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Light-activated ruthenium (II)-bicalutamide prodrugs for prostate cancer.
Journal of Inorganic Biochemistry 196 (2019) 110684
ContentslistsavailableatScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Light-activated ruthenium (II)-bicalutamide prodrugs for prostate cancer
T
Jian Zhaoa, Nannan Liua, Shuchen Suna, Shaohua Goua,⁎ , Xinyi Wanga, Zhimei Wanga,
Xiaoyan Lib, Wenjing Zhangb,⁎
aResearchCenterandSchoolofChemistryandChemicalEngineering,andJiangsuProvinceHi-TechKeyLaboratoryforBiomedicalResearch,SoutheastUniversity,
Nanjing211189,China
bTheCollegeofChemistryandMolecularEngineering,ZhengzhouUniversity,Zhengzhou,HenanProvince450001,China
ARTICLE INFO ABSTRACT
Keywords: Targeteddeliveryofclinicallyapprovedanticancerdrugtotumorsitesisaneffectivewaytoachieveenhanced
Ruthenium(II)complexes drugefficacyaswellasreducedsideeffectsandtoxicity.HerebicalutamideiscagedbytheRu(II)centerthrough
Photoactivatedchemotherapy thenitrilegroup,andthreephotoactiveRu(II)complexesweredesignedandsynthesized.Dockingstudyshowed
Bicalutamide that the ruthenium(II)fragmentscaneffectively blockthebinding of complexes1–3 with AR(androgenre-
Anticancer
ceptor)owingtothelargestericstructures,thusbicalutamideincomplexes1–3couldnotinteractwithAR-LBD
(ligandbindingdomain).Onceirradiationwithbluelight(465nm),complexes1–3canreleasebicalutamideand
anticancerRu(II)fragments,whichpossessesdual-actionofARbindingandDNAinteractionsimultaneously.In
vitrocytotoxicitystudyonthesecomplexesfurtherconfirmedthatcomplexes1–3exhibitedconsiderablecy-
totoxicityuponirradiationwithbluelight.Significantly,complex3couldbeactivatedat660nm,whichgreatly
increasesthescopeofcomplex3totreatdeeperwithintissue.Theoreticalcalculationsshowedthatthelowest
singletexcitationenergyofcomplex3islowerthanthoseofcomplexes1–2,whichexplainstheexperimental
resultswell.Moreover,the3MC(metalcentered)statesofthesecomplexesaremorestablethantheir3MLCT
(metaltoligandchargetransfer)states,indicatingthatthephotoactiveprocessesofthesecomplexesarelikelyto
resultinliganddissociation.
1. Introduction which enables maximum tissue penetration with minimum damage
[29,37–40]. Recently, ruthenium-based PACT agents with high se-
Prostatecancerisoneoftheleadingcausesofcancerdeathformen lectivityandlowtoxicityhavebeenattractingmuchattention[41–44],
worldwide [1]. The development and growth of prostate cancer is as they can be used to deliver a wide variety of bioactive molecules,
found to be initially dependent on androgens, and androgen receptor including enzyme inhibitors, neurotransmitters and so on [45–53]. A
(AR)islargelyinvolvedintheprogressionofthiscancer[2,3].There- majority of clinically approved anticancer drugs are small molecular
fore,therapeuticstrategiesbyregulatingtheARactivitycaneffectively compounds. Their efficacy has been well studied and established.
lead to a reduction of prostate cancer [4–9]. Because AR is widely However,thesecompoundstendtohavewidebiodistributionsamong
distributed in the human tissues, including the prostate and seminal various tissues after intravenous administration, which may result in
vesicles, skin, cardiac muscle, adrenal cortex, liver, and so on [10]. widespread systemic toxicity and suboptimal efficacy. Thus, targeted
Thus,improvingtheselectivityandactivityoftheARantagonistarethe delivery of clinically approved anticancer drugs to tumor sites is an
keypointsforthetreatmentofprostatecancer. effective way to achieve enhanced drug efficacy and to reduce side
Photoactivated chemotherapy (PACT) is a promising strategy for effectsandtoxicity.
tumortargeteddelivery[11–19],whichcanbetriggeredbyirradiation Inthisstudy,bicalutamide,themostwidelyusedARantagonistin
toreleasetheactivespecieswithinthetumor[20–32].Generally,such clinic, is chosen to coordinate to ruthenium(II) fragments through its
agentsasprodrugsarekineticallyinertandnontoxicintheabsenceof nitrilegroupwhichwasreportedasanimportantbioactive“warheads”
light, but rapidly give rise to toxic effects upon photoexcitation often engaging in key hydrogenbonding interaction with protein tar-
[33–36].Anidealcandidateforaphotoactivatableagentshouldhave gets [54]. Although the nitrile groups are widely present in medical
the ability to be activated in the “therapeutic window”, 600–850nm, agents,onlyafewstudiesregardingthecagingofthebioactivenitriles
⁎Correspondingauthors.
E-mailaddresses:sgou@seu.edu.cn(S.Gou),zhangwj@zzu.edu.cn(W.Zhang).
https://doi.org/10.1016/j.jinorgbio.2019.03.024
Received14January2019;Receivedinrevisedform25March2019;Accepted28March2019
Available online 30 March 2019
0162-0134/ © 2019 Elsevier Inc. All rights reserved.
J.Zhao,etal. Journal of Inorganic Biochemistry 196 (2019) 110684
Fig.1.Chemicalstructuresofcompoundsstudied.
throughRu(II)coordinationhavebeenreported[55–59].Herein,three whereascomplex3presentedobviouschangesunderthesamecondi-
light-activated ruthenium(II) complexes were designed, synthesized tion, confirming that complex 3 could be activated in the PDT (pho-
and biologically evaluated as potential PACT agents (Fig. 1). Ex- todynamictherapy)therapeuticwindow(Fig.S11).Thequantumyields
pectedly, the designed compounds can rapidly release bicalutamide ofthefirststepofcompounds1–3werefoundtobe0.021,0.022and
upon irradiation at the tumor site and leave the diaquaruthenium(II) 0.019inthesametestcondition(465nm),respectively.Thephotolysis
moiety to interact with DNA, exhibiting dual-actions against prostate of complexes 1 and 3 was further confirmed by 1H NMR as well.
cancercells. Complex1wasstableunder660nmirradiation,however,itunderwent
liganddissociationwithirradiationat465nm(Fig.S12).Unlikecom-
2. Resultsanddiscussion plex1, spectralchangeswere observedforcomplex3after15minof
irradiationat660nm,whichisconsistentwiththeUV–Visresults(Fig.
2.1. Synthesisandcharacterization S13).
Complexes1–3werepreparedbyrefluxingamixtureofcis-RuL2Cl2 2.3. Theoreticalcalculations
(L=2,2′-bipyridine, 1,10-phenanthroline and 2,2′-biquinoline), bica-
lutamide (4.0equiv) and silver(I) hexafluorophosphate (2.4equiv) in Theoretical calculations were performed to gain further under-
ethanol.Thetargetcomplexeswerecharacterizedby1Hand13CNMR standing of the photolysis of complexes 1–3. TDDFT (time-dependent
spectroscopy,electrosprayionizationmassspectrometry(ESI-MS)and density functionaltheory)calculationsshowedthat thelowest-energy
elemental analysis (Figs. S1–9). The ESI-MS diagrams of compounds excitedsingletstatesofcomplexes1–3are1MLLCT(MLLCT=metal-
1–3 showed the highest isotope at 637.06, 661.06 and 737.14, re- ligand-to-ligandchargetransfer)transitionsduetothecontributionsof
spectively, corresponding to the dication of [RuL2(bicalutamide)2]2+. bicalutamide(Fig.3)[62],andthecorrespondingexcitationenergiesof
Moreover,1Hand13CNMRspectraaswellaselementalanalysiswere complexes 1–3 are 2.76eV (449nm), 2.91eV (427nm) and 2.25eV
in good conformity with the proposed molecular structures of com- (551nm),respectively(TableS1),whichisinaccordwiththeexperi-
plexes1–3.ThelogPOW(theoctanol-waterpartitioncoefficient)values mental results that the absorption tail of complex 3 terminates at re-
of the ruthenium(II) complexes were measured using the shake-flask latively longer wavelength as compared with complexes 1–2. More
method, which were −0.46, −0.28 and −0.19 for complexes 1–3, intense absorption bands indicating the MLLCT transitions are pre-
respectively. The stabilityofcomplexes1–3wasevaluatedbyUV–vis dicted at 320nm (f=0.0476), 341nm (f=0.0420) and 478nm
spectral analysis at different times (Fig. S10) [60,61]. No detectable (f=0.0517) for complexes 1–3, respectively,which also corresponds
changesoftheabsorptionbandsofcomplexes1and2havebeenob- wellwiththeexperimentalresults.
served within 15h, while tiny changes were observed for complex 3, Itisgenerallyacceptedthatthecomplexesarefirstexcitedbythe
demonstratingthatcomplexes1and2weremorestablethancomplex desiredwavelengthoflightandthenundergointersystemcrossinginto
3. However, the changes of the absorption bands of complex 3 were a 3MLCT state, followed by internal conversion to a dissociative 3MC
verysmall,indicatingthatnegligiblehydrolysiswashappenedduring stateandreleaseofthebicalutamide.Thus,twolow-lyingtripletstates
thetesttime. of complexes 1–3 were optimized, corresponding to the 3MLCT and
3MC states (spin density surfaces). The 3MC states of complexes 1–3
2.2. Photolysisstudy corresponds to five-coordinate structures with a bicalutamide dis-
sociatedfromtheRucenter.Notably,therelativeenergiesof3MCstates
The photolysis of complexes 1–3 in 98% H2O/2% MeOH was stu- ofcomplexes1–3are0.35eV,0.45eVand0.28eVbelowtheir3MLCT
died under irradiation with blue light (465nm) by using UV–Vis stats, respectively, indicating that the photoactive process of these
spectroscopy. UV–Vis spectral changes were obviously observed for complexesismorelikelytoresultinliganddissociation[63,64].
these complexes after irradiation (Fig. 2). The metal-to-ligand charge
transfer (MLCT) bands at 336nm and 359nm for complexes 1 and 2 2.4. Dockingstudy
were graduallyreplacedby newMLCTbands at490nmand 480nm,
respectively.Forcomplexes1and2,noisosbesticpointwasobserved, Inordertoevaluatethelikelihoodofcomplexes1–3bindingtothe
indicatingthatthedissociationreactionwasmorethanonestep.Asfor androgen receptor, a docking study with crystal structure of AR-LBD
complex 3, the initial MLCT was observed at λmax=515nm, which (ligand binding domain) (PDB: 1z95) was performed [65]. Bicaluta-
showed bathochromic shift compared to complexes1 and 2 [44], de- midewasfirstlydockedwiththeAR-LBD,whichwassituatedinsidethe
monstrating that the application of biquinoline in complex 3 can de- bindingpocketofARandshowedgoodoverlapwiththeco-crystallized
crease the energy gap between the d-orbital of Ru(II) and the lowest bicalutamideintheARcrystalstructure,provingthatthemethodused
unoccupied molecular orbital (LUMO) levels of the ligand. Moreover, issuitablefordockingstudy(Figs.4a,S16a).However,complexes1–3
complexes 1–3 were studied with irradiation at longer wavelength. werefoundtobesituatedonthesurfaceoftheprotein(Figs.4b,S16b,
Neither1nor2showedanybandchangesuponirradiationat660nm, c), which could not enter theligand binding pocket ofAR due tothe
2
1.0
0.5 ↑
0.0
300 400 500 600 700
largestericstructuresofthecomplexes.Moreover,theinteractionen- bicalutamide as a positive control. According to the IC50 values
ergies of these complexes with AR were also calculated. As shown in (Table 1), bicalutamide showed moderate cytotoxicity against LNCaP
Table S2, the binding energy of the bicalutamide molecule was cells with an IC50 value of 45.0μM, which was not affected by irra-
−9.61kcal/mol,whichwasmuchlowerthanthoseofcomplexes1–3, diation.Complexes1–3werefoundtobelesscytotoxicinthedarkthan
indicating that the ruthenium(II) fragments decreased the binding af- bicalutamide against LNCaP cells. However, the antiproliferative ac-
finityofbicalutamidewithAR.Takentogether,thisstudyindicatedthat tivities of complexes 1–3 were greatly improved upon irradiation at
theruthenium(II)fragmentsofcomplexes1–3caneffectivelyblockthe 465nm with PI (phototoxicity index: the toxicity in the dark vs. the
binding of bicalutamide with AR owing to their large sizes, implying light) values of 9.6, 5.3 and 7.7, respectively. The ability of these
thatcomplexes1–3wouldbebiologicallyinactiveprecursors. complexes activated by red light (660nm) was much attractive, be-
causelongerwavelengthsoflightcanpenetratedeeperintothetissue
withlessnormal-tissuedamage.Thecytotoxicityofcomplexes1and2
2.5. ARbindingaffinity
greatly decreased upon irradiation at 660nm compared with irradia-
tionat465nm,whilethatofcomplex3wasslightlydecreasedwithaPI
TheARbindingaffinityofcomplexes1–3weredeterminedbythe
valueof5.8.AsforPC-3cells(AR-),bicalutamideshowednoapparent
fluorescence polarization based binding assay. According to the IC50
activity, exhibiting that the cytotoxicity of bicalutamide is dependent
values(Fig.4c,TableS3),complexes1–3showednegligibleARbinding
on the expression of AR. In addition, complexes 1–3 had negligible
affinity in the dark. In contrast, significant AR binding affinity was
cytotoxicity with or without irradiation (Table S4). Taken together,
observed forcomplexes1–3uponirradiationat465nm.Notably,the
complexes1–3asconfirmedcouldbeactivatedbylight,whichshowed
IC50valueofcomplex2waslowerthanthatofbicalutamide,demon-
considerable cytotoxicity against AR+ LNCaP cells. Especially, com-
strating that more than one equiv of bicalutamide was released from
plex3couldbeactivatedbyredlightthatisknowntopenetratetissue
complex 2. With irradiation at 660nm, the binding affinity of com-
deeper.
plexes1and2wasgreatlyweakened,whilecomplex3maintainedthe
AR binding affinity with an IC50 value of 9.2μM. The result further
indicated that complexes 1–3 are biologically inactive precursors,
2.7. DNAinteraction
whichcouldbeactivateduponirradiationwithlight.
The interaction of DNA with complex 3 was monitored by tapping
2.6. Invitrocytotoxicity modeatomicforcemicroscopy(AFM)[67].TheimageofpBR322DNA
withoutrutheniumcomplexesisshowninFig.5a.Theaverageheightof
The cytotoxicity and photocytotoxicity of complexes 1–3 were theDNAwas1.0nm(n=15).Whencomplex3wasaddedtothesolution
evaluatedagainstAR-negativePC-3andAR-positiveLNCaPcellswith of DNA, no obvious morphological changes were observed. Once
sbA
(a) (b)1.5
↓ 1.0
↓
0.5 ↑
0.0
300 400 500 600 700
Wavelength (nm)
sbA
0.4
0.3
0.2 ↑
↓ ↓
0.1
0.0
400 500 600 700 800
Wavelength (nm)
sbA
J.Zhao,etal. Journal of Inorganic Biochemistry 196 (2019) 110684
(c)
Wavelength (nm)
Fig. 2.Electronic absorptions of a) complex 1, b) complex 2, c) complex 3 (25μM, 98% H2O/2% MeOH) upon irradiation with blue light (λirr=465nm,
tirr=40min,10mWcm−2)every2min.
Fig. 3.Relative energies of the 1MLLCT, 3MLCT and 3MC
states for complexes 1–3, electron density difference maps
(EDDMs) of the lowest-lying singlet transitions (yellow in-
dicatesadecreaseinchargedensity,whileorangeindicatesan
increase)andspindensityofT1and3MCstatesforthecom-
plex 3. Both EDDMs and spin density were plotted by the
GaussView (version 5.0) program, and those for complexes
1–2weregiveninFigs.S14–S15.
3
J.Zhao,etal. Journal of Inorganic Biochemistry 196 (2019) 110684
Fig.4.Dockingstudiesofbicalutamideandcomplex
3 with AR-LBD (PDB: 1z95) using Autodock 4.2
[66].(a)Thedockingmodelofbicalutamideand(b)
complex3withAR,yellowdashlinesrepresentthe
H-bondinteractionbetweenbicalutamideandcom-
plex3withAR;(c)ARbindingaffinityofcomplexes
1–3andbicalutamidewithorwithoutlightirradia-
tion.
Table1 staining and flow cytometry assay. As shown in Fig. 7, when LNCaP
IC50values(μM)andphotoselectivityindexes(PI)ofcomplexes1–3inLNCaP cells were treated with Ru(II) complexes upon irradiation at 465nm,
cells. the typical morphological changes of cell apoptosis such as cell
Compound IC50(μM) PIa IC50(μM) PIa shrinkageandchromatincondensationwereobserved,whilenucleiof
thecellsretainedtheregularroundcontoursinthedark,indicatingthat
Dark 465nm 660m Ru(II) complexescausedlittlecell apoptosiswithout lightirradiation.
Theresultswerefurtherexaminedbyflowcytometryassay.Underdark
1 85.7±4.8 8.9±0.6 9.6 75.3±3.8 1.1
conditions, the apoptoticrates ofLNCaP cells treated withcomplexes
2 71.4±2.5 13.6±1.9 5.3 68.5±4.5 1.0
3 91.9±5.7 11.9±1.2 7.7 15.8±1.7 5.8 1–3 were 16.51%, 15.9%, and 14.42%, respectively, which were
Bicalutamide 45.0±4.9 42.3±3.4 1.0 47.4±2.1 1.0 slightlyincreasedascomparedwiththatoftheuntreatedcells(6.01%).
Onceirradiationwasconducted(465nm),theapoptoticratesinduced
a PI=darkIC50value/lightIC50value.
bycomplexes1–3weresignificantlyincreasedincontrasttothedark
group, which ranged from 70.29% to 72.56%. Particularly, when
irradiationwasconducted(465nmor660nm),theformationofreticular
complex 3 was irradiated with 660nm, the apoptotic rate reached
structures was observed. This suggested that complex 3 with light irra-
67.84%whichwasmuchhigherthanthecontrol(9.01%)(Fig.S17).
diationinducedcrosslinkingbetweenDNAmoleculesbyabridgingeffect,
whicharepresumablyattributedtoDNAcovalentbindingeffect.
3. Conclusions
2.8. Gelelectrophoresisstudy
Ruthenium(II) polypyridyl complexes are a kind of promising
photo-triggereddrugdeliverysystem,whichofferspatialandtemporal
The ability of complex 3 to bind DNA upon light activation was
controloverthereleaseofthebioactivedrugs.Herein,
furtherdeterminedbygelelectrophoresiswithpBR322plasmidDNA.
three light-activated Ru(II)-bicalutamide prodrugs were designed
AsshowninFig.6,adecreaseintherateofmigrationforclosedcircular
andsynthesized,whichpossessbothARbindingandDNAinteraction
DNA (form I) wasobserved upon 465nmand 660nmirradiation,in-
actions.Dockingstudiesshowedthatthesecomplexescouldnotenter
dicating the covalent binding of complex 3 with pBR322 DNA. How-
theligandbindingpocketofARduetothelargestericstructuresofthe
ever,nomigrationwasobservedforcomplex3inthedark.Moreover,
Ru(II) fragments, implying that they would be biologically inactive
theplasmidDNAgraduallydisappearedwithincreasingconcentrations
precursors.However,whenirradiatedwithbluelight(465nm),these
ofcomplex3,indicatingthatcomplex3inhibitedtheintercalationof
complexescouldreleasebothbicalutamideandanticancerRu(II)frag-
EtBrinplasmidDNAathighconcentrations.Thisstudyindicatesthat
mentstoinhibitthegrowthofcancercellsviadifferentmodesofaction.
complex3cancovalentlybindtoDNAuponirradiation.
Invitrocytotoxicitystudyconfirmedthatcomplexes1–3couldbeac-
tivated upon photoexcitation with blue light, exhibiting considerable
2.9. Apoptosisstudy cytotoxicity against the LNCaP (AR+) cells. Significantly, the antic-
ancer activity of complex 3 was also retained when irradiated by red
ThepotentialofthesecomplexestoinduceapoptosisinLNCaPcells light (660nm), hintingthat complex 3could be activatedin thePDT
wasperformedunderlightanddarkconditionsbyHoechst33342DNA therapeutic window. Theoretical calculations showed that the singlet
4
J.Zhao,etal. Journal of Inorganic Biochemistry 196 (2019) 110684
Fig.5.AFMimagesofpBR322plasmidDNAinHEPES(N-2-
hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer ad-
sorbedonpeeledmica:(a)imagesofpBR322plasmidalone;
(b) incubated with complex 3 in dark; (c) incubated with
complex3uponirradiationat465nm;and(d)incubatedwith
complex3uponirradiationat660nm.
4. Materialsandmethods
4.1. Materialsandinstrument
All chemicals and solvents were of analytical reagent grade and
used without further purification. Bicalutamide was obtained from
AladdinasaracemicmixtureoftheRandSenantiomersandusedas
received. Cis-RuL2Cl2 (L=2,2′-bipyridine, 1,10-phenanthroline and
2,2′-biquinoline)werepreparedaccordingtopreviousreports[37,68].
1H and 13C NMR spectra were measured on Bruker Avance III-HD
600MHz spectrometer. UV–Vis spectra and kinetic traces were re-
corded on a Shimadzu UV2600 instrument. Mass spectra were mea-
suredbyanAgilent6224ESI/TOFMS(electrospray-ionizationtime-of-
flightmassspectrometry)instrument.ElementalanalysisofC,H,andN
used a Vario MICRO CHNOS elemental analyzer (Elementar). Cancer
cells were obtained from Jiangsu KeyGEN BioTECH company(China).
Cell apoptosis experiments were measured by flow cytometry (FAC
Scan,BectonDickenson)andanalyzedbyCellQuestsoftware.
4.2. Preparationofcompounds
Fig.6.GelelectrophoreticmobilitypatternofpBR322plasmidDNAincubated
withvariousconcentrationsofcomplex3.Lane1–8(0,10,20,40,80,160,320, Generalprocedureforsynthesisofcomplexes1–3.Amethanolso-
640μM)+DNA.a)dark;b)irradiationat465nmfor;c)irradiationat660nm. lution (50mL) of RuL2Cl2 (0.20mM), AgPF6 (0.12g, 0.48mM) and
bicalutamide (0.34g, 0.8mM) was heated at reflux for 12h in the
excited energy of complex 3 is lower than those of complexes 1–2, absenceoflight.ThenthesolutionwasfilteredtoremoveAgClandthe
which can explain the experimental results well. Moreover, the 3MC reaction mixture was concentrated to 5mL. The crude product was
states of these complexes are more stable than their 3MLCT state, in- collected and recrystallized from methanol. Complexes 1–3 were iso-
dicating that the photoactive processes of these complexes are more latedasracemicmixtures(seesupportingFig.S18).
likelytocauseligand dissociation.Inall,ourstudyindicatedthatca- Complex1.Yield:0.16g(50.6%).Dark-redpowder.Anal.Calcd(%)
ging of bicalutamide through its nitrile group is an efficient way to forC56H44F20N8O8P2RuS2:C43.00,H2.84,N7.16.Found:C43.09,H
increasingitsselectivity.Therefore,photocagingofclinicallyapproved 2.88,N7.08;ESI-MS:m/z[M/2–PF6]+=637.06;1HNMR(600MHz,
anticancer drugs using ruthenium(II) polypyridyl complexes is an ef- DMSO‑d6)δ1.38(s,6H),3.72(s,1H),3.74(s,1H),3.90(s,1H),3.93(s,
fectivewaytodiscovernewclinicalagents. 1H), 6.46 (m, 2H), 7.36–7.39 (t, 4H, J=8.8Hz), 7.48–7.51 (t, 2H,
5
Control Complex 1 Complex 2 Complex 3 Bicalutamide
Control Complex 1 Complex 2 Complex 3 Bicalutamide
(b)
Control Complex 1 Complex 2 Complex 3 Bicalutamide
AnnexinV-FITC
Control Complex 1 Complex 2 Complex 3 Bicalutamide
J=6.7Hz), 7.78–7.79 (d, 2H, J=5.4Hz), 7.90–7.92 (m, 4H), 2.96,N6.37;ESI-MS:m/z[M/2–PF6]+=737.14;1HNMR(600MHz,
8.00–8.02(t,2H,J=6.5Hz),8.16–8.19(t,2H,J=7.9Hz),8.26(m, CD3OD)δ1.30(s,6H),3.45(s,1H),3.47(s,1H),3.85(s,1H),3.89(s,
4H),8.36(m,2H),8.45–8.48(m,2H),8.80–8.82(d,2H,J=8.2Hz), 1H), 6.77–6.84 (m, 4H), 7.04–7.08 (m, 4H), 7.44–7.46 (t, 2H,
8.93–8.94 (d, 2H, J=8.3Hz), 9.48–9.49 (d, 2H, J=5.4Hz), 10.44 J=7.4Hz), 7.77–7.80 (m, 4H), 7.86–7.87 (d, 2H, J=7.9Hz),
(m, 2H) ppm; 13C NMR (150MHz, DMSO‑d6) δ 16.97, 26.47, 56.94, 7.91–8.00(m,10H),8.09–8.10(m,2H),8.28–8.30(d,2H,J=8.0Hz),
63.60,73.22,100.81,115.66,115.81,117.46,121.29,122.41,122.48, 8.34–8.36(m,4H),8.69–8.70(d,2H,J=8.6Hz),9.62(m,2H)ppm;
123.10, 123.74, 124.09, 127.10, 128.05, 131.36, 131.43, 133.52, 13C NMR (150MHz, CD3OD) δ 26.43, 53.41, 63.51, 73.14, 100.64,
133.73, 136.87, 136.89, 137.12, 138.64, 138.97, 143.69, 151.71, 115.61, 115.76, 117.37, 118.84, 120.05, 121.00, 122.45, 122.81,
153.43,157.00,157.73,164.90,166.58,173.98ppm. 123.42, 127.51, 128.87, 129.09, 129.70, 130.00, 131.33, 131.40,
Complex2.Yield:0.17g(52.3%).Dark-redpowder.Anal.Calcd(%) 133.05, 133.27, 133.83, 136.74, 137.06, 140.05, 140.71, 143.88,
forC60H44F20N8O8P2RuS2:C44.70,H2.75,N6.95.Found:C44.79,H 149.17,150.43,160.36,160.59,164.85,166.53,173.86ppm.
2.74,N6.86;ESI-MS:m/z[M/2–PF6]+=661.06;1HNMR(600MHz,
CD3OD)δ1.32(s,6H),3.46(s,1H),3.49(s,1H),3.86(s,1H),3.89(s,
1H), 5.38 (m, 1H), 7.07–7.10 (m, 4H), 7.49–7.52 (d-d, 2H, J=5.3, 4.3. LogPOWdetermination
2.8Hz), 7.79–7.81 (m, 4H), 7.89–7.94 (m, 4H), 7.98–7.99 (d, 2H,
J=8.7Hz), 8.03–8.04 (m, 2H), 8.15–8.16 (d, 2H, J=8.9Hz), ThelogPOWdeterminationofcomplexes1–3wasperformedusing
8.23–8.26 (m, 2H), 8.28–8.29 (d, 2H, J=8.9Hz), 8.50–8.52 (d, 2H, the shake-flaskmethod. An excess ofcomplexes1–3 wasdissolved in
J=8.2Hz), 8.88–8.89 (m, 2H), 9.83–9.84 (d, 2H, J=5.1Hz) ppm; double-distilled water presaturated with n-octanol for 24h at 37°C.
13C NMR (150MHz, CD3OD) δ 26.45, 63.57, 73.19, 100.81, 115.63, Thesolutionwasfilteredtoremoveundissolvedrutheniumcomplexes.
115.79, 117.32, 121.04, 122.40, 122.62, 125.46, 126.59, 127.67, Subsequently,thesolutionwasaddedtoanequalvolumeofn-octanol
127.89, 130.77, 131.11, 131.34, 131.40, 133.41, 133.62, 136.87, (presaturatedwithwater).Theheterogeneousmixturewasshakenfor
137.04, 137.51, 137.95, 143.59, 147.71, 148.18, 152.77, 154.52, 2h before centrifuging for 15min to achieve phase separation. The
164.87,166.56,173.93ppm. initialandfinalconcentrationsofcompoundsinanaqueousphasewere
Complex3.Yield:0.22g(61.3%).Dark-redpowder.Anal.Calcd(%) determined by the UV–vis spectrum method, and the water-octanol
forC72H52F20N8O8P2RuS2:C49.01,H2.97,N6.35.Found:C49.12,H partitioncoefficients(logPOW)werecalculated.
IP
AnnexinV-FITC
IP
J.Zhao,etal. Journal of Inorganic Biochemistry 196 (2019) 110684
(a)
20 μm 20 μm 20 μm 20 μm 20 μm
20 μm 20 μm 20 μm 20 μm 20 μm
(c)
(d)
Fig.7.CellapoptosisinductiononLNCaPcellsaftertreatmentwithcomplexes1–3andcisplatinat30μM:morphologicalchangeswithHoechst33342stainingin
theabsence(a)andpresencebluelight(b);FlowcytometryanalysisforapoptosisofLNCaPcellsintheabsence(c)andpresencebluelight(d).
6
J.Zhao,etal. Journal of Inorganic Biochemistry 196 (2019) 110684
4.4. Photolysisstudy first and then diluted to 0.5mM with hepes buffer (the final con-
centration of DMF was less than 0.4%). The solutions of complex 3
Photolysisofcomplexes1–3wasrecordedonaShimadzuUV2600 (20μL)andpBR322(20μL)weremixturetogether,andirradiatedwith
instrument equipped with a thermostatically controlled cell holder. blue light (465nm, 30min) or red light (660nm, 1h). After 24h in-
Stock solutions of 1–3 were prepared in methanol and diluted with cubation,themixturewasdroppedontofreshlycleavedmica.Thenthe
watertogiveafinalwater/methanolcompositionof98:2.Thecuvette sampleswere rinsed for10s withdeionized waterand dried withni-
was irradiated with a directional LED light at 465 ± 10nm trogen gas. The images were obtained in air at room temperature on
(10mWcm−2)or660 ± 10nm(50mWcm−2),andtheoutputpower areasof5×5μm2.
density of the LED was controlled by an LED controller. UV–Vis ab-
sorbancespectrawererecordedatregularintervals(2minfor465nm 4.9. Gelelectrophoresisstudy
irradiation,5minfor660nm).Ferrioxalateactinometrywasusedasa
reference to determine the photon flux of our LED light source. The DNA binding properties of complex 3 was also investigated by
quantumyieldsofthefirststepofcomplexes1–3werecalculatedac- agarose gel electrophoresis. Desired concentrations of the complex 3
cordingtothemethoddescribedbefore[57] werepreparedbydilutionofthecompoundwithTris-H3PO4(100mm)
buffer. pBR322 DNA (5μL) was added to each tube. The mixtures of
4.5. DFTcalculation complex3andpBR322plasmidDNAweremixturetogether,andirra-
diatedwithbluelight(465nm,30min)orredlight(660nm,1h).After
AllcalculationswereperformedusingtheGaussian09suitofpro- 24h incubation, the agarose gel (made up to 1% w/v) was prepared
gram[69].Thegroundstate(S0),thelowest-lyingtripletstate(T1),and withTAbuffer(50mmTris-acetate,pH7.4).Themixtureswithloading
the dissociated structures of complexes 1–3 were optimized by using buffer (1mL) were submitted to electrophoresis in agarose gel in TA
theB3LYPdensityfunctional[70]withtheLanL2DZbasissetandef- bufferat100Vfor90min.Agarosegelswerethendyedwithethidium
fective core functional [71]used for the Ruatom while the 6-31G(d) bromide (0.5mg/L) for 20min. Bands were imaged by using a
basisset[72,73]fortheotheratoms.Allstructureswereconfirmedto MolecularImager(Bio-Rad,USA)underUVlight.
be minima by harmonic vibrational frequency calculations. The time-
dependent density functional theory (TD-DFT) [74–76] calculations 4.10. MTTassay
were performed at the same level to predict the singlet electronic
transitionsandtheUV–visiblespectra.The3MLCT,and3MCelectronic Cytotoxicityofcomplexes1–3andbicalutamideagainstLNCaPand
configurationswerecharacterizedbyexaminingthespindensities,and PC-3 cells was determined by means of the MTT (3-(4,5-di-
the 1MLLCT state were identified by analyzing the molecular orbital methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Cells
populations. The electron density difference maps (EDDMs) of the were cultured in RPMI-1640 medium with 10% fetal bovine serum
lowest-lyingsinglettransitionsofthethreecomplexeswereplottedby (FBS).Allmediawerealsosupplementedwith100mg/mLofpenicillin
theGaussView(version5.0). and 100mg/mL of streptomycin. Cells (105 per well) with better vi-
talitywereseededin96-wellplates.Thecompoundsweredissolvedby
4.6. Dockingstudy DMF and diluted with medium to various concentrations (the final
concentrationofDMFwaslessthan0.4%).Afterbeingincubatedinthe
Docking studies were carried out using Autodock Dock 4.2. The darkfor4h,cellswereirradiatedwithbluelight(465nm)for30min
crystalstructureofAR-LBD(ligandbindingdomain)wasobtainedfrom orredlight(660nm)for1h,andthenthecellswereincubatedinthe
theProteinDataBank(PDBID:1z95).Bicalutamidein1z95werere- dark for a further 48h. After that, cells were stained with 3-(4,5-di-
movedprior tothedockingby softwarePyMOL.Thedockingsimula- methyl-2-thiazolyl)-2,5-diphenyl-2Htetrazoliumbromide(MTT)(5mg/
tionwasperformedwiththeLamarckiangeneticalgorithmforasmuch mL) for another 5h, and then the medium was thrown away and re-
as150dockingruns.Eachrunofthedockingoperationwasterminated placedby150mLDMSO.Theinhibitionofcellgrowthinducedbythe
after a maximum of 2,500,000 energy evaluations. During docking tested complexes was detected by measuring the absorbance of each
studies,theproteinstructurewaskeptrigid.Rotationinthecomplexes wellat570/630nmusingenzymelabelinginstrument.TheIC50values
1–3waspermittedaboutallsinglebonds. werecalculatedbySPSSsoftwareafterthreeparallelexperiments.
4.7. ARligandbindingaffinity 4.11. ApoptosisassessmentbyHoechst33342staining
The fluorescence polarization technique was applied to study the LNCaPcellswereseededin24-wellplatesat1×105cells/welland
binding of complexes 1–3 and bicalutamide to the androgen receptor incubated overnight. Cells were incubated with 30μM of the tested
usingthePolarScreen™ARCompetitorAssay,Green(lifetechnologies, compounds,andthenirradiatedwithbluelight(465nm,30min)orred
A15880)accordingtothemanufacturer'sinstructions.Complexes1–3 light(660nm,1h).Thecellswereincubatedinthedarkforafurther
withdesiredconcentrationswereirradiatedwithadirectionalLEDlight 24h.Afterthat,thecellswererinsedtwiceinPBS(phosphate-buffered
at465nm(30min)or660nm(1h),andthenweretitratedagainsta saline)andstainedwithHoechst33342fluorescentdyefor10minin
preformed complex of Fluormone™ AL Green and the AR-LBD (GST). thedarkat37°C.Cellapoptosiswasexaminedunderthefluorescence
The assay mixture was incubated at room temperature for 4h, after microscopewithexcitationwavelengthof330–380nm.
which the fluorescence polarization values were measured in a
SpectraMax® Paradigm® Multi-Mode Detection Platform using an ex- 4.12. Apoptosisanalysisbyflowcytometry
citationwavelengthof485nmandanemissionwavelengthof535nm.
Data analysis for the ligand binding assays was performed using LNCaP cells were grown in a 6-well plate and cultured overnight.
GraphPadPrismsoftware. The tested complexes were added with the final concentration of
30μM.Afterincubationfor24h,cellswereirradiatedwithbluelight
4.8. Atomicforcemicroscopy(AFM) (465nm,30min)orredlight(660nm,1h).Afterthat,thecellswere
incubatedinthedarkforafurther24h,andcollectedbycentrifugation
ThepBR322plasmidDNA(200ng/μL)wasdilutedto5ng/μLwith (5min,25°C,2000rpm).Then,thecellswerewashedtwicewithcold
hepes (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer water, resuspended in binding buffer (10mM hepes, 140mM NaCl,
(40mMhepes,5mMMgCl2,pH7.4).Complex3wasdissolvedinDMF 2.5mMCaCl2,pH7.4).Thecellswerestainedwith5μLofAnnexinV-
7
J.Zhao,etal. Journal of Inorganic Biochemistry 196 (2019) 110684
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