Toxicology in Vitro 24 (2010) 178–183

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Toxicology in Vitro

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Titanium(IV) complexes: Cytotoxicity and cellular uptake of titanium(IV) complexeson caco-2 cell line

Ramón Hernández a,c, Janet Méndez b, José Lamboy a, Madeline Torres b,Féliz R. Román a, Enrique Meléndez a,*

a University of Puerto Rico, Department of Chemistry, P.O. Box 9019, Mayaguez, 00681, Puerto Ricob University of Puerto Rico, Department of Chemical Engineering, P.O. Box 9046, Mayaguez, 00681, Puerto Ricoc Pontifical Catholic University of Puerto Rico, Department of Chemistry, 2250 Ave. Las Américas Ponce, 00717-9997, Puerto Rico

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 July 2009Accepted 16 September 2009Available online 20 September 2009

Keywords:Caco-2 cellColon cancerTitanocene dichlorideMaltol

0887-2333/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.tiv.2009.09.010

* Corresponding author. Tel.: +1 (787) 832 4040x25E-mail address: enrique.melendez@upr.edu (E. Me

Replacement of the ancillary ligand in titanocene dichloride by amino acids provides titanocene specieswith high water solubility. As part of our research efforts in the area of titanium-based antitumor agents,we have investigated the cytotoxic activity of Cp2TiCl2 and three water soluble titanocene-amino acidcomplexes – [Cp2Ti(aa)2]Cl2 (aa = L-cysteine, L-methionine, and D-penicillamine) and one water solublecoordination compound, [Ti4(maltolato)8(l-O)4] on the human colon adenocarcinoma cell line, Caco-2.At pH of 7.4 all titanocene species decompose extensively while [Ti4(maltolato)8(l-O)4] is stable for overseven days. In terms of cytotoxicity, the [Cp2Ti(aa)2]Cl2 and [Ti4(maltolato)8(l-O)4] complexes exhibitedslightly higher toxicity than titanocene dichloride at 24 h, but at 72 h titanocene dichloride and[Ti4(maltolato)8(l-O)4] have higher cytotoxic activity. Cellular titanium uptake was quantified at varioustime intervals to investigate the possible relationship between Ti uptake and cellular toxicity. Resultsindicated that there was not a clear relationship between Ti uptake and cytotoxicity. A structure–activityrelationship is discussed.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The first non-platinum complex tested in clinical trials as anti-tumor agent was cis-[(CH3CH2O)2(bzac)2Ti(IV)], Scheme 1. Thiscomplex is active against a wide variety of ascites and solid tumors(Keppler et al., 1991; Clarke et al., 1999; Schilling et al., 1996).Other cis-[X2(bzac)2Ti(IV)] complexes have been investigatedexhibiting similar biological activity as the ethoxide complex.However, the interest for non-platinum complex was at a slowpace due to the remarkable and well-reputed antitumor propertiesof cis-platin. The discovery of metallocene-based organometallicanticancer agent, Cp2TiCl2, in 1979 by Köpf and Köpf-Maier(1979) stimulated much interest to investigate other non-platinumcomplexes with different mechanism of cancinostatic activity.

Immediately, in the following years, other metallocenes withthe general formula Cp2MX2, (Scheme 2), (M = Ti, V, Nb, Mo;X = halides and pseudo-halides), Cp2Fe+X�, main group (C5R5)2M(M = Sn, Ge; R = H, CH3) and ionic [Cp2TiL2]2+[Y]2 (L = anionic li-gand, amino acid) have been synthesized and investigated for anti-tumor activity (Köpf-Maier, 1994; Köpf-Maier and Köpf, 1987,1988, 1994; Harding and Mokdsi, 2000; Meléndez, 2002). In gen-

ll rights reserved.

24.léndez).

eral, these complexes demonstrated to be efficient antineoplasticagents with less toxic effect than the well-reputed cis-platin(Köpf-Maier, 1994; Köpf-Maier and Köpf, 1987, 1988, 1994; Har-ding and Mokdsi, 2000; Meléndez, 2002). Among all the metallo-cenes tested, Cp2TiCl2 was the most active reaching phase I and IIclinical trials (Berdel et al., 1994; Korfel et al., 1998; Luemmenet al., 1998; Christodoulou et al., 1998; Kröger et al., 2000; Baum-gart et al., 2000). However, titanocene dichloride is unstable atphysiological pH due to extensive hydrolysis (Toney and Marks,1985). To circumvent this, Cp2TiCl2 is dissolved in Me2SO/H2O (sal-ine) mixture for in vivo experiments (Köpf-Maier, 1994; Köpf-Ma-ier and Köpf, 1987, 1988, 1994; Harding and Mokdsi, 2000;Meléndez, 2002). Nevertheless, many mechanistic details are un-known, which hinders its use as a chemotherapeutic agent.

Modification of ligands in titanocene dichloride is an active areaof research since, among the metallocenes, titanocene dichloride isthe most effective species (Boyles et al., 2001; Valadares et al.,2003, 2006; Connor et al., 2006; Allen et al., 2004; Potter et al.,2007; Pampillón et al., 2007; Sweeney et al., 2005, 2006; Rehmannet al., 2005; Gómez-Ruiz et al., 2007; Top et al., 2002; Meléndezet al., 2000; Pérez et al., 2005; Gao et al., 2007; Hernández et al.,2008; Gansäuer et al., 2005, 2008a,b; Weber et al., 2008; Obersch-midt et al., 2007; O’Connor et al., 2006). The modification on the Cprings and ancillary ligands are aimed to improve antitumor activity

TiOC2H5

OC2H5

OH3C

O

O

CH3

O

Scheme 1. Structure of cis-[(CH3CH2O)2(bzac)2Ti(IV)].

M

X

X

Scheme 2. Structure of metallocenes dihalide.

R. Hernández et al. / Toxicology in Vitro 24 (2010) 178–183 179

and water solubility. On way to improve titanocene water solubil-ity is to replace the chloride group for hydrophilic ligands such asamino acids (Köpf-Maier, 1994; Köpf-Maier and Köpf, 1987, 1988,1994; Harding and Mokdsi, 2000; Meléndez, 2002; Pérez et al.,2005). Another approach is to synthesize robust, water stableTi(IV) species coordinated by oxygen containing ligands. In thisregard, we have synthesized [Ti4(maltolato)8(l-O)4] (maltolato =3-hydroxy-2-methyl-4-pyronato) complex which is stable at andabove physiological pH (Lamboy et al., 2007). As part of ourresearch efforts, we have studied the cytotoxic activity and cellularuptake of two types of titanocene derivatives and the [Ti4(maltola-to)8(l-O)4] compound on the colon cancer cell line, Caco-2.

Caco-2 cells are human colon carcinoma cells that are able toexpress differentiation features of mature intestinal cells (Artursonand Borchardt, 1997). It is an excellent model to study intestinaldrug absorption and metabolism since Caco-2 cells express mostdrug transporters of the intestine (Arturson and Borchardt, 1997).Currently, this cell line is being used in many pharmaceutical com-panies as an initial screening test. Therefore, we have examined thecytotoxic activities and titanium uptake of the subject complexesin Caco-2 cells line. To our knowledge, this is the first report oncytotoxic properties and cellular uptake of Ti(IV) complexes on co-lon cancer cells.

2. Experimental

2.1. Methods and materials

Titanocene dichloride salts were handled under dried nitrogen inSchlenk anaerobic lines. Cp2TiCl2 was obtained from Aldrich andused without further purification. [Cp2Ti(L-cysteine)2]Cl2, [Cp2Ti(D-penicillamine)2]Cl2, [Cp2Ti(L-methionine)2]Cl2 and [Ti4(maltola-to)8(l-O)4] were prepared by published procedures (Pérez et al.,2005; Lamboy et al., 2007). The purity of titanium complexes waschecked by IR and/or by 1H NMR spectroscopy.

2.2. Physical measurements

FTIR spectra were recorded on a Bruker Vector-22 spectropho-tometer with the samples as compressed KBr pellets. 1H spectra

were recorded on a 300 MHz Varian Gemini and 500 MHz AvanceBruker spectrometers under controlled temperature. TPPS was in-serted in a sealed capillary tube inside the NMR tube and used asan internal reference.

2.3. Cell culture

Caco-2 cells were purchased from the American Tissue CultureCollection (Rockville, MD). The cells were cultivated on 75 cm2

flasks (Beckman Diagnostics, Franklin Lakes, NJ) using Dulbecco’smodified Eagle medium (DMEM) (Sigma, St. Louis, MO) containing10% fetal bovine serum (Life Technologies, Gaithersburg, MD), 1%nonessential amino acids (Life Technologies, Gaithersburg, MD),100 units/mL of penicillin, and 100 lg/mL streptomycin (Sigma,St. Louis, MO). Cells were maintained on a controlled atmosphereat 37 �C, 95% relative humidity, and 5% CO2. Culture medium waschanged every other day for approximately 5–6 days until cellsreached approximately 80–90% confluency.

2.3.1. Cytotoxicity studiesThe biological activity was determined using the CellTiter-

BlueTM assay. Cells with a concentration of 10,000 cells/cm2 wereseeded in 96 well assay plates with an area of 0.71 cm2/well(3603, Costar, Corning, NY). They were cultivated at 37 �C and 5%of CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supple-mented with 10% fetal bovine serum (FBS) (Sigma–Aldrich, St.Louis, MO). Prior to the doubling time, cells were washed twicewith Hank’s Balanced Salts (HBSS) (Sigma–Aldrich, St. Louis, MO)to remove the phenol red containing culture media and sampleswere placed. DMEM was used as a negative control and 1.5% hypo-chlorite solution was used as a positive control. Samples wereprepared to contain the desired concentration of [Ti4(maltola-to)8(l-O)4], titanocene-D-penicillamine, titanocene-L-methonine,and titanocene-L-cysteine using a concentration range between312.5 lM and 2500 lM. Titanocene dichloride, was used as a com-parison known drug model and was tested in the concentrationrange between 19.53 lM and 2500 lM. Cells were incubated for2, 24 and 72 h with the drugs. After the end of the contact period,solutions were removed. The cells were allowed to recuperate untila total of one week of cultivation was achieved. At this point, cellswere washed and incubated at 37 �C with CellTiter-Blue (Promega,Madison, WI) for the appropriate time. Cell viability was analyzedwith a spectrofluorometer (SpectraMax Gemini EM, Molecular De-vices, Sunnyvale, CA) by measuring the fluorescence emitted withan excitation of 560 nm and an emission of 590 nm. Wells with ahigh concentration of viable cells possessed the highest fluores-cence. Cytotoxicity results (obtained from sixteen independentmeasurements) were presented by normalizing all the relativefluorescent unit values with the relative fluorescent unit value ofthe negative control (cells with DMEM). Concentrations of com-pounds required to inhibit cell proliferation by 50% (IC50) were cal-culated by fitting data to a four-parameter logistic plot by means ofthe SigmaPlot software from SPSS.

2.4. Quantitative study of titanium cellular uptake by InductiveCoupled Plasma-Atomic Emission Spectroscopy

Cells at a concentration of 50,000 cells/cm2 were seeded in ster-ile 6 well plate (3502, Falcon, Corning, NY) and cultivated for oneweek in supplemented DMEM with phenol red at 37 �C and 5% ofCO2. After one week of cultivation, the cells were placed in contactwith drug solutions with a concentration of 312.5 lM. This concen-tration was found to be non toxic for aforementioned cells for allthe compounds. Solutions were prepared in DMEM for and placedin contact with the cells for 5, 24, 48, 72 h. The compound’s solu-tion was then removed and the cells were rinsed with ice-cold

180 R. Hernández et al. / Toxicology in Vitro 24 (2010) 178–183

BRS to removed excess drug. The cell monolayer was trypsinizedfor 20 min in the incubator at 37 �C. Detached cells were countedand then centrifuged. The resulting pellet was solubilized with0.5% Triton-X 100 (Sigma, St. Louis, MO) in BRS for 30 min at37 �C. Finally, BRS was added to a final volume of 1 mL. The sam-ples were analyzed using Inductive Couple Plasma Mass Spectrom-eter (7500, Agilent, CA).

2.5. Ethidium bromide displacement assay by fluorescencespectroscopy

A 6.6 � 10�6 M lyophilized calf-thymus DNA (Sigma) solutionwas freshly prepared in RNase- and DNase-free water. Prior tothe interactions, the titanium–maltolato complex’s fluorescencewas verified to be non-existent in aqueous solution. Ethidium bro-mide was added to a final concentration of 6.31 � 10�7 M. The tita-nium–maltolato complex was added to a 1:1 ratio with EtBr andspectra were recorded every ten minutes for 1 h. The experimentwas performed with the complex to EtBr ratio at 2:1 and 45:1and all measurements were recorded at 28 �C. No changes in thefluorescence were observed at all ratios studied.

2.6. Titanium–maltolato complex interaction with DNA monitored by 1

H NMR spectroscopy

The titanium–maltolato complex was dissolved in 25 mM Tris-d11/10 mM NaCl (D2O) buffer at pH 7.4 to a concentration of1.05 � 10�4 M for each (monomer and tetramer). The resultingsolution had a tetramer:monomer 1:1 ratio and the pH was 7.4which was not adjusted. To the titanium–maltolato solution wasadded aliquots of 50 lL of DNA, 3.1 � 10�5 M (in mM Tris-d11/10 mM NaCl (D2O) buffer at pH 7.4) at room temperature andthe spectra recorded after each aliquot.

3. Results and discussion

The syntheses of these three new water soluble titanocene-ami-no acid complexes ([Cp2TiL2]Cl2, L = L-cysteine, D-penicillamine andL-methionine) and [Ti4(maltolato)8(l-O)4] have been previously re-ported (Pérez et al., 2005; Lamboy et al., 2007), but this is the firstreport on the cytotoxic properties and titanium cellular uptake ofthese species in the colon cancer Caco-2 cell line. The titanocene-amino acid complexes have been previously characterized by 1Hand IR spectroscopy and the amino acids are engaged in Ti–O(car-boxylate) coordination, according to the structure presented inFig. 1. [Ti4(maltolato)8(l-O)4] has been characterized crystallo-graphically (Lamboy et al., 2007). It is a tetranuclear species con-taining bridging oxygens forming a Ti4O4 cyclic unit, seeSupplementary Material.

Previous kinetic studies in water, at pH = 3, showed that[Cp2TiL2]Cl2, L = L-cysteine and L-methionine) complexes are less

R

Rn

Cl2

R

R

n

Ti

O2C NH 3+

O2C NH 3+

SR

SR

Fig. 1. Proposed structure for titanocene-aminoacid complexes.

stable than titanocene dichloride while [Cp2Ti(D-penicilla-mine)2]Cl2 has similar stability to Cp2TiCl2 (Pérez et al., 2005).However, increasing the pH to 7.4 leads to the formation of a yel-low solution, which eventually vanishes. Further decompositionwas observed as evidenced by the formation of cloudiness. Similarresults were reported by Marks and co-workers where theyproposed the formation of a series of titanium species such asTi(Cp)0.31O0.30(OH) Toney and Marks, 1985. Further decompositionof this species into TiO2, at high pH, has been proposed (Toney andMarks, 1985).

The scenario is different for [Ti4(maltolato)8(l-O)4]. This speciesis very stable at pH of 7.4 and above, under buffer conditions, with-out any detectable decomposition (Lamboy et al., 2007). The Ti–Ooctahedral coordination sphere and the bulkiness of the maltol li-gands could be responsible for its stability. Therefore, formation ofTiO2 can be ruled out.

The in vitro cytotoxicity of the titanocene-aminoacids, titano-cene dichloride and [Ti4(maltolato)8(l-O)4] in Caco-2 cell linewas determined using the commercially available cell viability as-say (Cell Titer-Blue from Promega) at 24 and 72 h drug exposureperiod. Titanium complexes were tested in concentrations thatranged from 313–2500 lM. Initial cytotoxic studies at 2 h drugexposure were performed in concentrations between 0.01 and10 mM to find the IC50 range. Since titanocene dichloride has alonger intracellular activation period, it is usually tested at a timeinterval of 72 h, as was done in our study. Table 1 summarizes theIC50 data for the titanocene complexes.

Upon examination of Table 1 it is evident that all the titano-cene-amino acid and [Ti4(maltolato)8(l-O)4] complexes demon-strated slightly higher cytotoxic activity than titanocenedichloride (Cp2TiCl2) at 24 h drug exposure. However, the apparent(albeit slight) enhancement in cytotoxicity by replacing the chlo-rides by sulfur-containing amino acids or by [Ti4(maltolato)8(l-O)4] is not significant since all the IC50 values have the same orderof magnitude.

The cytotoxicity studies were performed at longer drug expo-sure period of 72 h. Fig. 2 presents the IC50 curves for the com-plexes at 72 h of drug exposure. This time period is sufficient toallow titanocene dichloride to express its biological activity. Ingeneral, it is clear that all the titanocene complexes as well as[Ti4(maltolato)8(l-O)4] showed somehow enhanced cytotoxicactivity at 72 h as compared to 24 h. Furthermore, titanocenedichloride and [Ti4(maltolato)8(l-O)4] showed to be one order ofmagnitude more cytotoxic than titanocene-aminoacid complexes.The cytotoxicities of titanocene dichloride and [Ti4(maltolato)8(l-O)4] at 72 h are also one order of magnitude higher than their cyto-toxicities at 24 h.

In order to correlate and understand the cytotoxic responses ofthese titanium complexes as function of structure, we monitoredthe titanium uptake process on Caco-2 cell line at different drugexposure times using Inductive Coupled Plasma-Atomic EmissionSpectroscopy (ICP-AES). Three representative complexes werestudied: Cp2TiCl2, [Cp2Ti(L-cysteine)2]Cl2 and [Ti4(maltolato)8

Table 1IC50 values for the complexes studied in the Caco-2 cell line, determined using theavailable cell viability assay Cell Titer-Blue. IC values are the average of sixteenindependent measurements.

Complex IC50 (mM)

24 h 72 h

Cp2TiCl2 6.9(6) 0.109(8)[Cp2Ti(L-cysteine)2]Cl2 2.9(6) 1.7(3)[Cp2Ti(L-methionine)2]Cl2 3.16(1) 1.2(8)[Cp2Ti(D-penicillamine)2]Cl2 3.29(0) 1.193(1)[Ti4(maltolato)8(l-O4)] 2.50(5) 0.214(5)

Fig. 2. Dose response curves of top: [Cp2Ti(L-cysteine)2]Cl2 (diamonds), [Cp2Ti(L-methionine)2]Cl2 (triangles) and [Cp2Ti(D-penicillamine)2]Cl2 (squares); bottom: Cp2TiCl2

(squares) and [Ti4(maltolato)8(l-O4)] (diamonds) against Caco-2 cell line at 72 h drug exposure, determined using cell viability assay Cell Titer-Blue.

Table 2Titanium uptake by Caco-2 colon cancer cells. Concentration values are average ofthree independent experiments, expressed as pgTi/cell. Std in parenthesis.

Exposure time 5 h 24 h 48 h 72 hCompound (pgTi/cell) (pgTi/cell) (pgTi/cell) (pgTi/cell)

Cp2TiCl2 0.016(2) 0.032(5) 0.049(2) 0.066(7)[Cp2Ti(cysteine)2]Cl2 0.116(2) 0.203(2) 0.27(2) 0.31(2)[Ti4(maltolato)8(l-O4)] 0.144(2) 0.3(1) 0.11(1) 0.17(1)

Fig. 3. Comparison of intracellular titanium uptake by Caco-2 cells. Cp2TiCl2

(triangles), [Cp2Ti(cysteine)2]Cl2 (squares) and [Ti4(maltolato)8(l-O4)] (diamonds).

R. Hernández et al. / Toxicology in Vitro 24 (2010) 178–183 181

(l-O4)]. The Ti uptake by Caco-2 cell line was measured at 5, 24, 48and 72 h (drug exposure), see Table 2 and Fig. 3. Upon examinationof Table 2, it can be observed that [Ti4(maltolato)8(l-O4)] has the

higher Ti uptake by Caco-2 cells at 5 and 24 h but it decreases atlonger times. On the other hand, [Cp2Ti(L-cysteine)2]Cl2 andCp2TiCl2 showed a constant increase in Ti uptake as time evolved.[Cp2Ti(L-cysteine)2]Cl2 showed higher cellular uptake by Caco-2cells when compared to titanocene dichloride. Interestingly, theTi uptake quantified for the three titanium complexes demon-strated no correlation to their cytotoxic activities. For instance at72 h of drug exposure, [Cp2Ti(L-cysteine)2]Cl2 has the highest IC50

value (lower cytotoxic activity) than Cp2TiCl2 and [Ti4(maltola-to)8(l-O4)] but its Ti concentration on Caco-2 cells is higher.

The Ti uptake on Caco-2 cell by [Ti4(maltolato)8(l-O4)] deservesespecial attention. At shorter period of drug exposure it has thehigher Ti uptake but as we increase the drug exposure time theamount of Ti uptaken by Caco-2 cells decreases. This suggests that[Ti4(maltolato)8(l-O4)] interaction inside the cell is weak and mustlikely reversible and as a result the cell metabolize and removetitanium from the cell. This result is in agreement with the struc-tural features of [Ti4(maltolato)8(l-O4)], as described below.

[Ti4(maltolato)8(l-O4)] is a tetranuclear species, inert, waterstable and it is not uptaken by transferrin as do the titanocenecomplexes (Pérez et al., 2005). This implies that [Ti4(maltola-to)8(l-O4)] enters into the cell as a tetranuclear species and sinceits coordination sphere is saturated, it can partially intercalate be-tween DNA bases rather than undergoing DNA (phosphate andnitrogen) coordination. In fact, the distance between the adjacentmaltol rings is 3.6 Å which can be envisioned to intercalate withinthe DNA bases but its interaction is weak. To explore this possibil-ity, we performed [Ti4(maltolato)8(l-O4)]-DNA interaction studiesusing 1H NMR spectroscopy.

At physiological pH, [Ti4(maltolato)8(l-O4)] is the predominantspecies, but since Ti(maltolato)2(OH)2 may exist as a minor prod-uct, the 1H NMR spectroscopic DNA binding interaction studies

Fig. 4. 1H NMR binding studies of [Ti4(maltolato)8(l-O4)] and its monomeric species with calf-thymus DNA in 25 mM Tris-d11/10 mM NaCl (D2O) buffer at pH 7.4 and roomtemperature. (a) Mixture of 1:1 monomer:tetramer, (b) after addition of 50 lL of DNA and (c) after addition of 100 lL of DNA.

182 R. Hernández et al. / Toxicology in Vitro 24 (2010) 178–183

were performed in a sample that contained both the monomericand tetrameric species at pH of 7.4 in Tris-d11 buffer, using calf-thymus DNA as a model. Upon addition of calf-thymus DNA intothe Ti-maltol solution, (Fig. 4), we observed that the maltol signalsH-5 and H-6 of the tetrameric species broadened and move upfieldand then collapsed into the baseline, while the monomeric speciesremained intact. This strongly suggests that [Ti4(maltolato)8(l-O4)] is intercalating between the DNA bases. However, the interca-lation is weak since in fluorescence spectroscopy experiments[Ti4(maltolato)8(l-O4)] was not able to replace ethidium bromidefrom calf-thymus DNA.

Thus, this data could explain why [Ti4(maltolato)8(l-O4)] is re-moved from the cell at long drug exposures (48 and 72 h). Finally,the monomeric species does not show any interaction with calf-thymus DNA since it is coordinatively saturated and does not con-tain parallel rings separated by 3.6 Å.

4. Concluding remarks

In this study, we have presented the cytotoxicity and Ti uptakeon the Caco-2 cell line of a selected group of titanocene complexes(three highly water soluble titanocene-aminoacid complexes andslightly soluble titanocene dichloride) and a robust coordinationcompound [Ti4(maltolato)8(l-O4)]. Titanocene-aminoacid speciesas well as titanocene dichloride are considerably stable in wateras long as the pH is kept below 3. Thus, even though the aminoacidligand imparts water solubility to their complexes, it does not nec-essarily impart more long term hydrolytic stability at low pH. Atphysiological pH all complexes degrade substantially. What possi-ble effects will induce the presence of an aminoacid as ancillary li-gand and their outcome in terms of cytotoxicity? We can infer thatthe increased solubility of [Cp2Ti(aa)2]Cl2 species make themslightly more accessible to the cells and slightly more cytotoxic.Apparently, this scenario applies at least at 24 h of drug exposure.At 72 h of drug exposure, Cp2TiCl2 is more cytotoxic than[Cp2Ti(aa)2]Cl2 species and slightly more cytotoxic than [Ti4(malto-lato)8(l-O4)]. On the other hand, [Ti4(maltolato)8(l-O4)], a highlysoluble and robust species at physiological conditions demon-strated to be more active at shorter drug exposure period but lessactive than titanocene dichloride at 72 h. The initial response on[Ti4(maltolato)8(l-O4)] at 24 h could be explained as a result of

higher content of Ti/cell but in marked contrast, its IC50 value con-tinues to decrease (improving its cytotoxicity) at 72 h even thoughthe concentration of Ti/cell decreases. Thus there is no simple cor-relation between cytotoxic activity and Ti uptake by Caco-2 cells.On the other hand, titanocene dichloride showed less concentra-tion of Ti/cell than [Cp2Ti(L-cysteine)2]Cl2 at all time intervals (5,24, 48, 72 h) but at 72 h exhibited higher cytotoxic activity. Second,in contrast to [Ti4(maltolato)8(l-O)4], both titanocene dichlorideand [Cp2Ti(L-cysteine)2]Cl2 continue to increase the Ti uptake/cellas time evolves and their cytotoxic activities improve along.

Comparing titanocene dichloride versus [Cp2Ti(L-cysteine)2]Cl2,the fact that the Caco-2 cell line is known to express cation mem-brane transporters (also express most drug transporters of theintestine) (Arturson and Borchardt, 1997; Yeung et al., 2005) couldexplain these differences, because even though both, Cp2TiCl2 and[Cp2Ti(aa)2]Cl2, complexes are expected to form cationic species,their stabilities in water are different. We may invoke the forma-tion of [Cp2Ti(aa)2]2+ versus [Cp2Ti(OH)Cl]+ or [Cp2Ti(H2O)(OH)]+,keeping in mind that these species predominate at low pH. The dif-ference in Ti uptake may rely in the amino acid ligands. They areweakly coordinated to Ti(IV) and as a result, the titanocene–aminoacid complex is more unstable, and it becomes coordinativelyunsaturated easily, forming rapidly [Cp2Ti(aa)2]2+. Although atphysiological pH species like Ti(Cp)0.31O0.30(OH) will predominate(Toney and Marks, 1985), the small amount of [Cp2Ti(aa)2]2+ thatremain in solution will be more effectively transported inside thecells than the remaining amount of [Cp2Ti(OH)Cl]+ or [Cp2Ti(-H2O)(OH)]+, as a result of its di-cationic nature. In any event, withthis experimentation we cannot elucidate the mechanistic detailsof their cytotoxicity. But what our experimental results showedis that there is no correlation between Ti uptake on Caco-2 cell lineand the cytotoxicity of the titanium complexes. Also, the water sol-ubility as a vital property for these complexes to improve the anti-proliferative activity remains questionable.

To our knowledge, there are no previous studies on Ti uptake bycancer cells using titanocenes. There is one report on molybdenoc-enes where Mo uptake by cancer cells is monitored by atomicabsorption (Waern et al., 2005). Harding and co-workers foundthat there is no correlation between the amount of molybdenumuptaken by the cancer cells and cytotoxicity. Also, there is no cor-relation between the molybdenocene hydrophobic character and

R. Hernández et al. / Toxicology in Vitro 24 (2010) 178–183 183

cytotoxic activity. We have found similar results with Ti(IV)complexes.

Acknowledgements

E.M. acknowledges the NIH-MBRS SCORE Programs at the Uni-versity of Puerto Rico Mayagüez for financial support and NSF-MRIfor providing funds for the purchase of the 500 MHz NMR.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.tiv.2009.09.010.

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