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Mono- and Dinuclear Phosphorescent Rhenium(I) Complexes: Impact of Subcellular Localization on Anticancer Mechanisms.
DOI: 10.1002/chem.201505160 Full Paper
&Cancer
Mono- and Dinuclear Phosphorescent Rhenium(I) Complexes:
Impact of Subcellular Localization on Anticancer Mechanisms
Rui-Rong Ye, Cai-Ping Tan,* Mu-He Chen, Liang Hao, Liang-Nian Ji, and Zong-Wan Mao*[a]
Abstract: Elucidation of relationship among chemical struc- phorescence. We found that increased lipophilicity of the
ture, cellular uptake, localization, and biological activity of bidentate ligands could enhance their cellular uptake, lead-
anticancermetalcomplexesisimportantfortheunderstand- ingtoimprovedanticancerefficacy.Thedinuclearcomplexes
ingoftheirmechanismsofaction.Organometallicrhenium(I) were more potent than the mononuclear counterparts. The
tricarbonyl compounds have emerged as potential multi- molecular anticancer mechanisms of action evoked by Re3
functional anticancer drug candidates that can integrate andRe4wereexploredindetail.Re3withalowerlipophilic-
therapeutic and imaging capabilities in a single molecule. ity localizes to lysosomes and induces caspase-independent
Herein, two mononuclear phosphorescent rhenium(I) com- apoptosis,whereasRe4withhigherlipophilicityspeciallyac-
plexes (Re1 and Re2), along with their corresponding dinu- cumulates in mitochondria and induces caspase-independ-
clear complexes (Re3 and Re4), were designed and synthe- ent paraptosis in cancer cells. Our study demonstrates that
sizedas potentanticanceragents. Thesubcellular accumula- subcellular localization is crucial for the anticancer mecha-
tion of Re1–Re4 was conveniently analyzed by confocal mi- nismsofthesephosphorescentrhenium(I)complexes.
croscopy in situ in live cells by utilizing their intrinsic phos-
Introduction
The intracellular sequestration of anticancer agents is of
therapeutic interest and subcellular organelles-targeted cancer
Rhenium(I) organometallic compounds, especially phosphores- therapy has gained much attention.[4] The mitochondrion is
cent rhenium(I) tricarbonyl complexes, have been widely used considered to be the power house of the cell as it provides
as luminescent probes for cell imaging due to their intriguing adenosinetriphosphate(ATP)tothecellbytheprocessofoxi-
photophysical and photochemical properties, including long- dativephosphorylation.[5]Lysosomesareinvolvedinthedegra-
lived excited states, large Stokes shifts, high quantum yields, dationandturnoverofintracellularcytosolicmoleculesandor-
resistance to photobleaching, and environment-sensitive emis- ganelles.[6] Both mitochondrion and lysosome play essential
sion.[1] Because these rhenium(I) complexes are luminescent, roles in cell death signaling and the malfunctioning of these
theiraccumulationinsubcellularorganellescanbeconvenient- structuresisassociatedwiththeimmortalityofcancercells.[7]
lyanalyzedbyconfocalmicroscopy.Ontheotherhand,agrow- Formetalcomplexes,targeting tospecific subcellularorgan-
ing number of studies have recently revealed the potential of elles can be realized by regulating their charge, lipophilicity,
organometallic rhenium(I) complexes as anticancer agents.[2] molecular size, and so on.[8] Barton and co-workers found that
Although in many cases, organometallic rhenium(I) complexes nuclear-localized RhIII complexes showed higher mismatch
display potent anticancer activity comparable to that of cispla- repair-deficient cell-selective biological activities compared
tin,veryfewexplorationsofanticancermechanismsofcytotox- with the complexes accumulated in mitochondria.[9] Lippard
ic organometallic rhenium(I) compounds can be found in the and co-workers demonstrated that OsVI nitrido compounds
literature.[2b]Moreover,comparedwithmononuclearrhenium(I) bearing different bidentate ligands induced completely differ-
carbonyl complexes, the potential of the biological applica- entcellularresponsesincludinggenomicDNAdamageanden-
tions of dinuclear rhenium(I) complexes has rarely been ad- doplasmic reticulum (ER) stress.[10] Up to now,most of therhe-
dressed.[3] As far as we know, the cytotoxic mechanisms of di- nium(I) tricarbonyl complexes, especially for those with highly
nuclearrhenium(I)complexeshavenotyetbeenreported. lipophilic ligands (e.g., 4,7-diphenyl-1,10-phenanthroline
(DIP)),[11] are reported to specially localize in mitochondria.[12]
However, it is still a challenge to direct rhenium complexes to
[a] R.-R.Ye,Dr.C.-P.Tan,M.-H.Chen,L.Hao,Prof.L.-N.Ji,Prof.Z.-W.Mao
MOEKeyLaboratoryofBioinorganicandSyntheticChemistry otherdifferentorganelles.
SchoolofChemistryandChemicalEngineering In this study, a series of phosphorescent rhenium(I) com-
SunYat-SenUniversity,Guangzhou,510275(P.R.China) plexes Re1–Re4 (Figure1A) have been designed and synthe-
E-mail:cesmzw@mail.sysu.edu.cn
sized. The primary aim is to illustrate the correlations among
tancaip@mail.sysu.edu.cn
molecular structures, subcellular localizations, and anticancer
Supportinginformationforthisarticlecanbefoundunderhttp://
dx.doi.org/10.1002/chem.201505160. mechanisms of phosphorescent rhenium(I) complexes. The cy-
Chem.Eur.J.2016,22,7800–7809 7800 (cid:211)2016Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim
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lular localization of Re1–Re4 was studied by confocal micros-
copy. Furthermore, the differences in mechanisms of action of
Re3 and Re4 were explored utilizing a variety of cell-based
assays.Ourstudydemonstratesthatsimplestructuralmodifica-
tion can influence the subcellular localizations of phosphores-
cent rhenium(I) complexes, which is crucial for their antitumor
mechanisms.
Results and Discussion
Synthesisandcharacterization
Re1 was synthesized by treating [Re(N¢N)(CO)(CHCN)](PF)
3 3 6
(N¢N=1,10-phenanthroline (phen)) with 1,2-bis(4-pyridyl)-
ethane(L)underaninertatmosphere ofnitrogenaccordingto
literature methods.[13] Re2 was synthesized by adopting similar
methods. Re3 and Re4 were obtained by treating [Re(N¢
N)(CO)(CHCN)](PF) (Re3: N¢N=phen; Re4: N¢N=DIP) with
3 3 6
L at a 2:1 molar ratio. All the complexes were purified by re-
crystallization, which gave the desired product in high yields.
Re1–Re4 were characterized by 1HNMR spectroscopy, ESI-MS,
and elemental analysis. The structure of Re4 was also deter-
mined by X-ray crystallography (Figure1B, the Supporting In-
formation,TablesS1andS2).
TheUV/VisabsorptionspectraofRe1–Re4inphosphate-buf-
fered saline (PBS), CHCN, and CHCl at 298K are shown in
3 2 2
Figure2A. The complexes display intense absorption bands at
approximately 250–320nm attributed to intraligand (p!p*)
Figure1.A)ThestructuresofRe1–Re4;B)X-raycrystalstructureofRe4.The
transitions and relatively weak bands at approximately 320–
hydrogenatomsandcounterionsareomittedforclarity.
450nm assigned to metal-to-ligand charge-transfer absorp-
totoxicity of Re1–Re4 was assessed by 3-(4,5-dimethylthiazol- tion.[14] The photophysical properties of Re1–Re4 are summar-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The subcel- ized in TableS3 (the Supporting Information). Re1–Re4 exhibit
Figure2.A)UV/VisspectraofRe1–Re4(2(cid:213)10¢5m)measuredina)degassedPBS;b)degassedCHCN;andc)degassedCHCl at298K;B)Emissionspectraof
3 2 2
Re1–Re4(2(cid:213)10¢5m)measuredina)degassedPBS;b)degassedCHCN;andc) degassedCHCl at298K.
3 2 2
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strong yellow emission in PBS, CHCN, and CHCl under ambi- certain selectivity toward human cancer cells over non-cancer-
3 2 2
entconditionsuponexcitationat405nm(Figure2B). ous cells. Re2, Re3, and Re4 show approximately 5-fold lower
cytotoxicityagainstLO2cellsthanagainstHepG2cells.
Cellularuptake
Cellularlocalization
The lipophilicity (log P) and molecular size of a compound will
affect its cellular uptake, localization, cytotoxicity, and antican- The studies on cellular localization of phosphorescent metal
cer action mechanisms.[9,15] As estimated, the log P values ob- complexes can not only provide clues for the investigations of
tained for rhenium(I) complexes are in the following order: anticancer mechanisms, but also offer the possibility of devel-
Re1 (¢1.09)<Re3 (¢0.67)<Re2 (2.86)<Re4 (3.12). The lipo- oping multifunctional theranostic agents.[12b,16] As shown in
philicity is increased with the lipophilicity of bidentate ligands Figure3, Re1–Re4 can be effectively taken up by HeLa cells
(phen<DIP), and the dinuclear complexes is more lipophilic and exhibit obvious organelle accumulation after 2h incuba-
thanthecorrespondingmononuclearcomplexes. tion. Interestingly, co-localization experiment shows that the
As rhenium is an exogenous element, the cellular uptake phosphorescence of Re1 and Re3 is highly overlapped with
levels of rhenium can be quantitatively determined by ICP-MS. the fluorescence of LysoTracker Deep Red FM (LTDR), whereas
After incubation with Re1 (30mm), Re2 (15mm), Re3 (30mm), the phosphorescence of Re2 and Re4 is colocalized with the
and Re4 (15mm) for 4h, the intracellular rhenium contents of fluorescence of MitoTracker Deep Red FM (MTDR). The Pear-
Re1–Re4 are (167.17(cid:156)16.52), (988.89(cid:156)96.05), (580.00(cid:156)56.80), son’s correlation coefficients obtained for Re1 and Re3 with
and (1057.22(cid:156)100.23)ng per 106cells, respectively. The capa- LTDR are 0.81 and 0.80, respectively. The Pearson’s correlation
bility of these complexes to penetrate the cell membrane fol- coefficients calculated for Re2 and Re4 with MTDR are 0.86
lows the order of Re1<Re3 and Re2<Re4. Based on these and 0.84, respectively. Meanwhile, minimal colocalization was
observations, it can be concluded that besides lipophilicity, observed for Re1 or Re3 with MTDR and little colocalization is
otherfactors,suchasmolecularsizeandcharge,mayalsocon- observedforRe2orRe4withLTDR(Figure3).
tributetothecellularuptakeefficaciesofReIcomplexes. Ithasbeenreportedthattheorganelleselectivityofamole-
cule can be predicted by analyzing its log P value and ionic
number.AmoleculewithalogPvaluebetween¢5and0and
Invitrocytotoxicity a cation number more than 0 shows high probability to local-
ize in lysosomes.[17] Mitochondrial accumulation is usually ob-
The cytotoxicities of Re1–Re4 were evaluated in human cervi-
served for molecules with log P values falling between 0 and
cal cancer (HeLa), human lung adenocarcinoma epithelial
+5.[18] Based on these observations, the lipophilicity of Re1–
(A549), cisplatin-resistant A549 (A549R), human hepatocellular
Re4 seems to play a vital role in their subcellular localizations.
liver carcinoma (HepG2), and human normal liver (LO2) cells.
Re1 and Re3 with lower lipophilicity tend to accumulate in ly-
As shown in Table1, the anticancer activities of Re1–Re4 are
sosomes, whereas Re2 and Re4 with higher lipophilicity tend
correlatedwiththeircellularuptakeefficacies.Generally,thedi-
tolocalizeinmitochondria.
nuclearcomplexesRe3andRe4aremoreactivethantheircor-
responding mononuclear complexes Re1 and Re2. Re1 is basi-
cally inactive against all the cell lines tested, whereas Re3 is
MorphologicalcharacterizationofReI-inducedcelldeath
more active than cisplatin in the cancer cells tested. The cyto-
toxicity of Re2 and Re4 is comparable, and both of them ex- The difference in the cellular localization of metal complexes
hibithigherinvitroanticanceractivitiesthanRe1,Re3,andcis- may affect their anticancer action mechanism.[9,10] As the dinu-
platin. Notably, Re2, Re3, and Re4 show marked activity clear complexes Re3andRe4 weremore activethan themon-
against the cisplatin-resistant A549R cells, with Re4 displaying onuclearcomplexesRe1andRe2,theywerechosenforfurther
approximately 38-fold higher anticancer efficacy than cisplatin investigations of their anticancer mechanisms. Apoptosis is
in killing A549R cells, which indicates that these complexes characterized by several morphological alternations including
can overcome cisplatin resistance. Furthermore, they exhibit cell shrinkage, membrane blebbing, chromatin condensation,
Table1. IC valuesoftestedcompoundstowardsdifferentcelllines.[a]
50
Compound IC [mm]
50
HeLa A549 A549R HepG2 LO2
Re1 44.7(cid:156)4.0 >100 >100 39.8(cid:156)3.7 >100
Re2 2.0(cid:156)0.2 4.6(cid:156)0.3 1.6(cid:156)0.1 3.1(cid:156)0.3 12.4(cid:156)1.2
Re3 9.1(cid:156)0.8 11.7(cid:156)4.1 11.5(cid:156)1.0 12.0(cid:156)1.1 56.2(cid:156)4.7
Re4 1.7(cid:156)0.1 2.0(cid:156)0.2 1.9(cid:156)0.1 2.1(cid:156)0.2 10.2(cid:156)1.0
cisplatin 23.8(cid:156)2.1 13.8(cid:156)1.5 72.6(cid:156)6.9 16.2(cid:156)1.6 38.1(cid:156)3.0
[a]Dataarepresentedasthemeans(cid:156)standarddeviations(SD),andcellviabilitywasassessedafter48hofincubation.
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Figure3.A)ConfocalmicroscopicimagesofHeLacellsco-labeledwithRe1–Re4(20mm,2h)andLTDR(50nm,0.5h).B)ConfocalmicroscopyimagesofHeLa
cellsco-labeledwithRe1–Re4(20mm,2h)andMTDR(150nm,0.5h).Scalebar:20mm.
and formation of apoptotic body.[19] Previously, we found that
a ReI complex can induce a type of non-apoptotic cell death
known as paraptosis.[20] Unlike apoptosis, the features of para-
ptosis include extensive cytosolic vacuolization and intact
nuclei.[21] In some cases, swelling of mitochondria is also pre-
sentedduringparaptosis.[20,22]
First, the ultrastructural changes in HeLa cells treated with
Re3andRe4wereanalyzedbytransmissionelectronmicrosco-
py (TEM). Compared with vehicle-treated control cells (Fig-
ure4a),Re3-treated cellsshow morphologicalchanges typical-
ly associated with apoptosis, such as condensation of chroma-
tin and fragmentation of the nucleus (Figure4b). In contrast,
cells incubated with Re4 show large cytoplasmic vacuoles,
whereas the plasma membrane and nuclei are intact (Fig-
ure4c). These morphological changes are the typical features
of paraptosis. Massive swollen mitochondria with disrupted
cristae are observed, which indicates that Re4 causes mito-
chondrialdestruction(Figure4d).
ThemorphologicalchangesofHeLacellscausedbyRe3and
Re4 treatment were also examined with 2’-(4-ethoxyphenyl)-5-
(4-methyl-1-piperazinyl)-2,5’-bi-1H-benzimidazole trihydrochlor-
ide (Hoechst 33342) staining. As shown in Figure5, vehicle- Figure4.RepresentativeTEMimagesshowingthemorphologicalfeaturesof
HeLacellstreatedwithvehicle(A),40mmRe3(B)and6mmRe4(CandDis
treatedcontrolcellsshownormaloverallmorphologyandaho-
theenlargedviewofCinsidethesquare)for24h.Whitearrows:thenuclear
mogeneousnuclearstainingpattern.AftertreatmentwithRe3,
membraneisaffected,whereasthenucleusisintact;blackarrows:swollen
cells displaying typical apoptotic changes, for example, cell mitochondria;*:thevacuolesaremostlyempty.Scalebars:5mm.
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Figure5.Hoechst33342stainedHeLacellsaftertreatmentwithRe3and
Re4attheindicatedconcentrationsfor24h.Scalebar:20mm.
shrinkage, membrane bubbling, fragmented nuclei and apo-
ptotic bodies, increase gradually in a concentration-dependent
manner.However,noobviouschangesinnucleiofcellstreated
with Re4 are observed, which is also suggestive of paraptotic
celldeath.[21]
Figure6.A)Theactivationofcaspase-3/7inHeLacellstreatedwithRe3,
Re4,orcisplatinattheindicatedconcentrationsfor6h;B)Theimpactof
z-VAD-FMKonthecytotoxicityofRe3andRe4.HeLacellsweretreatedwith
Re3andRe4for48hattheindicatedconcentrationsintheabsenceor
presenceofz-VAD-FMK.CellviabilitywasmeasuredbyMTTassay.
Activationofcaspase-3/7
Caspases frequently viewed as central components of the ma-
Impactonmitochondrialmembranepotential(MMP)
chinery responsible for apoptosis.[23] However, apoptosis can
also occur through caspase-independent pathways.[24] Parapto- Mitochondriaareuniqueorganellesinvolvedinenergyproduc-
sis-like cell death typically does not involve activation of cas- tion and cell death regulation. The execution of apoptosis can
pases,soparaptosisdoesnotresponsetocaspaseinhibitors.[25] occur through mitochondria-dependent or mitochondria-inde-
We next investigated whether the activation of caspase-3/7 pendentpathways.[27]Asparaptoticcelldeathisusuallycharac-
was involved in the cell death induced by Re3 and Re4 by terized by physical enlargement of the mitochondria or endo-
a Caspase-Glo assay.[26] As shown in Figure6A, the activities of plasmic reticulum (ER), mitochondrial damage is demonstrated
caspase-3/7 do not increase upon treatment of Re3 and Re4, to be closely associated with paraptosis.[28] The impact of Re3
whereas the positive control cisplatin (25mm, 6h) causes an andRe4 onMMPwasassessedby5,5’,6,6’-tetrachloro-1,1’-3,3’-
approximately 6.1-fold increase in caspase-3/7 activity. Further- tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) staining.
more,thegrowthinhibitoryeffectofRe3andRe4isnotaffect- As shown in Figure7, compared with the vehicle-treated cells,
ed by the pan caspase inhibitor z-VAD-fmk (Figure6B). These the impact of Re3 on MMP is not obvious. In contrast, treat-
resultscollectivelyrevealthatRe3causescaspase-independent mentofRe4(10mm)for6hdecreasestheJC-1red/greenfluo-
apoptosis, whereas Re4 causes caspase-independent parapto- rescence ratio from 10.0(cid:156)1.0 to 3.1(cid:156)0.3, which indicates mi-
sis. tochondrialmembranedepolarization.Theresultindicatesthat
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cellular ROS levels, whereas cells treated with Re4 (10mm;
meanfluorescenceintensity(MFI):366(cid:156)35)displaya4-foldin-
creaseinDCFfluorescenceascomparedwiththevehicle-treat-
ed cells (MFI: 92(cid:156)8). Additionally, pretreatment of cells with
N-acetylcysteine (NAC, a ROS scavenger) remarkably reduces
thecytotoxicityofRe4(theSupportingInformation,FigureS5).
TheresultsdemonstratethatgenerationofROSisnotinvolved
in Re3-induced apoptosis, whereas ROS play a vital role in
Re4-inducedparaptosis-likecelldeath.
Geneexpressionprofilingandconnectivity-mapanalyses
Both Re3 and Re4 are highly cytotoxic towards the cancer cell
lines. To study their underlying cytotoxic mechanisms, altera-
tion of the gene expression after treatment of HeLa cells with
Re3 and Re4 was examined by microarray experiments using
the Affymetrix GeneChip HTA2.0 platform. HeLa cells were
treated with Re3 (20mm) or Re4 (4mm) for 24h in triplicate
runs. For Re3, 340 and 334 probe sets were up- and down-
regulated by more than 2-fold, respectively. In Re4-treated
HeLa cells, 359 and 244 probe sets were identified as up- and
Figure7.EffectsofRe3andRe4onMMP.Cellsweretreatedwithvehicle,
Re3,andRe4attheindicatedconcentrationsfor6h. down-regulated by more than 2-fold, respectively. Complete
lists of regulated genes are summarized in the Supporting In-
formation(TablesS4andS5).
Re3 induces apoptosis through mitochondria-independent The activation of cell death processes by Re3 or Re4 has
pathways, whereas mitochondrial dysfunction is involved in also been revealed by analysis of the regulated genes using
Re4-inducedparaptosis-likecelldeath. another web-based bioinformatics tool DAVID (database for
annotation, visualization, and integrated discovery),[29] and the
findings are summarized in the TableS6 (the Supporting Infor-
mation).ThefindingssuggestedthatbothRe3andRe4affect-
Elevationofintracellularreactiveoxygenspecies(ROS)
ed the “p53 signaling pathway”. Activation the p53 pathway
levels
plays a significant roles in both apoptotic and paraptotic cell
ROS are mainly generated inside mitochondria, and mitochon- death.[30] It is worth mentioning that Re3 affects “endocytosis”,
dria are the main source of ROS. We next tested the possible and lysosomes are the last compartment of the endocytic
involvement of ROS in ReI-induced apoptosis and paraptosis. pathway.Re4affects“tricarboxylicacid(TCA)cycle”thatoccurs
Based on our previous reports, ReI complexes generate ROS in the matrix of the mitochondrion, which indicates that Re4
during paraptosis.[20] The effects of Re3 and Re4 on intracellu- displays its antitumor activity by affecting mitochondrial func-
larROSlevelswereexaminedby2’,7’-dichlorofluorescein(DCF) tion.
fluorescence assay measured by flow cytometry (Figure8). The NextweusedCmap(theConnectivityMapdatabase),which
results show that Re3 (60mm) has no obvious effects on intra- contains gene expression profiles for 1309 compounds, to
characterize the cellular response of Re3 and Re4 in HeLa
cells.[31] A high positive connectivity score indicates positive
correlation with the drugs and vice versa. Results of the Cmap
analysis in the cases of Re3 and Re4 are summarized in
Table2.BothRe3andRe4showhighpositivescorewiththap-
sigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2+
ATPase (SERCA) that is involved in cell death.[32] The gene ex-
pression profile of Re4-treated cells is similar to that observed
withdisulfiram[33]andvalinomycin,[34]bothofwhicharereport-
edtocausemitochondrialdysfunction.
Conclusions
A series of phosphorescent rhenium(I) carbonyl complexes in-
Figure8.EffectsofRe3andRe4onROSgeneration.HeLacellsincubated
cluding two mononuclear complexes (Re1 and Re2) and their
withRe3andRe4at378Cfor6h,afterwhichtheywerelabeledwith
dinuclear counterparts (Re3 and Re4) were designed and syn-
HDCFDAandanalyzedbyflowcytometry(reflectedbyMFIofDCF;excita-
2
tionat488nmandemissionat525nm). thesized.Interestingly,wefoundthatthesmallvariationofmo-
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nologies), MTT(Sigma Aldrich), Hoechst 33342 (Sigma Aldrich),JC-
Table2. Connectivity-mapanalysisshowingthehighest rankedcorrelat-
1(SigmaAldrich),HDCFDA(2’,7’-dichlorodihydrofluoresceindiace-
edbioactivecompounds. 2
tate, Sigma Aldrich), z-VAD-fmk (Sigma Aldrich), NAC (Sigma Al-
Re3 Re4 drich). Caspase-3/7 activity assay kit was purchased from Promega
(USA). All the tested compounds were dissolved in DMSO just
Rank Compound Score Compound Score
before the experiments, and the concentration of DMSO was 1%
Cellline Cellline
(v/v). The solutions of Re1–Re4 in PBS were proved to be stable
Concentration Concentration
foratleast48hatroomtemperatureasmonitoredbyUV/Visspec-
1 Puromycin 0.775 Disulfiram 0.871 troscopyandElectrospraymassspectra(ESI-MS).
MCF7[a] MCF7[a]
7mm 13mm
2 Lycorine 0.773 Thapsigargin 0.852 Instrumentation
HL60[b] MCF7[a]
12mm 100nm NMR spectra were recorded on a Bruker Avance400 spectrometer
3 Thapsigargin 0.769 Puromycin 0.817 (Germany). Shifts were referenced relative to the internal solvent
MCF7[a] MCF7[a] signals.ESI-MSwererecordedonaThermoFinniganLCQDECAXP
100nm 7mm spectrometer(USA).Thequotedm/zvaluesrepresented themajor
4 Lycorine 0.768 Spiperone 0.815
peaks in the isotopic distribution. Microanalysis (C, H, and N) was
PC3[c] PC3[c]
carried out using an Elemental Vario EL CHNS analyzer (Germany).
12mm 10mm
UV/VisspectrawererecordedonaVarianCary300spectrophotom-
5 MG-132 0.741 Astemizole 0.779
MCF7[a] MCF7[a] eter(USA).EmissionmeasurementswereconductedonanFLS920
21mm 9mm combined fluorescence lifetime and steady state spectrometer
6 Lycorine 0.741 Mefloquine 0.759 (Japan). Quantum yields of luminescence at room temperature
MCF7[a] MCF7[a] were calculated according to literature procedures using
12mm 10mm [Ru(bpy)](PF) asthereference.[35]
3 62
7 Spiperone 0.738 Methylbenzethoniumchloride 0.758
PC3[c] PC3[c]
10mm 9mm
Synthesisofthecorrespondingrhenium(I)complexes
8 Anisomycin 0.732 Valinomycin 0.755
MCF7[a] PC3[c]
Synthetic procedure of [Re(N¢N)(CO)(CHCN)](PF) ((N¢N)=
15mm 100nm 3 3 6
phen, DIP): The [Re(phen)(CO)Cl][36] and [Re(DIP)(CO)Cl][37] com-
9 Celastrol 0.715 Pararosaniline 0.749 3 3
plexes were prepared according to a literature procedure. They
MCF7[a] MCF7[a]
3mm 10mm were converted to [Re(N¢N)(CO) 3 (CF 3 SO 3 )] following a standard
10 Anisomycin 0.705 Celastrol 0.748 method with slight modifications.[38] AgCFSO (0.80mmol) was
3 3
HL60[b] MCF7[a] added to a suspension of [Re(N¢N)(CO)Cl] (0.80mmol) in CHCN
3 3
15mm 3mm (200mL).Themixturewasheatedatrefluxundernitrogenfor24h
in the dark. After removed off-white AgCl precipitate, a 6-fold
[a]MCF-7:humanbreastadenocarcinomacellline.[b]HL60:humanpro-
myelocyticleukemiacellline.[c]PC3:humanprostatecancercellline. excess of NH 4 PF 6 was added, the resulting yellow precipitate was
collected by centrifugation and washed with water then with di-
ethyl ether, which can be used directly for the next step without
furtherpurification.
lecularstructurehadaprofoundimpactonsubcellularlocaliza- SyntheticprocedureofmononuclearcomplexesRe1[13]andRe2:
tions and anticancer mechanisms of Re1–Re4. Complexes Re1 A mixture of [Re(N¢N)(CO)(CHCN)](PF) (0.50mmol) and ligand L
3 3 6
andRe3withlowerlipophilicitylocalizetolysosomes,whereas (0.5mmol) in THF (50mL) was heated at reflux under an inert at-
Re2 and Re4 with higher lipophilicity localize to mitochondria. mosphereofnitrogenfor4h.Themixturewasthenevaporatedto
dryness, and then subsequently recrystallized from a mixture of
Further investigation ofanticancermechanisms ofactionshow
CHCNanddiethylether.
that the different localizations between rhenium(I) complexes 3
SyntheticprocedureofdinuclearcomplexesRe3andRe4:Amix-
inducedifferentanticancermechanisms.Re3localizesinthely-
ture of [Re(N¢N)(CO)(CHCN)](PF) (0.50mmol) and ligand L
sosomes and induces caspase-independent apoptosis. In con- 3 3 6
(0.25mmol)inTHF(50mL)washeatedatrefluxunderaninertat-
trast, Re4 specially localizes to mitochondria and induces cas-
mosphere of nitrogen for 4h. The yellow solid was separated by
pase-independent paraptosis. These results give insight into filtration, washed with THFand then with diethyl ether. Thecrude
the rational design of anticancer metal complexes targeting product was recrystallized from a mixture of CHCN and diethyl
3
specificsubcellularorganelles. ether.
[Re(phen)(CO)(L)](PF) (Re1):[13] Complex Re1 was synthesized ac-
3 6
cording to the synthetic procedure described above, giving the
product as a light-yellow powder. Yield: 0.292g (75%). 1HNMR
Experimental Section
(400MHz, [D]DMSO): d=9.73 (dd, J=5.1, 1.2Hz, 2H; CH-11 and
6
CH-18),9.02(dd,J=8.3,1.2Hz,2H;CH-8andCH-10),8.26(m,8H;
Materials
CH-1, CH-3, CH-12, CH-13, CH-14, CH-15, CH-16 and CH-17), 7.14
Re(CO)Cl (Sigma Aldrich), phen (Sigma Aldrich), DIP (Sigma Al- (d,J=6.6Hz,2H;CH-7andCH-9),7.02(d,J=6.0Hz,2H;CH-2and
5
drich), ligand L (Sigma Aldrich), Silver trifluoromethanesulfonate CH-4), 2.76ppm (td, J=7.2, 2.6Hz, 4H; CH-5, 6); MS (ESI): m/z:
2
(Sigma Aldrich), cisplatin (Sigma Aldrich), NHPF (Alfa Aesar), 635.1 [M¢PF]+; elemental analysis calcd (%) for C H FNOPRe:
4 6 6 27 20 6 4 3
DMSO (Sigma Aldrich), MTDR (Life Technologies), LTDR (Life Tech- C41.59,H2.59,N7.19;found:C41.55,H2.62,N7.11.
Chem.Eur.J.2016,22,7800–7809 www.chemeurj.org 7806 (cid:211)2016Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim
Full Paper
[Re(DIP)(CO)(L)](PF)(Re2):ComplexRe2wassynthesizedaccord- Cytotoxicityassay
3 6
ing to the synthetic procedure described above, giving the prod-
Growthinhibitioneffectofthetestedcompoundswasdetermined
uct as a yellow powder. Yield: 0.377g (81%). 1HNMR (400MHz,
by MTTassay as previously described.[40] For the cytotoxicity assay
[D]DMSO): d=9.81 (d, J=5.4Hz, 2H; CH-11 and CH-26), 8.43 (d,
6 in the presence of the inhibitors, HeLa cells were preincubated
J=6.5Hz, 2H; CH-8 and CH-10), 8.35 (d, J=5.9Hz, 2H; CH-1 and
with 50mm z-VAD-FMK or 10mm NAC for 1h before the com-
CH-3),8.20(d,J=5.4Hz,2H;CH-12andCH-25),8.16(s,2H;CH-18
plexes were added. Percentage of cell viability was determined by
andCH-19),7.69(m,10H;CH-13,14,15,16,17,20,21,22,23,24),
MTT.
7.26(d,J=6.4Hz,2H;CH-7andCH-9),7.12(d,J=5.8Hz,2H;CH-
2 and CH-4), 2.85ppm (dq, J=8.7, 6.1Hz, 4H; CH-5, 6). MS (ESI):
2
m/z: 787.2 [M¢PF]+; elemental analysis calcd (%) for Colocalizationassay
6
C H FNOPRe·2HO: C 48.40, H 3.33, N 5.79, found: C 48.32, H
39 28 6 4 3 2 HeLacellswereseededin35mmdishesfor24handthenincubat-
2.95,N5.43.
ed with Re1–Re4 (20mm) at 378C for 1.5h. The cells were further
[(phen)(CO)Re(L)Re(CO)(phen)](PF) (Re3): Complex Re3 was
3 3 62 co-incubated with MTDR (150nm) or LTDR (50nm) at 378C for
synthesizedaccordingtothesyntheticproceduredescribedabove,
30min. Cells were washed three times with PBS and visualized by
giving the product as a light-yellow powder. Yield: 0.275g (80%).
a confocal microscope (LSM 710, Carl Zeiss, Gçttingen, Germany)
1HNMR (400MHz, [D]DMSO): d=9.74 (dd, J=5.1, 1.1Hz, 4H; CH-
6 immediately. Re1–Re4 were excited at 405nm, MTDR and LTDR
6(cid:213)2 and CH-13(cid:213)2), 9.02 (dd, J=8.3, 1.1Hz, 4H; CH-3(cid:213)2 and CH-
were excited at 633nm. Emission was collected at (560(cid:156)20)nm
5(cid:213)2), 8.31–8.27 (m, 8H; CH-8(cid:213)2, CH-9(cid:213)2, CH-10(cid:213)2 and CH-11(cid:213)
forRe1–Re4,(660(cid:156)20)nmforMTDRandLTDR.
2),8.24(dd,J=8.3,5.1Hz,4H;CH-7(cid:213)2andCH-12(cid:213)2),7.11(d,J=
6.6Hz, 4H; CH-2(cid:213)2 and CH-4(cid:213)2), 2.62ppm (s, 4H; CH-1(cid:213)2); MS
2 Transmissionelectronmicroscopy
(ESI): m/z: 543.2 [M¢2PF]2+; elemental analysis calcd (%) for
6
C 42 H 28 F 12 N 6 O 6 P 2 Re 2 ·CH 3 CN: C 37.32, H 2.21, N 6.92; found: C 37.22, HeLa cells were treated with Re3 (40mm) and Re4 (6mm) at 378C
H2.25,N7.03. for 24h. Cells were washed twice and fixed overnight at 48C in
[(DIP)(CO)Re(L)Re(CO)(DIP)](PF) (Re4): Complex Re4 was syn- phosphate buffer (pH7.4) containing 2.5% glutaraldehyde. The
3 3 62
thesized according to the synthetic procedure described above, cellswerethentreatedwithosmiumtetroxide,stainedwithuranyl
giving the product as a yellow powder. Yield: 0.315g (75%). acetate and lead citrate, and visualized under a transmission elec-
1HNMR (400MHz, [D]DMSO): d=9.79 (d, J=5.4Hz, 4H; CH-6(cid:213)2 tron microscope (JEM100CX, JEOL, Tokyo, Japan). Images were
6
and CH-21(cid:213)2), 8.43 (d, J=6.5Hz, 4H; CH-3(cid:213)2 and CH-5(cid:213)2), 8.18 photographedusingtheEversmartJazzprogram(Scitex).
(d, J=5.4Hz, 4H; CH-7(cid:213)2 and CH-20(cid:213)2), 8.13 (s, 4H; CH-13(cid:213)2
and CH-14(cid:213)2), 7.67 (m, 20H; CH-8(cid:213)2, CH-9(cid:213)2, CH-10(cid:213)2, CH-11(cid:213)
Hoechststaining
2, CH-12(cid:213)2, CH-15(cid:213)2, CH-16(cid:213)2, CH-17(cid:213)2, CH-18(cid:213)2 and CH-19(cid:213)
2), 7.24 (d, J=6.5Hz, 4H; CH-2(cid:213)2 and CH-4(cid:213)2), 2.73ppm (s, 4H; The morphological changes of HeLa cells treated with Re3 and
CH-1(cid:213)2); MS (ESI): m/z: 695.4 [M¢2PF]2+; elemental analysis Re4 at the indicated concentrations were studied by Hoechst
2 6
calcd (%) for C H F NOPRe·CHCN: C 47.47, H 2.75, N 5.70; 33342stainingaccordingtothemethodpreviouslyreported.[40]
66 44 12 6 6 2 2 3
found:C47.37,H2.90,N5.46.
Caspase-3/7activityassay
Crystallographicstructuredetermination
Caspase-3/7 activity was measured using the Caspase-Glo(cid:210) Assay
Crystals of Re4 qualified for X-ray analysis were obtained by slow kit (Promega, Madison, WI, USA) according to the manufacturer’s
diffusionofdiethylethertotheCHCNsolution.Thedatawerecol- instructions. Briefly, cells were cultured in 48-well plates and treat-
3
lected at 293K on a Rigaku Pilatus diffractometer equipped with ed with different concentration of Re3 and Re4 for 6h, and then
Mo radiation (l=0.71073(cid:230)). The crystal structure of Re4 was 50mLcelllysate wasadded to eachwell, followedbytheaddition
Ka
solved by direct methods with program SHELXS and refined using of 50mL Caspase-Glo(cid:210) 3/7 reagent. The mixture was incubated at
the full-matrix least-squares program SHELXL.[39] Crystallographic roomtemperaturefor30minandthentheluminescencewasmea-
data and details of data collection and structure refinements were suredusingaTECANInfiniteM200station.
listed in TableS1 (the Supporting Information). Selected bond
lengths and angles were listed in TableS2. The structural plots AnalysisofMMP
weredrawnusingthexppackageinSHELXTLata30%thermalel-
lipsoidsprobabilitylevel. TheimpactofRe3andRe4onMMPwasdeterminedaspreviously
described.[40] Briefly, HeLa cells were treated with Re3 and Re4 at
Celllinesandcultureconditions the indicated concentrations for 6h. The cells were then collected
and stained with 5mgmL¢1 JC-1, then analyzed immediately in
HeLa, A549, A549R, HepG2 and LO2 cells were obtained from Ex- a flow cytometer (FACSCaliburTM, Becton Dickinson, Franklin Lakes,
perimental Animal Center of Sun Yat-Sen University (Guangzhou, NJ, USA). Red and green mean fluorescence intensities were ana-
China). The Cells were maintained in DMEM (Dulbecco’s modified lyzed using FlowJo7.6 software (Tree Star, OR, USA). 10000 events
Eagle’s medium, Gibco BRL) or RPMI1640 (Roswell Park Memorial wereacquiredforeachsample.
Institute1640, Gibco BRL) medium containing 10% FBS (fetal
bovine serum, Gibco BRL), 100mgmL¢1 streptomycin, and
MeasurementofintracellularROS
100UmL¢1 penicillin (Gibco BRL). The cells were cultured in a hu-
midified incubator at 378C under 5% CO. A549R cells were cul- TheimpactofRe3andRe4onROSlevelswasdeterminedasprevi-
2
tured in a medium containing increasing concentrations of cispla- ously described.[40] Briefly, cells were treated with Re3 and Re4 at
tin to maintain the resistance. In each experiment, cells treated the indicated concentrations for 6h and then incubated with
with vehicle only DMSO (1%, v/v) were used as the reference 10mm HDCFDA in serum-free DMEM for 15min at 378C in the
2
group. dark.Thefluorescenceintensityofthecellswasmeasuredimmedi-
Chem.Eur.J.2016,22,7800–7809 www.chemeurj.org 7807 (cid:211)2016Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim
Full Paper
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