated at 37 
°C for 4 h. Afterward, 100 μL of acidified isopropanol was added to each well and incu-
bated at 37 °C for 30 min. Absorbance was read at 570 nm with a Varioskan™ LUX multi-
mode microplate reader (Thermo Fisher Scientific, Carlsbad, CA, USA). From the absorb-
ance values, percent cell viabilities were calculated. 
4.4. Colony Formation/Clonogenic Assay 
To assess the ability of the compounds to impact colony formation, the cells were 
seeded into 6-well plates at a density of 2 × 103 cells/well and incubated at 37 °C for 24 h. 
The cells in the WNT3a, WNT3a-cryptolepine, and WNT3a-XAV 939 groups were treated 
 
Pharmaceuticals 2023, 16, 1026 13 of 16
microplate reader (Thermo Fisher Scientific, Carlsbad, CA, USA). From the absorbance
values, percent cell viabilities were calculated.
4.4. Colony Formation/Clonogenic Assay
To assess the ability of the compounds to impact colony formation, the cells were
seeded into 6-well plates at a density of 2 × 103 cells/well and incubated at 37 ◦C for
24 h. The cells in the WNT3a, WNT3a-cryptolepine, and WNT3a-XAV 939 groups were
treated with 100 ng/mL of WNT3a for 24 h. Following this, the cells were treated or
untreated with cryptolepine or XAV 939 for 72 h. At 3 days post-treatment, the culture
medium was removed and fresh medium without drugs was added. The medium was
changed every 3 days for 14 days. Then, the cells were washed with 1× PBS, fixed with 4%
paraformaldehyde for 10 min, and stained with crystal violet dye (0.1% w/v) for 15 min.
Excess dye was washed with 1× PBS and images were taken with an OPTIKA® microscope
(OPTIKA, Ponteranica, Italy). ColonyArea plugin in ImageJ (NIH, Bethesda, MD, USA) was
used to determine clonogenic cell growth [59].
4.5. Wound Healing Assay
A wound healing assay was performed to determine the effect of the compounds
on cell migration. Briefly, 70 µL of cell suspension containing 2 × 104 cells were seeded
into each chamber of a 35 mm µ-dish with culture-insert (Ibidi GmbH, Munich, Germany)
placed in a 6-well plate, and incubated at 37 ◦C for 24 h. The cells in the WNT3a, WNT3a-
cryptolepine, and WNT3a-XAV 939 groups were treated with 100 ng/mL of WNT3a for 24 h.
Then, the inserts were removed and the cells were treated or untreated with cryptolepine or
XAV 939 for 48 h. Cell migration was monitored with an OPTIKA® microscope (OPTIKA,
Ponteranica, Italy) at 0, 24, and 48 h. The images were analyzed with the Wound Healing
Size Tool plugin in ImageJ (NIH, Bethesda, MD, USA) to determine the percentage closure
of the wound [60].
4.6. Reverse-Transcription Quantitative PCR (RT-qPCR)
COLO205 and DLD1 cells were seeded into 6-well plates at a density of 1 × 106
cells/well and treated as described. Total RNA was extracted using a Quick-RNA™
MiniPrep Plus Kit (Cat. No. R1057: Zymo Research, Irvine, CA, USA) per the manu-
facturer’s protocol. Using a Luna Universal One-Step RT-qPCR kit (New England Biolabs,
Ipswich, MA, USA), RT-qPCR was performed to determine the mRNA expression of β-
catenin, c-MYC, WISP1, CD133, OCT4, SNAIL1, TWIST1, MMP2, and MMP9 genes. A
QuantStudio™ 5 RT-PCR System (Thermo Fisher Scientific, Carlsbad, CA, USA) was used
for reverse transcription and amplification. Thermocycling conditions were reverse tran-
scription (55 ◦C for 15 min), initial denaturation (95 ◦C for 1 min), 40 cycles of denaturation
(95 ◦C for 15 s), annealing (different temperatures depending on target gene for 15 s), and
extension (60 ◦C for 1 min). The housekeeping gene GAPDH was used as an internal
control. Sequences and annealing temperatures of primers are listed in Supplementary
Table S1. QuantStudio™ Design & Analysis Software (Life Technologies, Carlsbad, CA,
USA) was used to obtain CT values, and the 2−∆∆CT method [61] was used to determine
the relative expression of each gene.
4.7. Statistical Analyses
Data were analyzed using GraphPad Prism 9.1.2 (GraphPad Software, San Diego,
CA, USA). One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc
test was used to compare differences between multiple groups. Data are presented
as the mean ± standard error of the mean (SEM) of at least three independent experi-
ments performed in triplicate. Group differences were considered statistically significant
when p ≤ 0.05.
Pharmaceuticals 2023, 16, 1026 14 of 16
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/ph16071026/s1, Table S1: Sequences and annealing tem-
peratures of primers.
Author Contributions: Conceptualization, J.T.Q. and A.R.A.; methodology, J.T.Q., K.F., N.A.O.
and A.K.S.; writing—original draft preparation, J.T.Q.; writing—review and editing, O.Q. and
A.R.A.; supervision, O.Q. and A.R.A. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by funds from a World Bank African Centres of Excellence
grant (WACCBIP+NCDs: Awandare) and a DELTAS Africa grant (DEL-15-007: Awandare). This
research was funded in whole, or in part, by the Wellcome Trust [DEL-15-007] and the UK Foreign,
Commonwealth & Development Office, with support from the Developing Excellence in Leadership,
Training and Science in Africa (DELTAS Africa) programme.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and supple-
mentary material.
Acknowledgments: The authors would like to thank Alberta Serwaa, Nelson Edu, and Evans
Armaah-Vedjesu of the Department of Biochemistry Cell and Molecular Biology at the University of
Ghana for their technical support in running the RT-qPCR experiments.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
CRC: colorectal cancer, CSC: cancer stem cell, ECM: extracellular matrix, EMT: epithelial-
mesenchymal transition; FZD: frizzled, ISC: intestinal stem cell, MMP: matrix metalloproteinase,
OCT4: octamer-binding transcription factor 4, TCF/LEF: T cell factor/lymphoid enhancer factor,
TGF-β: transforming growth factor-β, WISP1: WNT-induced secreted protein 1, WNT: wingless
integrated type-1.
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