A Photoactive Platinum(IV) Anticancer Complex Inhibits Thioredoxin

2 days ago - figure. Scheme 1. Chemical Structures of Complex 1 (trans,trans,trans-[Pt(N3)2(OH)2(py)2]) and the Pt-Containing Residues Arising from It...
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Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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A Photoactive Platinum(IV) Anticancer Complex Inhibits Thioredoxin−Thioredoxin Reductase System Activity by Induced Oxidization of the Protein Jun Du,† Yuanyuan Wei,†,‡ Yao Zhao,*,‡ Fengmin Xu,†,‡ Yuanyuan Wang,‡,§ Wei Zheng,‡ Qun Luo,‡,§ Ming Wang,‡,§ and Fuyi Wang*,‡,§ †

College of Chemistry and Materials Science, Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Laboratory of Molecular-Based Materials, Anhui Normal University, Wuhu 241000, People’s Republic of China ‡ Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, National Centre for Mass Spectrometry in Beijing, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Thioredoxin (Trx) is an important enzyme in the redox signaling pathway and is usually overexpressed in tumor cells. We demonstrate herein that the photoactive platinum(IV) anticancer complex trans,trans,trans-[Pt(N3)2(OH)2(Py)2] (1) can bind to His, Glu, and Gln residues of Trx upon the irradiation of blue light. More importantly, complex 1 can also induce the oxidation of Met, Trp, and the Cys catalytic sites to form disulfide bonds by generating reactive oxygen species (ROS) upon photoactivation. These eventually lead to inhibition of activity of Trx enzyme and the Trx system and further increase in the cellular ROS level. We speculate that the oxidative damage not only inhibits Trx activity but also greatly contributes to the anticancer action of complex 1.

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gold(III),11 ruthenium(II),12 and platinum(II)13,14 that can inhibit TrxR activity have also been extensively studied.15,16 However, the interactions between metal complexes and Trx have been rarely explored. To the best of our knowledge, only a few examples concerning the interaction between Trx and platinum(II) or gold(I) complexes have been reported.17 The interaction of the platinum(IV) complex ormaplatin ([Pt(dach)Cl4], dach = (1R,2R)-(−)-1,2-diaminocyclohexane) with Trx was also explored using 3,6-dioxa-1,8-octanedithiol (DODT) as a model compound to mimic the active site of Trx. The oxidation of the thiols in DODT upon reductive activation of the PtIV complex was confirmed.18 A recent report described that Ag+ exhibits its antibacterial activity by disrupting the Trx system.19 In a previous work, trans,trans,trans-[Pt(N3)2(OH)2(Py)2] (1) was developed as a photoactive complex.20 It is noncytotoxic in the dark but becomes a potent anticancer agent upon irradiation with blue or UVA (ultraviolet A type, λmax 365 nm) light. Previous studies showed that, upon irradiation, the PtIV of 1 was reduced to PtII and the pyridine ligands remain intact but one or two of the azide groups may dissociate and be replaced by nucleophiles (Scheme 1). Further study discovered that complexes of the type trans,trans,trans-[Pt(N3)2(OH)2(am1)(am2)] (am = ammines or imines) have low cross-resistance toward cisplatin-

INTRODUCTION Thioredoxin (Trx) is a type of multifunctional protein widely existing in eukaryotic and prokaryotic organisms and has been found to regulate gene transcription and cell growth and to inhibit cell apoptosis.1 It is a general protein disulfide reductase that is closely related to many diseases with reference to DNA synthesis, oxidative stress defense, apoptosis, or redox signaling.1 Overexpression of Trx can be commonly observed in MCF-7 breast cancer cells,2 pancreatic ductal adenocarcinoma,3 non-small-cell lung carcinomas,4 and other cancer cells. It has been shown that increased levels of Trx in cancer cells may reduce their susceptibility to cisplatin or other anticancer drugs,5 suggesting that drug resistance may be associated with the increased level of Trx.6 The Trx system contains Trx, thioredoxin reductase (TrxR), and NADPH. The homostasis of this system has also been found to be altered in many diseases, including virus infection, inflammation, cancer, diabetes, and cardiovascular diseases.7 The activity of the Trx system is directly dependent on the activity of Trx. Therefore, studying the interactions between Trx and anticancer drugs may help to explore the mechanism of action of the drugs and therefore promote the research of the treatment of certain cancers and other diseases. During the last 40 years, metal-based anticancer complexes have been successfully applied in clinical treatment of cancers and their mechanisms of action have been deeply explored.8 Metal complexes such as those of silver(I),9 gold(I)9,10 © XXXX American Chemical Society

Received: February 28, 2018

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DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX

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P3, (19)ADGAILVDFWAEWC*GPC*K(36), were found to fully form disulfide in the native protein. Another oxidation modification found for the native protein was the monooxidation of peptide P4 ((37)MIAPILDEIADEYQGK(52), Table S2). Usually, Cys, Met, and Trp residues are believed to be possible oxidation sites.23 In P4, Met37 residue is the only possible oxidation site, and this oxidative modification is usually observed during the MS analysis of Met-containing peptides. Similar results were also found for native Trx irradiated with blue light (λ 460 nm, 1 h) and Trx incubated with 10 mol equiv of 1 in the dark for 24 h (Table S2). These results show that the bottom-up strategy of mass spectrometry can characterize Trx and its modifications very well. When Trx was incubated with complex 1 (50 mol equiv) upon irradiation with blue light (λ 460 nm, 1 h), a series of Pt residues bound to peptides and oxidized peptides were found by MS analysis in comparison with the native protein (Table 1). The sample with nTrx:n1 = 1:10 gave results similar to those obtained in the sample with nTrx:n1 = 1:50, but fewer Pt-bound peptides were found (Table S3). Oxidative modifications were found in P3 and P4 arising from both samples. In comparison to the data for the native Trx samples described above, Met37 in P4 was partially doubly oxidized (Figure 1a,b). This is consistent with previous reports that, in neutral buffers, the sulfur atom of Met could be oxidized to sulfoxide and sulfone by PtIV complexes upon photoactivation.23,24 This might be one of the reasons that no Pt residues binding to Met37 were observed as found in the reaction of cisplatin and proteins.25 The 2+ ion of tryptic P3 derived from the photoactivated 1 modified Trx was showed to be 15.9883 Da larger in mass than that derived from the native Trx, the two cysteines of which form a disulfide bond. (Table 1 and Table S1). This means that this peptide was doubly oxidized (Figure 1C). There are two Trp and two Cys residues in the peptide P3 (19)ADGAILVDFWAEWCGPCK(36), which are all possibly oxidized. In order to identify which residue of the peptide is oxidized, tandem MS analysis of this peptide was performed with highresolution FT-ICR-MS. As shown in Figure 2 and Table S4, a series of fragment ions were observed in the MS/MS spectrum. The detection of y6°°* ion containing doubly oxidative modification and y5* ion containing no oxidative modification unambiguously suggested that the Trp31 residue was doubly oxidized. According to the solution structure of Trx (PDB code: 1XOA)26 (Figure S2), Trp28 is located in the interior but Trp31 is located on the surface of the protein. Therefore, it is

Scheme 1. Chemical Structures of Complex 1 (trans,trans,trans-[Pt(N3)2(OH)2(py)2]) and the PtContaining Residues Arising from Its Photoactivation

resistant cancer cells21 and the photoactivation of these PtIV complexes may generate reactive oxygen species.22 Considering the reducing activity of Trx and the oxidative intermediates generated by trans,trans,trans-[Pt(N3)2(OH)2(am1)(am2)] complexes, it is worth exploring the interaction between them and Trx. Indeed, we reveal in this work that complex 1 could not only bind to Trx upon photoactivation but also induce oxidation of the amino acid residues, which further block the Trx-mediated redox circulation, by generating reactive oxygen species (ROS).

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RESULTS AND DISCUSSION Trx was mixed with 10 or 50 mol equiv of complex 1 in PBS. After irradiation with blue light of λ 460 nm for 1 h at 310 K, the mixture was incubated for another 23 h in the dark. Unbound platinum complexes including the photolytic residues were removed by ultrafiltration with a 3 kDa cutoff filter. Then the buffer was replaced with NH4HCO3 (20 mM) before tryptic digestion of the modified protein. Negative controls were prepared by incubation of intact Trx with/without irradiation or Trx with complex 1 without irradiation following the procedures similar to those described above. LC-ESI-MS analysis for the intact Trx was performed first. Both the reduced form and oxidized form (with Cys32 and Cys35 disulfide bonds) were found in the mass spectra (Figure S1). This suggests that native Trx is present in both reduced form and oxidized form in solution. The native Trx, incubated in the dark, was then digested by trypsin, and the tryptic peptides were separated with a C18 column, followed by online ESI-MS analysis. Nine out of the twelve tryptic peptides were identified, as shown in Table S1. The protein sequence coverage was 86.1%. Without use of alkylating agents to modify the thiols of Cys32 and Cys35, these two cysteine residues in

Table 1. Modified Tryptic Peptides Observed by Mass Spectrometry Analysis for Trx Incubated with Complex 1 (50 mol equiv) upon Irradiation of λ 460 nm for 1 h Followed by 23 h of Incubation in the Dark peptide no.

peptide sequencec

modification

charge

potential modification site

P3 P3 P4 P4 P2 P2 P5 + P6 P9 + P10 P10 + P11 P11 + P12

(19)ADGAILVDFWAEWC*GPC*K (36) (19)ADGAILVDFWAEWC*GPC*K (36) (37)MIAPILDEIADEYQGK(52) (37)MIAPILDEIADEYQGK(52) (4)IIHLTDDSFDTDVLK(18) (4)IIHLTDDSFDTDVLK(18) (53)LTVAKLNIDQNPGTAPK(69) (83)NGEVAATKVGALSK(96) (91)VGALSKGQLK(100) (97)GQLKEFLDANLA(108)

+2O +4O +O +2O +Pt(py)2−2H +Pt(N3)(py)2−H +Pt(N3)(py)2−H +Pt(N3)(py)2−H +Pt(N3)(py)2−H +Pt(N3)(py)2−H

2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+

Trp31 Trp28, Trp31 Met37 Met37 His6 His6 Gln62 Glu85 Gln98 Glu101

m/z obs (theor)a 1006.4339 1022.4406 911.4462 919.4468 1042.4543 1063.9645 1088.0154 870.3862 698.3332 857.3834

(1006.4418) (1022.4368) (911.4511) (919.4486) (1042.4620) (1063.9706) (1088.0287) (870.4045) (698.3378) (857.3805)

δb/ppm −7.85 3.72 −5.38 −1.96 −7.39 −5.73 −12.2 −21.0 −6.59 3.38

The observed (obs) and theoretical (theor) mass to charge ratios of the most abundant isotopomers. bδ = [(m/z)obs − (m/z)theor]/(m/z)theor. cThe asterisk denotes that there is a disulfide bond between Cys32 and Cys35. a

B

DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Mass spectra (black lines) and theoretical isotope patterns (red dots) for the oxidative modified peptides of Trx incubated with complex 1 (50 mol equiv) upon the irradiation with blue light (λ 460 nm) for 1 h: (a) [M°IAPILDEIADEYQGK]2+; (b) [M°°IAPILDEIADEYQGK]2+; (c) [ADGAILVDFWAEW°°C*GPC*K]2+; (d) [ADGAILVDFW°°AEW°°C*GPC*K]2+. Legend: (°) the residue was mono-oxidized; (°°) the residue was doubly oxidized; (*) a disulfide bond between Cys32 and Cys35.

Figure 2. MS/MS spectrum obtained by ultrahigh-resolution mass spectrometry (FT-ICR-MS) and assigned fragments of the peptide ADGAILVDFWAEW°°C*GPC*K. Legend: (°°) the fragment was doubly oxidized; (*) disulfide bond between Cys32 and Cys35. The full peak list is given in Table S4.

The oxidation of thiols to disulfide bonds by platinum(IV) complexes has been reported. For example, cis-[Pt(NH3)2Cl4], [Pt(dach)Cl4], and trans-[PtCl2(CN)4]2− were found to be able to induce the oxidation of thiols in a model compound of Trx to form an intramolecular disulfide bond.18 Those reactions were carried out at 298 K under unspecified light conditions. By comparison, the light-induced reaction in this work is much faster. Furthermore, apart from the formation of disulfide bonds, oxidation of amino acids was also discovered. The discovery of the double oxidation of Trp31 and Trp28 in Trx

reasonable that Trp31 was doubly oxidized before the oxidation of Trp28. Cys32 and Cys35 were shown to be oxidized to form a disulfide bond, which is the possible reason why they were not further oxidized. Quadruply oxidized peptide P3 was also found (Table 1 and Figure 1d), but the signal was too low to carry out tandem MS analysis. As the disulfide bond between Cys32 and Cys35 inhibited their oxidation, it is reasonable to assign that Trp28 and Trp31 were both doubly oxidized in this case. C

DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 3. Mass spectra (black lines) and theoretical isotope patterns (red dots) for the platinated peptides derived from Trx incubated with complex 1 (50 mol equiv) upon irradiation with blue light (λ 460 nm) for 1 h: (a) [IIH†LTDDSFDTDVLK]2+; (b) [IIH‡LTDDSFDTDVLK]2+; (c) [LTVAKLNIDQ‡NPGTAPK]2+; (d) [NGE‡VAATKVGALSK]2+; (e) [VGALSKGQ‡LK]2+; (f) [GQLKE‡FLDANLA]2+. Legend: (†) [Pt(py)22H]; (‡) [Pt(N3)(py)2-H].

glutamic acid (Glu) residues were also reported to be possible binding sites for PtII fragments30a,31 via amide−Pt and carboxylate−Pt coordination, Gln62, Glu85, and Gln98 are probably platination sites in peptides P5 + P6, P9 + P10, and P10 + P11, respectively. There are both Gln98 and Glu101 in the peptide P11 + P12 ((97)GQLKEFLDANLA(108)), but only one [Pt(N3)(py)2]+ modification was found on it. The calculated solvent-accessible surface area (SASA) of the sidechain amide N atom of Gln98 is 11.47 Å2, much smaller than those (41.9 and 20.9 Å2) for the nonprotonated O atoms in the carboxyl group of Glu101; thus, the modification was assigned to Glu101. The tryptic cleavage of K100 was missed when Trx was platinated, further evidencing that Glu101 was platinated, as a consequence, hampering the hydrolysis of the peptide bond between K100 and E101. When the molar ratio of Trx to 1 in the reaction mixture decreased to 1:10, only His6 was observed to bind to platinum residues (Table S3 and Figure S3), indicating that the PtII fragment has higher affinity to His6 than to Glu and Gln residues, consistent with previous reports.25a,30,31 It has been widely accepted that PtII complexes such as cisplatin react easily with the sulfur atoms of cysteine and methionine.30a,31a,32 However, in this work, platinum-modified Cys and Met were not found. Because the reducing agent DTT was not added during trypsin digestion, a disulfide bond between Cys32 and Cys35 was formed in both native Trx and platinated Trx. Moreover, the SASAs of the sulfur atoms of

upon the photolysis of complex 1 is very important, as the tryptophan radical is known to be an efficient intermediate in biological electron transfer. 27 It was reported that the photoactivation of trans,trans,trans-[Pt(N3)2(OH)2(methylamine)(pyridine)],22 a derivative of 1, may generate reactive oxygen species such as singlet oxygen and azidyl radicals. The latter could oxidize the Trp to Trp• radicals.28 In other words, Trp can quench the azidyl radicals generated by 1 upon photoactivation.29 Moreover, singlet oxygen is also a powerful oxidative agent. Therefore, it is reasonable to say that the photoactivation of 1 caused the oxidation of tryptophan in addition to accelerating the oxidation of cysteine residues in Trx. Trx platination was also found in reaction mixtures of Trx and complex 1. Once it is irradiated by light of a suitable wavelength, complex 1 can be reduced and produce platinum(II)-containing residues such as [Pt(py)2]2+ and [Pt(N3)(py)2]+ (Scheme 1). These residues were used as modifiers to the protein to search for platinated peptides arising from digestion of modified Trx. Upon the reaction of Trx with a 50fold excess of complex 1, peptides P2, P5 + P6, P9 + P10, P10 + P11, and P11 + P12 were platinated by the PtII fragment [Pt(py)2]2+ or [Pt(N3)(py)2]+ (Table 1 and Figure 3). His, Met, and Cys are frequent binding sites for metal complexes;25a,30 thus, the only His6 residue in P2 could be the binding site of PtII residues. There are no Met, Cys, and His residues in the rest of the platinated peptides. As glutamine (Gln) and D

DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 4. Absorbance at λ 650 nm for the reduction reaction of insulin by DTT: (a) insulin (0.13 mM) and DTT (0.33 mM) added after the mixture of Trx (7.8 μM) and 1 in 0.1 M PBS (pH 7.2) was irradiated with blue light at λ 460 nm for 1 h at 310 K; (b) sample under the same conditions as described in (a) except identical amounts of AA and 1 were added before light irradiation. The curves are plotted on the basis of the average of three independent experiments.

Figure 5. Time-dependent absorbance at 340 nm for the reaction mixtures of Trx (3 μM) + TrxR (0.36 unit/mL) + NADPH (200 μM) in the presence or in the absence of complex 1 in PBS (50 mM, pH 7.2) at 310 K. The decrease in absorbance indicates the oxidation of NADPH to NADP+. Trx and 1 were mixed and (a) irradiated by light at λ 460 nm for 1 h or (b) incubated in the dark at 298 K before addition of TrxR and NADPH. The curves were plotted on the basis of the average of two independent experimental data.

Cys32 and Cys35 are very small or nonexistent (1.06 and 0 Å2, respectively), indicating that both cysteines are hardly accessible for PtII fragments in aqueous solution. Met37, the only Met in Trx, was doubly oxidized, as described earlier. The sulfur atom has no available lone pair electrons; thus, it cannot coordinate to PtII. These results also imply that Cys32, Cys35, and Met37 residues are more susceptible to oxidation than to platination. ICP-MS was performed to determine the binding stoichiometry of photolytically reduced complex 1 and Trx. The concentration of Trx was determined by BCA method as described in the Supporting Information. After a mixture of Trx and 1 (10 mol equiv) was irradiated with light of λ 460 nm for 1 h and incubated for further 23 h in the dark, 2.8 ± 0.2 mol of Pt was found binding to 1 mol of Trx. Since complex 1 binds to and oxidizes Trx under the irradiation conditions, it is therefore necessary to explore whether 1 can inhibit the activity of the enzyme. The activity of Trx is usually assessed according to the reduction rate of insulin by dithiothreitol (DTT) upon the catalysis of Trx.33 The reduction of insulin forms a precipitate in aqueous buffer (pH 7.5) at 25 °C and can be monitored by increased absorption at λ 650 nm of the resulting suspension. In the control experiment, Trx alone did not reduce insulin within 2 h (Figure S4); if only DTT was added, insulin was slowly reduced and the absorbance of λ 650 nm reached its maximum in ca. 50 min (t1/2 = 31.1 min). When Trx and DTT were both used, the reduction of insulin was significantly promoted and the A650 value reached its maximum in ca. 10 min (t1/2 = 4.1 min), indicating the catalytic activity of Trx toward the reduction reaction of insulin by DTT (Figure S4).

When a mixture of Trx with 5 mol equiv of complex 1 was irradiated with blue light of λ 460 nm for 1 h before insulin and DTT were added, only a minor decrease in the reduction rate of insulin was observed, as shown in Figure 4a. However, when more complex 1, e.g. 10 or 50 mol equiv, was added, the reduction reaction rate was significantly decreased. Dark controls were also carried out, and there were only minor differences between the catalytic activity of Trx with and without addition of 5−50 mol equiv of complex 1 (Figure S5). These results suggest that the photoactivation of 1 could substantially inhibit the activity of Trx. The IC50 value, which is the concentration of 1 required for 50% inhibition against Trx (3.9 μM), was found to be 21.1 ± 2.7 μM (Figure S6). It is usually believed that the binding of metal ions to amino acid residues in the proteins may inhibit the activity of the target proteins.8b,10b,25a,31a,34 However, in this work, apart from producing reactive PtII residues, the photoactivation of complex 1 also generated ROS. Therefore, it is necessary to figure out if the resulting ROS accounts for the inhibition of Trx. To this end, L-ascorbic acid (AA) was used as an ROS scavenger to carry out the above experiment again. As shown in Figure 4b, in the presence of equal amounts of AA and complex 1, insulin was quickly reduced by DTT due to Trx catalysis, and the excess of complex 1 upon irradiation with blue light did not reduce the catalytic activity of Trx significantly. These results reveal that the principal reason that complex 1 inhibits the catalytic activity of Trx upon irradiation is the generation of ROS which induced oxidation of Trx and that the platination of Trx is less important. The inhibition of Trx by complex 1 may further influence the activity of the entire Trx system. In the Trx system, TrxR E

DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry catalyzes the reduction of Trx(S2) to Trx(SH)2 by NADPH, where NADPH is oxidized to NADP+. The oxidation rate of NADPH to NADP+ can be measured by the decrease in absorbance at λ 340 nm.17d,35 To find out if complex 1 can inhibit the activity of the Trx system, TrxR (0.36 unit/mL) and NADPH (200 μM) were added after the irradiation of the mixture of Trx (3 μM) and complex 1 (1 or 8 mol equiv with respect to Trx) at λ 460 nm for 1 h. As shown in Figure 5a, the oxidation rate of NADPH was slower in comparison to that in the absence of 1. In contrast, when the mixture of Trx and 1 was not irradiated with light prior to addition of TrxR and NADPH, no significant change was observed in the oxidation rate of NADPH both in the presence and in the absence of 1 (Figure 5b). These results verify that the photoactivation of 1 reduces the activity of the entire Trx system. Since the Trx system is one of the most important redox systems in cells, the inhibition of the Trx system may further disturb the maintenance of cellular ROS level. With regard to this, the fluorescent probe DCFH-DA was used to determine the ROS level in non-small-cell lung carcinoma A549 cells.20b Before the DCFH-DA assay, the time-dependent cellular uptake of complex 1 in A549 cells and the antiproliferation activity of 1 against A549 were determined. As shown in Figure S7, the amount of Pt accumulated in A549 cells significantly increased along with an increase in incubation time and reached 41.1 ng/106 cells when the cells were exposed to complex 1 for 24 h. Therefore, 24 h was used as a standard incubation time for cells with complex 1 for the ROS assays. The half-maximum antiproliferative concentration (IC50) for 1 against the A549 cell line upon 1 h of irradiation (λ 460 nm) and 23 h of further incubation in the dark was measured to be 21.1 μM. The cytotoxicity of 1 without irradiation was much lower; the IC50 values are over 70 μM, showing a large gap between dark and light cytotoxicity. For the DCFH-DA assay, 21.1 μM of 1 was used to treat the A549 cells according to its half-maximum antiproliferative concentration. After incubation with 1 for 24 h, the cells were irradiated with light of λ 460 nm for a certain time, and DCFHDA was added 40 min before the fluorescence intensity was measured. As shown in Figure S8, a significant increase in the fluorescence intensity for the irradiated cells was observed, in comparison to the cells incubated in the dark or in the absence of 1. The duration of irradiation did not make much difference. ROSup is a standard ROS generator which was used as a positive control. The ROS levels of the cells after the photoactivation of 1 were similar to those for the positive control. The ROS levels of the cells incubated in the absence of 1 upon irradiation with light were similar to those in the dark. These results suggest that the photolysis of 1 could directly raise the level of cellular ROS. Furthermore, in order to examine the disturbance of the cellular ROS balance after the photoactivation of 1 in cells, the ROS level in A549 cells was monitored for up to 48 h. As shown in Figure 6, the ROS level in the cells after the photoactivation of 1 was high and was further increased during the next 2 h. The fluorescence intensities slightly decreased after 5 h but were still similar to that of the positive control. By comparison, the ROS levels for the cells incubated in the absence of 1 were significantly lower. These results suggest that the photoactivation of complex 1 could raise the level of ROS in A549 cells and this effect continues for at least 48 h after irradiation. Cancer cells are vulnerable to the disturbance of

Figure 6. Fluorescence intensity of A549 cells probed by DCFH-DA which indicates the ROS level of the cells. Black columns show the fluorescence intensity of A549 cells incubated with complex 1 for various times following the photoactivation (λ 460 nm, 1 h) of 1. Red columns show the fluorescence intensity of A549 cells incubated without 1 for various times following irradiation with light at λ 460 nm for 1 h. ROSup denotes an ROS-generating agent, and blank denotes a negative control where cells were incubated without 1 and light irradiation.

ROS;36 therefore, the oxidative stress induced cell death may contribute to the anticancer activity. Our studies described earlier demonstrated that the ROS quencher ascorbic acid could largely rescue the activity of Trx. Considering that Cys32 and Cys35, the previously believed catalytic sites of Trx, was fully oxidized upon light irradiation of complex 1 to form disulfide bonds and no Pt binding to the sites was observed, we propose that the mechanism of inhibition of Trx and Trx system by complex 1 is the oxidation of Trx, including oxidation of Trp and Cys residues due to the generation of ROS induced by photolysis of complex 1. Specifically, the Cys32 and Cys35 residues are catalytic sites of Trx, and the formation of disulfide bonds between them as the consequence of oxidation deactivates this enzyme. Hence, the induced formation of disulfide bonds between Cys32 and Cys35 by complex 1 under irradiation is most likely the major reason for inhibition of Trx by complex 1. Tryptophan and methionine residues in proteins are efficient intermediates in biological electron transfer,24,27 and the Trp28, Trp31, and Met37 residues are located close to Cys32 and Cys35 in Trx. Therefore, the oxidation of Trp31, Trp28, and Met37 may contribute to the oxidation of nearby Cys and as a consequence has a minor but non-negligible effect in the activity of Trx. By comparison, as His6, Gln62, Glu85, Gln98, and Glu101 residues are far away from the active center in Trx, we speculate that the platination of these residues does not substantially affect the activity of Trx. The oxidation of proteins has been overlooked in the recent study of the interactions between metal-based anticancer complexes and proteins. Previous work discovered that an organometallic Ru anticancer complex oxidized a free cysteine thiolate to sulfinate.37 Few studies have reported the oxidation of proteins by the ROS generated from metal complexes, except for a recent work showing that the two-photon activation of an Ir anticancer complex induced oxidation of histidine in specific proteins.38 As a matter of fact, anticancer complexes that can induce oxidative damage to the cellular proteins implied that the oxidative stress may play an important role in their anticancer mechanism. F

DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

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flow rate of the LC system was always 0.2 mL min−1. The salt was removed from the tryptic digestion solution by holding the B phase at 3% within the first 2 min, and then the peptides were eluted with a 18 min linear gradient from 3% to 60% of B, with the eluent directly infused into the mass spectrometer. The desolvation temperature was 623 K, and the source temperature was 373 K. The cone voltage was 35 V. Nitrogen was used as desolvation gas with a flow rate of 600 L h−1. The collision energy was set at 10 eV. All spectra were acquired in the range of m/z 200−2000. The “MS-Digest” program (http:// prospector.ucsf.edu) was used to simulate the digestion of Trx to obtain the peptide mass fingerprint of the protein. The parameters were set to multiple charge, one missed cleavage, and variable modifications include oxidation of Cys, Met, and Tyr, and Ptcontaining residues binding on Cys, Met, His, Glu, and Gln. Mass Lynx (ver. 4.0) and Biopharmalynx (ver. 1.3.3) software was used for analyzing the tryptic peptides of Trx and platinated Trx. The mass error is given as δ = [(m/z)obs − (m/z)theor]/(m/z)theor. FT-ICR Mass Spectrometry Analysis. Trx was mixed with complex 1 at a molar ratio of 1:10; after 1 h of irradiation at λ 460 nm, the mixture of Trx and 1 was incubated at 310 K for another 23 h in the dark. The sample was then dialyzed with 50 mM NH4CO3 buffer solution with a D-Tube dialyzer Midi (MWCO 3.5 kDa, Merck Millipore) and was digest with 1/40 trypsin at 310 K for 16 h. Finally, the sample was directly injected into a 9.4 T Bruker Solarix FT-ICRESI mass spectrometer at 60 μM in H2O/MeCN (6/5) with 0.1% formic acid. ESI-MS and MSMS analyses were then performed. For collision-induced dissociation (CID), the parent ions were isolated with the first quadrupole (Q1) and disrupted in the collision cell with a collision energy of 22 eV and then transferred into the ICR for detection. Binding Stoichiometry of Complex 1 and Trx. The recombinant Trx in PBS was mixed with 10 mol equiv of 1. After irradiation with light of λ 460 nm for 1 h, the mixture was incubated at 310 K for another 23 h in the dark. Unbound Pt fragment ions were dialyzed with 10 mM PBS and removed by a 3.5 kDa D-Tube dialyzer Midi. After that, the sample was concentrated to 50 μL with PEG. Half of that sample was used to measure the Trx concentration using the BCA Protein Assay Kit (TIANGEN Biotech (Beijing) Co., Ltd.). The second half was diluted with 1% dilute nitric acid to measure the Pt concentration on an Agilent 7700x ICP-MS instrument. The control sample was prepared by Trx alone following the same procedure above. Three sets of parallel tests were performed, and the results were expressed as mean ± SD. Thioredoxin Activity Assay. The Trx activity assay was performed according to the product information and a previously described method33 with minor modification. An insulin solution (2 mg/mL, pH 7.2) was prepared according to the product specification, and 24 mM DTT was prepared and stored at 253 K. An aliquot of 7.8 μM Trx and different concentrations of 1 in 0.1 M PBS (pH 7.2) were placed in Eppendorf tubes to make nTrx:n1 = 1:5, 1:10, and 1:50, respectively. The mixtures were irradiated with light of λ 460 nm or incubated in the dark for 1 h at 298 K. Then insulin was placed in each tube (final concentration 0.13 mM) and the samples were transferred to quartz cuvettes. The reaction was then initialized by adding DTT (final concentration 0.33 mM) and deionized water to give a final volume of 200 μL in a 1 × 10 mm quartz cuvette and monitored by a UV-2550 UV−vis spectrophotometer (Shimadzu) at λ 650 nm every 0.5 min for 120 min at 310 K. The blank control was carried out without incubation with 1; the ROS scavenger AA (nAA:n1 = 1:1) was also added to repeat the above experiment. Protein Inhibition Assay. This set of experiments was carried out on the basis of the above Trx enzyme activity assays. The concentration of Trx was 3.9 μM, and the molar ratios of Trx to 1 were 1:0, 1:0.01, 1:0.1, 1:1, 1:5, 1:8, 1:10, and 1:15, respectively. The mixture was irradiated with light of λ 460 nm at 298 K for 1 h. Then the buffer was adjusted to pH 7.5, the temperature was changed to 310 K, and insulin was added for the reduction reaction. The absorbance at λ 650 nm was recorded immediately after addition of insulin and then at 20 min. The activity of Trx was defined by the following equation: Trx activity (%) = (Ai − A0)/A0 × 100%, where Ai represents the

CONCLUSIONS In conclusion, the photoactive platinum(IV) anticancer complex trans,trans,trans-[Pt(N3)2(OH)2(Py)2] (1) can interact with Trx upon irradiation with blue light in two ways. First, the photolytic PtII residues bind to amino acid residues such as His, Gln, and Glu. Second, amino acid residues such as Cys, Met, and Trp can be oxidized by the reactive oxygen intermediates generated by the photolysis of 1. Our results indicated that Met and Cys residues are more susceptible to induced oxidation by 1 than to platination by PtII residues and that the induced oxidation of the residues, in particular the catalytic Cys residues, may be the major reason for the inhibition of the activity of Trx and thereby affect the cycling of the Trx system, which further raises the ROS level in cancer cells. This effect persisted for more than 48 h, and the accumulated oxidative stress may be one of the possible reasons that complex 1 has potent anticancer activity.

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EXPERIMENTAL SECTION

Materials. trans,trans,trans-[Pt(N3)2(OH)2(py)2] (1) was synthesized following methods reported in the literature.20a Chemicals were obtained from commercial suppliers and used without further purification. The recombinant Trx from E. coli (T0910), recombinant Trx human (T8690), dithiothreitol (DTT), β-nicotinamide adenine dinucleotide 2′ (NADPH), Trx reductase from rat liver (TrxR), and bovine insulin were purchased from Sigma-Aldrich. Trypsin (sequence grade) was purchased from Promega. DCFH-DA was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Irradiation was carried out with an LED array of 12 blue 5630 SMD LEDs (3 W; λ 460 nm). The HPLC grade acetonitrile, formic acid, and water were obtained from Thermo Fisher. Microcon centrifugal filtration units with a 3 kDa molecular weight cutoff were purchased from Millipore. Aqueous solutions were prepared using Milli-Q water (Milli-Q Reagent Water System). Other synthetic reagents used in the experiment were obtained from Beijing Ouhe Technology Company. C haracteriza tion Dat a f or trans,trans,trans -[P t(N3)2(OH)2(py)2] (1). 1H NMR (D2O, 400 MHz) δ (ppm): 8.80 (dd, 4H, 3JPt−H = 26 Hz, 3JH−H = 6 Hz); 8.28 (t, 2H, 3JH−H = 8 Hz); 7.82 (t, 4H, 3J = 6 Hz). ESI-MS (m/z): found 472.0775 ([M + H]+, C10H13N8O2Pt requires 472.0809). Anal. Calcd for C10H12N8O2Pt: C, 25.48; H, 2.57; N, 23.77. Found: C, 25.41; H, 2.32; N, 23.48. Reaction of Trx with Complex 1. The recombinant Trx from E. coli (400 μM) in PBS (50 mM, pH 7.4) was mixed with 1 at a molar ratio of 1:10 or 1:50. After irradiation with a blue LED light of λ 460 nm for 1 h, the mixture was incubated at 310 K for another 23 h in the dark. Unbound platinum complexes were removed by ultrafiltration three times with a 3 kDa cutoff filter at 7500 rpm for 20 min at room temperature. Then platinated-Trx was washed three times with 200 μL of NH4HCO3. The final concentration of Trx in the reaction mixture was about 40 μM. For the dark control, Trx was mixed with complex 1 at a molar ratio of 1:10 without irradiation but was incubated for 24 h at 310 K in the dark. Free Trx samples were also prepared by following the same procedure described above. Tryptic Digestion of Trx and Trx-1 Adduct. Considering that Trx has only one pair of disulfide bonds, denaturation, disulfide bond reduction, and other pretreatment steps were not involved, and samples were treated by only direct trypsin digestion. The recombinant Trx from E. coli or platinated-Trx (100 μL, 40 μM counted in Trx) was mixed with 1 μg of trypsin (1/40), respectively, and then incubated at 310 K for 16 h for digestion. HPLC-ESI-MS Analysis. Positive-ion electrospray ionization mass spectra were carried out with a Xevo G2 Q-TOF mass spectrometer (Waters) coupled to a Waters ACQUITY UPLC system. The tryptic digests of free Trx and Trx-PtII adducts were separated on a UHPLC XB-C18 column (2.1 × 50 mm, 1.8 μm, Welch). Mobile phases were (A) 95% H2O containing 4.9% acetonitrile and 0.1% formic acid and (B) 95% acetonitrile containing 4.9% H2O and 0.1% formic acid. The G

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Inorganic Chemistry absorbance at 20 min and A0 the absorbance at the beginning. The relative activity of Trx (%) is referenced to the activity of Trx with no complex 1 added. The half-maximum inhibitory concentration (IC50) of 1 against Trx was calculated on the basis of the inhibition curve shown in Figure S6. Inhibition on Trx System. The assay was based on a previously published method17d,35 with minor modifications. The stock solution containing PBS (50 mM, pH 7.2), insulin (100 μM), and NADPH (0.2 mM) was diluted with deionized water to make a final volume of 10 mL. Different concentrations of 1 were added to 3 μM Trx (nTrx:n1 = 1:0, 1:1, 1:8) to give a 25 μL mixture. The Trx-1 mixture was incubated upon irradiation of λ 460 nm or in the dark for 1 h at 298 K. Then 250 μL of the stock solution mentioned above and 0.1 unit TrxR were added to the Trx-1 mixture to initialize the redox reaction. The inhibition potency of complex 1 against this redox cycle was evaluated by measuring the change in absorption of the reaction mixture at 340 nm at 310 K. In Vitro Cytotoxicity Assay. A suspension of cells was placed in 96-well plates at densities of A549 20000 mL−1 and A549/DDP 50000 mL−1 and cultured for 24 h, and then eight different concentrations of complex 1 were added to the corresponding wells. After incubation at 310 K for 48 h, the culture medium was removed, the cells were washed with PBS, and then 100 μL of MTT (3-(4,5-dimethylthiazol-2yl)-2,5-tetrazolium bromide; 0.5 mg/mL) solution was added to each well. The MTT medium was removed carefully after incubation at 310 K for 4 h, 100 μL of DMSO was added to each well to dissolve the purple crystals and the plate was shaken for 10 min. The 96-well plate was then transferred to a microplate reader (SpectraMax M5, Molecular Devices Corporation), and the absorption at 590 nm was recorded. For experiments under irradiation conditions, the 96-well plate was incubated with 1 for 24 h and then exposed to blue light (λ 460 nm) for 1 h and incubated at 310 K for another 23 h, followed by other procedures as described above. Accumulation in A549 Cells. Complex 1 was dissolved in deionized water and filtered to give a 1 mM stock solution. A549 cells were seeded in 12 Corning cellar culture dishes (60 mm × 15 mm) containing 3 mL of growth medium; when the cell coverage reached about 90% and the cell health was good, 3 mL of 21.1 μM 1 was added to nine dishes, and each group containing three dishes was incubated for 1, 6, and 24 h, respectively, at 310 K with 5% CO2 in an incubator. To the remaining three dishes were added the same volume of culture medium without 1 as the control group, and these dishes were also cultured for further 24 h. After incubation for a certain time, the medium was discarded, and the cells were washed three times with PBS and then detached with trypsin. Next, 1 mL of culture medium was added and dishes were shaken until the cells were suspended in the medium evenly. An automated cell counter (Luna Logos Biosystems, Korea) was used to count the number of cells. Another 1 mL of culture medium was added, and the cells were transferred to a 2 mL centrifuge tube and centrifuged for 1 min. The supernatant was removed, and the cells were washed three times with 1 mL of PBS. Cells were then transferred to digestion vials followed by addition of 20% nitric acid and digestion at 200 °C until the samples were completely dried. The solid residue was redissolved in 1% HNO3 and made up to 2 mL. Each sample was filtered through an aqueous-phase filter, and the platinum was determined by ICP-MS. The accumulation of 1 in A549 cells is expressed as the mass (ng) of Pt per 106 cells. Effect on Reactive Oxygen Species Levels in A549 Cells. For the positive control group, A549 cells were seeded in Corning Costar 3603 black-walled 96-well plates (5 × 103 cells/well). After 24 h, the culture medium was discarded and washed with PBS. An aliquot (100 μL) of serum-free medium with positive reagent ROSup was added to each well, and the plate was incubated at 310 K for 1 h. Then, the excessed ROSup was removed and washed with PBS, and 20 μM DCFH-DA was added to each well followed by incubating for 40 min in the dark. The medium was removed, and the cells were washed thoroughly with PBS before measuring the fluorescence of each well on a Synergy H1m fluorescence spectrometer with λex 488 nm and λem 525 nm. The negative (blank) control group was carried out using

only A549 cells without add ROSup or 1, and the other procedures are the same as above. For the experimental groups, A549 cells were incubated as described above. A 21.1 μM amount of complex 1 in culture medium was added, and the samples were incubated for 24 h in the dark. The plate was irradiated with blue light for different times (10, 20, 40, and 60 min). Another experimental group was carried out with irradiation of blue light (λ 460 nm) for 1 h and then incubation for different times (0, 1, 2, 5, 24, 48 h) in the dark. Then each well was washed with PBS, and 20 μM DCFH-DA was added followed by incubation for 40 min. The following steps are the same as those described above. The procedures of the experimental control group were the same as those for the experimental groups except that complex 1 was not added.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b00529. Tables S1−S4 and Figures S1−S8 as described in the text (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for Y.Z.: [email protected]. *E-mail for F.W.: [email protected]. ORCID

Yao Zhao: 0000-0003-0613-8708 Ming Wang: 0000-0002-2783-9426 Fuyi Wang: 0000-0003-0962-1260 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 21301181, 21371006, 21790390, 21790392, 21575145, 21635008, and 21621062). Y.Z. also thanks the Youth Innovation Promotion Association of CAS (No. 2017051).

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ABBREVIATIONS Trx,thioredoxin; TrxR,thioredoxin reductase; UVA,ultraviolet A type, λmax 365 nm; PBS,phosphate buffer saline; ROS,reactive oxygen species; NADPH,β-nicotinamide adenine dinucleotide 2′; AA,L-ascorbic acid; RF,resistance factor; ICP-MS,inductively coupled plasma mass spectrometry; DTT,dithiothreitol; py,pyridine; ESI-MS,electrospray ionization mass spectrometry; FT-ICR-MS,Fourier-transform ion-cyclotron resonance mass spectrometry; PDB,Protein Data Bank; SASAs,solvent accessible surface areas; DCFH-DA,dichlorofluorescin diacetate; DTT,dithiothreitol; DODT,3,6-dioxa-1,8-octanedithiol; dach, (1R,2R)-(−)-1,2-diaminocyclohexane

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REFERENCES

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DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.8b00529 Inorg. Chem. XXXX, XXX, XXX−XXX