A Phosphorescent Rhenium(I) Tricarbonyl Polypyridine Complex Appended with a Fructose Pendant That Exhibits Photocytotoxicity and Enhanced Uptake by Breast Cancer Cells

Article pubs.acs.org/Organometallics A Phosphorescent Rhenium(I) Tricarbonyl Polypyridine Complex Appended with a Fructose Pendant That Exhibits Photocytotoxicity and Enhanced Uptake by Breast Cancer Cells * Kenneth Yin Zhang, Karson Ka-Shun Tso, Man-Wai Louie, Hua-Wei Liu, and Kenneth Kam-Wing Lo DepartmentofBiologyandChemistry,CityUniversityofHongKong,TatCheeAvenue,Kowloon,HongKong,People’sRepublicof China * S Supporting Information ABSTRACT: We demonstrated that the cytotoxicity of a phosphorescent rhenium(I) polypyridine complex with a fructose pendantwasenhanceduponirradiationandthecellularuptakeofthe complex was mediated by fructose transporters and inhibited by unmodified fructose but was independent of glucose-specific transporters. This complex has been used to image breast cancer cells, where fructose transporters are overexpressed. ■ INTRODUCTION cells is about double that of D-fructose and NBDF due to the preference of GLUT5 on the cyclic furanose mimic over the Glucose transporters (GLUTs) are a group of transmembrane furano/pyrano mixture of fructose conformers.9,10 In the past proteins that are involved in the cellular uptake of several years, the cellular uptake of phosphorescent transition- carbohydrates such as glucose and the maintenance of a metalcomplexeshasattractedmuchinterest.11Phosphorescent constantsupplyofcarbohydratesforcellularmetabolism.1They rhenium(I) tricarbonyl polypyridine complexes are among the differ from each other in substrate affinities, transport kinetics, mostextensivelystudiedsystems;forexample,Cooganandco- and tissue-specific expression. Since cancer cells have been workers have shown that the intracellular localization of found to show a high expression level of GLUTs,2 glucose rhenium(I) complexes depends highly on their lipophilicity conjugates with radiation,3 fluorescence,4 and phosphores- andformalcharges.12Also,Fordandco-workershavedesigned cence5 properties have been designed to monitor cellular a rhenium(I) complex as carbon monoxide releasing reagent glucoseuptakeandimagecancercells.Itisnoteworthythatthe and demonstrated its interesting photoactive carbon monoxide substrates of GLUTs are not limited to glucose; for example, releasing behavior in live cells.13 Since the coordination GLUT5 selectively facilitates uptake of fructose.6 This trans- chemistryofthegroupVIIcongenersrheniumandtechnetium porter is overexpressed in breast cancer tissues such as MCF-7 is similar, the same set of polypyridine ligands, especially andMDA-MB-231cells,butitsexpressioninothercancercells tridentate ligands, can be coordinated to the [Re(CO) ]+ and andnormalbreasttissues isverylimited.7Thus,fructose-based [99mTc(CO) ]+ cores to yield phosphorescent prob 3 es and 3 probes may serve as an alternative targeting strategy for radiopharmaceuticals, respectively. With our interest in the diagnosis of breast cancers. Fructose conjugates 1-Cy5.5-DF development of phosphorescent rhenium(I) complexes as and 1-NBDF functionalized with the fluorescent dyes Cy5.5 biological probes,14 we anticipate that modification of these and 7-nitro-1,2,3-benzadiazole (NBD), respectively, have been complexeswithafructosemoietywillgeneratecellularfructose usedtoimagebreastcancercells.8However,1-Cy5.5-DFshows uptakeindicatorsthatmaybeutilizedasselectivebreastcancer efficient uptake by both breast and liver cancer cells, implying imaging reagents.15 ■ thattheinternalizationisnotmediatedbyfructosetransporters. Although1-NBDFisselectivelytakenupbybreastcancercells, RESULTS AND DISCUSSION there is a lack of quantitative evidence on the inhibited uptake by unmodified fructose, and the delivery of the probe through Herein we report the new phosphorescent rhenium(I) fructose transporters remains inconclusive.8 Recently, a polypyridine fructose complex [Re(Ph 2 -phen)(CO) 3 (py- fluorescent 1-amino-2,5-anhydro-D-mannitol-based probe (NBDM) has been applied to investigate GLUT-mediated Received: June 26, 2013 cellularuptake.9TheuptakeefficiencyofthisprobebyMCF-7 ©XXXXAmericanChemicalSociety A dx.doi.org/10.1021/om400612f|OrganometallicsXXXX,XXX,XXX−XXX Organometallics Article Chart 1. Structures of Complexes 1 and 2 fructose)](CF SO ) (1; Ph -phen = 4,7-diphenyl-1,10-phenan- 3 3 2 throline, py-fructose = 3-(N-(1-deoxy-D-fructos-1-yl)- aminocarbonyl)pyridine) and its fructose-free counterpart Figure 1. Confocal microscopy images of MCF-7 cells costained by [Re(Ph -phen)(CO) (py-3-Et)](CF SO ) (2; py-3-Et = 3-(N- 2 3 3 3 (left)complexes1(top)and2(bottom),(middle)MitoTrackerDeep ethylaminocarbonyl)pyridine)14a (Chart 1). The synthetic Red FM, and(right) overlaid. route of the py-fructose ligand is shown in Scheme S1 (Supporting Information). Complex 1 was obtained by refluxing a mixture of [Re(Ph -phen)(CO) (CH CN)]- resistance of MCF-7 cells.17 Upon irradiation of complex- 2 3 3 (CF SO ) and py-fructose in MeOH/THF under an inert stained MCF-7 cells at λ >365 nm for 30 min, the IC values 3 3 50 atmosphere of nitrogen (see the Supporting Information for decreased considerably to 2.0 and 0.3 μM, respectively (Table characterization and spectroscopic data). Upon photoexcita- 1). Since the photoinduced cytotoxicity of transition-metal tion,thecomplexdisplayedintenseandlong-livedtripletmetal- complexes may be associated with their singlet oxygen to-ligand charge-transfer (3MLCT) emission at 505−553 nm sensitizing properties,18 we have examined the possibility of (Table S2 and Figure S3, Supporting Information). Mod- singlet oxygen generation using 1,5-dihydroxynaphthalene ification of the rhenium(I) polypyridine core with a fructose (DHN) as an indicator. Excitation of a mixture of each unit did not cause noticeable effects to the phosphorescence complex and DHN resulted in hypochromicity and hyper- properties, as evidenced by the similar photophysical data of chromicity of the absorption features at ca. 301 and 427 nm, complexes 1 and 2 (Table S2, Supporting Information). respectively (Figure S4, Supporting Information), indicative of The hydrophilic fructose pendant of complex 1 rendered it theoxidationofDHNtoJuglonebysingletoxygen.TheDHN less lipophilic than complex 2 (Table 1), which subsequently photooxidation yields were determined to be ca. 70% for both led to significant differences in the cellular uptake and complexes (Table 1). While 1O appears to play a role in the 2 cytotoxicity of the complexes. ICP-MS measurements revealed photoinduced cell death, it is noteworthy that the photo- that the fructose complex 1 entered MCF-7 cells 4.4-fold less induced cytotoxicity of transition-metal complexes has been efficiently than the fructose-free complex 2 (Table 1). The related to other causes such as DNA modification19 and NO intracellular rhenium concentrations of both complexes were and CO release.19,20 muchhigherthanthatinthemedium(50μM),suggestingthat After the uptake and cytotoxicity of the complexes were thecomplexeswereconcentratedwithinthecells.Treatmentof established, we investigated the possible selectivity in the the cells with complexes 1 and 2 at 4 °C resulted in reduction cellular uptake of complex 1. Six cell lines were used in this of uptake by 65 and 72%, respectively, indicating that the study, which included two human breast adenocarcinoma cell translocation of the complexes across the membrane is an lines (MCF-7 and MDA-MB-231), two nonbreast cancer cell energy-requiring process. Costaining experiments involving lines (human lung epithelial carcinoma A549 and human MitoTrackerDeepRedFMrevealedthatbothcomplexeswere hepatocarcinoma HepG2), and two nontransformed cell lines localizedinmitochondria,withcolocalizationcoefficientsofca. (mouse embryonic fibroblast NIH/3T3 andhuman embryonic 87 and 80%, respectively (Figure 1). The mitochondrial kidney-293 HEK293T). These cell lines were selected because targeting properties of both complexes have been attributed the expression levels of fructose transporters in these cells are to the lipophilic nature of the rhenium(I) tricarbonyl very different.7 The MTT assay results confirmed that polypyridinecore andtheir positive formalcharge. The former incubation with both complexes 1 and 2 for 1 h did not facilitates the access of the complexes to the mitochondrial cause detectable cell death (cellviability >98%) for allofthese membrane, while the latter helps their accumulation in this cell lines under our experimental conditions. The intracellular organelle.16 The cytotoxic activity of the complexes toward amountsofrheniumtakenupbythecellsuponincubationwith MCF-7inthedarkwasdeterminedbytheMTTassay,andthe the complexes (50 μM, 37 °C, 1 h) were determined. The IC values of complexes 1 and 2 were ca. 9.6 and 3.9 μM, intracellular amounts of the fructose complex 1 in all six cell 50 respectively (Table 1). Under the same experimental lines were lower than those of the fructose-free complex 2 conditions, cisplatin exhibited a much larger IC value (73.0 (Figure 2). This is reasonable because the more lipophilic 50 ± 9.2 μM), which is reasonable in view of the high cisplatin complex 2 underwent internalization through a more efficient, Table 1. Lipophilicity, Cellular Uptake, and Cytotoxicity (Dark and Light), and Photooxidation Yields of Complexes 1 and 2 complex logP [Re]/mMa IC /μM(dark)b IC /μM(light)b DHNphotooxidationyield/%c o/w 50 50 1 2.79 0.42±0.01 9.6±0.6 2.0±0.04 69.7 2 3.63 1.83±0.08 3.9±0.3 0.3±0.01 67.1 aConcentration of rhenium associated with an average MCF-7 cell (mean volume of 3.1 pL) upon incubation with the complexes (50 μM) in a sugar-freemediumat37°Cfor1h,asdeterminedbyICP-MS.bMCF-7cells,incubationinhighglucoseDMEMfor48h.cIrradiationtime4h. B dx.doi.org/10.1021/om400612f|OrganometallicsXXXX,XXX,XXX−XXX Organometallics Article phosphorescent iridium(III) fructose complexes15 exhibit similar uptake inhibition, as revealed by confocal microscopy, this is the first report on the use of a nonradioactive phosphorescent compound to quantitatively monitor changes ofuptakecausedbynativefructose.Importantly,wealsofound that this fructose-dependent cellular uptake of complex 1 was an energy-requiring process, since no significant inhibitory effectofexogenousfructoseonthecellularuptakewasobserved at 4 °C (data not shown). Althoughtheaboveexperimentsindicatedthattheuptakeof complex1ismediatedbyfructosetransporters,itisinteresting to know whether there are any glucose-specific GLUTs that Figure 2. Cellular uptake of rhenium by an average cell upon assist in the uptake. Treatment of cells with glucose (50 mM) incubationwithcomplexes1(left)and2(right)(50μM,37°C,1h). in a sugar-free cell culture medium or the glucose-uptake inhibitorsfasentin(80μM)andcytochalasinB(10μM)21for1 hhadnegligibleeffectsontheuptakeofbothcomplexes1and nonspecific diffusion uptake pathway. Interestingly, we found 2 (uptake amounts within ± 5%). Thus, the internalization of thattheuptakeofthefructosecomplex1byMCF-7andMDA- the complexes did not occur via a glucose-specific GLUT- MB-231 was much more efficient than that by A549, HepG2, mediated pathway. This is consistent with the results that NIH/3T3, and HEK293T cells (Figure 2, left). This is in complex1showedhighselectivityforbreasttumorsoverother accordancewiththefactthatthebreastcancercellsMCF-7and nonbreast cancers or nontransformed cells, even if the latter MDA-MB-231 overexpress fructose transporters, while non- overexpressed glucose-specific GLUTs. breast cancer and normal cells show minimal expression.7 In To further investigate the selectivity of complex 1 for breast sharp contrast, the uptake of complex 2 did not show any cancercells,wehavedesignedacocultureexperiment,inwhich dependenceonthecelllines(Figure2,right).Thecytotoxicity cancerous MCF-7 and nontransformed HEK293T cells were ofcomplexes1and2towardthesamesetofcelllineshasbeen grown in the same culture dish and incubated with complex 1 examined (Table 2 and Figures S5 and S6, Supporting (50 μM, 37 °C, 1 h). The confocal microscopy image clearly Information). Importantly, the fructose complex 1 exhibited showedthattheemissionoftheMCF-7cellsduetocomplex1 higher cytotoxic activity toward breast adenocarcinoma among was much higher than that of HEK293T cells (Figure 5). This the four cancer cell lines (Table 2 and Figure S5, Supporting highlights the possible use of the fructose complex 1 as a Information), while complex 2 did not show similar selectivity biological imaging reagent for breast cancer cells and a (Table 2 and Figure S6, Supporting Information). These phosphorescent fructose-uptake indicator. These results, interesting results illustrate that the fructose pendant of together with the mitochondrial targeting property and complex 1 enables it to be recognized and taken up more photocytotoxic activity of complex 1, are anticipated to inspire efficiently by the breast cancer cell lines, causing higher ■the development of phototherapeutics for breast cancers. cytotoxicity. To confirm that the cellular internalization process of CONCLUSION complex 1 was indeed mediated by fructose transporters, we In summary, we have prepared a phosphorescent rhenium(I)- performed the following uptake competition experiments. Unmodified fructose (0−50 mM) was added to the sugar-free based fructose-uptake indicator and breast cancer cell imaging reagent. This complex was localized in the mitochondria and cell culture medium. If the uptake of complex 1 involves exhibited photocytotoxic activity. Also, it showed selective fructose transporters such as GLUT5, there should be a accumulation in MCF-7 and MDA-MB-231 breast cancer cells competitionbetweencomplex1andexogenousfructoseforthe overother cancerandnoncancer cells.Theinhibiteduptakeof same transporter. Confocal microscopy images showed that additionof50mMofexogenousfructosesignificantlyinhibited the complex by native fructose clearly indicated the involvement of fructose transporters. Related studies of other the uptake ofcomplex 1 bythe breastcancer cellsMCF-7 and phosphorescent transition-metal fructose conjugates are in MDA-MB-231 but not by the other four types of cells (Figure p■rogress. 3). Also, the uptake of complex 2 by all six cell lines was independentofthepresenceofunmodifiedfructose(FigureS7, EXPERIMENTAL SECTION SupportingInformation).ICP-MSmeasurementsquantitatively showed that the inhibition of the uptake of complex 1 by the Allsolventswereofanalyticalreagentgradeandpurifiedaccordingto standard procedures.22 Chemicals for the synthesis of ligands and breast cancer cells was dependent on the concentration of complexeswerepurchasedfromAcrosorAldrich.MCF-7,MDA-MB- exogenousfructose,andabout40%oftheuptakewasinhibited 231, A549, HEK293T, HepG2, and NIH/3T3 cells were obtained at[fructose]=50mM(Figure4).Again,fortheotherfourcell fromtheAmericanTypeCultureCollection.HighglucoseDulbecco’s lines, the uptake of both complexes was independent of modifiedEagle’smedium(DMEM),phosphate-bufferedsaline(PBS), exogenous fructose (Figure S8, Supporting Information). fetalbovineserum(FBS),trypsin-EDTA,andpenicillin/streptomycin Although the fluorescent fructose conjugate 1-NBDF8 and were purchased from Invitrogen. Table 2. Cytotoxicity (IC values/μM, 48 h) of Complexes 1 and 2 toward Six Different Cell Lines 50 complex MCF-7 MDA-MB-231 A549 HepG2 NIH/3T3 HEK293T 1 9.6±0.6 4.9±0.4 26.8±2.1 33.9±0.9 2.1±0.3 6.7±0.7 2 3.9±0.3 2.3±0.2 2.6±0.3 5.7±0.4 1.8±0.3 2.1±0.2 C dx.doi.org/10.1021/om400612f|OrganometallicsXXXX,XXX,XXX−XXX Organometallics Article Figure3.Laser-scanningconfocalmicroscopyimagesofsixdifferenttypesofcellsuponincubationofcomplex1(50μM,37°C,1h)intheabsence or presence of 50 mM fructose. Note that the emission intensity can only be considered qualitatively for comparison purposes, and readers are referred to the ICP-MS data(Figures 2 and 4and FigureS8, Supporting In■formation) for quantitative information. ASSOCIATED CONTENT * S Supporting Information Text, a scheme, tables, and figures giving details of the synthesis, characterization, spectroscopic and photophysical properties, cellular uptake, cytotoxicity, and singlet oxygen generationofcomplexes1and2.Thismaterialisavailablefree o■f charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Figure 4. Relative cellular uptake of rhenium by an average MCF-7 Corresponding Author (left)andMDA-MB-231(right)celluponincubationwithcomplexes 1 (shaded) and 2 (empty) (50 μM, 37 °C, 1 h) in a medium *K.K.-W.L.: e-mail, bhkenlo@cityu.edu.hk; fax, +852 3442 containing various concentrations of fructose. The uptake of the 0522. complexeswasrelativetotheircorrespondinguptakeat[fructose]=0 Notes mM. T■he authors declare no competing financial interest. ACKNOWLEDGMENTS We thank The Hong Kong Research Grants Council (Project No.CityU102212)andCityUniversityofHongKong(Project No. 9667081) for financial support. We thank Mr. Kenneth King-Kwan Lau, Mr. Michael Wai-Lun Chiang, and Mr. Ho- Figure5.Fluorescence(left),brightfield(middle),andoverlaid(right) H■ang Chan for their assistance. confocal microscopy images of a coculture of MCF-7 and HEK293T cells treated with complex1 (50 μM,37 °C, 1 h). REFERENCES (1) (a) Bell, G. I.; Burant, C. F.; Takeda, J.; Gould, G. W. J. Biol. Chem.1993,268,19161.(b)Mueckler,M.Eur.J.Biochem.1994,219, Synthesisof[Re(Ph -phen)(CO) (py-fructose)](CF SO )(1).A 713.(c)Medina,R.A.;Owen,G.I.Biol.Res.2002,35,9.(d)Macheda, 2 3 3 3 mixture of [Re(Ph-phen)(CO)(CHCN)](CFSO) (198 mg, 0.25 M. L.;Rogers, S.;Best, J. D.J. Cell. Physiol. 2005,202, 654. 2 3 3 3 3 mmol) and py-fructose (70 mg, 0.25 mmol) was refluxed in THF/ (2) Warburg, O. Science 1956, 123,309. MeOH(20mL,1:1,v/v) undernitrogen for12 h.The solution was (3)Waki,A.;Kato,H.;Yano,R.;Sadato,N.;Yokoyama,A.;Ishii,Y.; evaporated to dryness to give a yellow solid. Recrystallization of the Fujibayashi, Y.; Yonekura, Y. Nucl. 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