Anticancer and Antiangiogenic Iron(II) Complexes That Target

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Anticancer and antiangiogenic iron(II) complexes that target thioredoxin reductase to trigger cancer cell apoptosis Lina Xie, Zuandi Luo, Zhennan Zhao, and Tianfeng Chen J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00917 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Journal of Medicinal Chemistry

Anticancer and antiangiogenic iron(II) complexes that target thioredoxin reductase to trigger cancer cell apoptosis Lina Xie, Zuandi Luo, Zhennan Zhao, Tianfeng Chen*

Department of Chemistry, Jinan University, Guangzhou 510632, China

*Corresponding author

KEYWORDS: Iron complex • TrxR • apoptosis • anticancer • antiangiogenesis

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ABSTRACT: Thioredoxin reductase (TrxR) is a selenoenzyme that could regulate intracellular oxidative balance and found to be overexpressed in many human tumor cells. Due to its important role in cancer progression, TrxR is becoming an attractive target in chemotherapeutic drug design. In this study, a new class of Fe(II) complexes with phenanthroline derivatives as ligands were synthesized and characterized. The mechanism of cell death induced by complex 3 revealed that the growth of cancer cells was suppressed by apoptosis and specifically inhibited the activities of TrxR. Furthermore, complex 3 exhibited brilliant antiangiogenic activity against HUVEC cells and inhibited cell migration and invasion. In addition, results of hematological analysis and H&E staining demonstrated that complex 3 has negligible toxicity on function of the major organs of mice. Taken together, this study provides a strategy for drug design to exploit Fe-based phenanthroline derivative as chemotherapeutic agent in cancer treatment.


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INTRODUCTION As powerful σ donor ligands, phenanthroline derivatives could enable the formation of stable M-N bond(s) which could stabilize central mental ions against precipitation into metal aggregates in physiological environment, while maintaining the appreciate strength to carry bioactive metal ions to cellular target.1-4 Numerous reports have reported that metal compounds with phenanthroline derivatives exhibited potent antitumor activities against different cancer cells and have been recognized as potential candidates of chemotherapeutic agents in cancer treatment.5-10 Iron is an essential element for the human body and enzymes containing iron could catalyze organic oxidation reactions.11-14 In fact, a number of synthetic and natural iron complexes exhibiting potent anticancer activities have been reported. For instance, bleomycin which was used in the treatment of testicular carcinoma showed high cure rates.15,16 Moreover, our previous studies disclosed that hydrophilic Fe(II) complexes passed through cancer cell membrane by transferrin receptor (TfR)-mediated endocytosis and exhibited strong anticancer efficacy.17 Recent studies revealed that thioredoxin reductase (TrxR) plays an important role in regulating the redox balance and intracellular signaling pathways, which made it a potential target for cancer treatment.18-22 For instance, alkynyl phosphine gold complex displays strong inhibitory effect on TrxR activities and proliferation of cancer cells. Curcumin, daily consumed by millions of people, exhibited potent irreversible inhibition on TrxR activity in a dose- and time-dependent manner.23,24 Though a great number of complexes (mostly gold compounds) have been reported to exhibit TrxR inhibition 3 ACS Paragon Plus Environment


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and anti-proliferative properties, iron complexes were rarely taken in consideration in this area. 25-28 Frontier researches have demonstrated that the biological response with TrxR was mainly depend on the chelate ability of the ligand on the complex, causing different rate of ligand-exchange of complex.29 However, high binding affinity of Fe(II/III) complexes with thiol group may result their easy binding with blood thiols, including glutathione (GSH) and albumin (BSA), which limited its bioavailability to cancer cells.30-32 Therefore, minimize the binding rate between blood thiols and Fe(II/III) complexes but maintaining its inhibition activity to TrxR is important in design of new anticancer complexes. The strong M-N coordinate bonds between central metal and phenanthroline derivatives could stabilize the bivalent iron under the physiological conditions and then deliver bioactive metal ion to cellular TrxR. Angiogenesis plays a fundamental role in carcinogenesis, metastasis and cancer progress.33-35 Vessels, connecting to tumors, could not only deliver essential nutrients and oxygen, but also facilitate the dormant small cancer into metastasis and invasive form.36,37 Recently, studies have proposed that anti-angiogenesis was an efficient strategy to inhibit tumor growth and metastasis.38,39 Moreover,

several metal-based

drugs which have been developed lately shown antiangiogenic efficacy in vitro and in vivo.40,41 In this study, we have synthesized a series of Fe(II) complexes and elucidated the relationship between their chemical structure and anticancer efficacy. The results showed that, the introduction of methoxy group to the Fe(II) complex significantly enhanced the selectivity between the cancer cells and normal cells. The underlying mechanisms revealed that different cytotoxicity of complex 3 may be 4 ACS Paragon Plus Environment

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caused by different cellular uptake and inhibition of TrxR activity. Interestingly, complex 3 also exhibited brilliant antiangiogenic activity. Moreover, results of hematological analysis and H&E staining suggested that complex 3 has little impact on the function of main organs in mice. Taken together, this study demonstrates that Fe(II) complex with proper ligands could be promising chemotherapeutic agent for cancer treatment.

RESULTS AND DISCUSSION Synthesis and anticancer activity evaluation of Fe(II) phenanthroline derivative complexes In this work, a new class of Fe(II) complexes with phenanthroline derivative as ligand, which was abbreviated as [Fe(II)(pip)3]2+ (1), [Fe(II)(pip-CH3)3]2+ (2), [Fe(II)(pip-OCH3)3]2+ (3), [Fe(II)(pip-NO2)3]2+ (4), [Fe(II)(pip-COOH)3]2+ (5), [Fe(II)(pip-OH)3]2+ (6), were successfully synthesized and characterized by ESI-MS, 1

H NMR and element analysis (see the experiment section and supporting information

for details ).

Their structures were illustrated in Scheme 1. The experimental and

theoretical results revealed that properties of metal complexes was closely related with the coordination ligand, such as changing the substituent groups or even its relative position on the ligand will cause great properties variation of the complexes.22 The cytotoxicity of Fe(II) complexes toward human cervical cancer cell lines (Caski, SiHa and HeLa), glioma cancer cell lines (U87 and C-6) and normal cell lines (Chem-5 and L02) were examined, cisplatin was used as positive control drug. As 5 ACS Paragon Plus Environment


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shown in Table 1, as the electron withdrawal capability of substituent on the ligand increasing, the cytotoxicity of Fe(II) complexes toward cancer cell lines increased correspondingly. For example, complex 4 showed higher cytotoxicity than complex 5. While by enhancing the electron donating ability substituent on the ligand, the cytotoxicity of complexes did not increase (IC50 of complex 6 was higher than complex 1). In general, complex 1, 2 and 4 showed high cytotoxicity toward all the cell lines tested in this study but low selectivity between cancer and normal cells. Worth mentioning, complex 3 showed high sensitivity toward Caski cell but low cytotoxicity toward other cervical cancer cell lines and normal cells. The IC50 value of complex 3 toward Caski cells was 0.75 µM, and 23.71 µM for L02 cells. Therefore, the safer index of complex 3 was calculated at 31.61, much higher than that of cisplatin (as shown in Figure. S1). The reason why complex 3 displayed the highest selectivity among the different cervical cancer cell lines was explored in the following study. It is well known that lipophilicity of a chemotherapy agent has a vital influence on its cytotoxicity. Thus, the lipophilicity of the synthesized Fe(II) complexes was evaluated. As indicated in Figure. 1, the complex with high lipophilicity exhibited certain anticancer effect toward Caski cells. Complex 3 possess high lipophilicity (Log P = 1.02) and low IC50 value (0.75 µM) in Caski cells. Complex with higher lipophilicity may more easily cross cell membrane and lead to more cellular uptake than other complex with lower lipophilicity. As mentioned above, complex 3 displayed the highest inhibition on Caski cells growth among three cervical cancer 6 ACS Paragon Plus Environment

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carcinomas. Therefore, the anti-proliferative mechanism and the cellular uptake of complex 3 in the different cervical cancer cells were determined. As shown in Figure. 2B, the cellular uptake of complex 3 in Caski cells was the highest compared to that in HeLa and SiHa cells at the same conditions, which could be the reason leading to its higher cytotoxicity towards Caski cells. Our previous studies showed that hydrophilic Fe(II) complexes can pass through cancer cell membrane by transferrin receptor (TfR)-mediated endocytosis and displayed anticancer efficacy. To figure out the underlying mechanism that complex 3 has high selectivity to different cells, the TfR expression in three cervical cancer cells was determined. The results exhibited that TfR expression is at the higher level in Caski and SiHa cells than that in HeLa cells (Figure. 2C). This trend was consistent with the cytotoxicity of the complexes against the tested cell lines. Moreover, the three cervical cancer cells were pre-treated with TfR antibody (anti-TfR), and then incubated with complex 3 to evaluate the effects of anti-TfR on the cellular uptake. The results (Figure. 2D) indicated that, the cellular uptake in Caski and SiHa decreased significantly compared with the cellular uptake in HeLa, which is in consistent with our hypothesis.

Fe(II) complex inhibit cancer cells proliferation by targeting TrxR. A number of studies indicated that metal complexes could strongly inhibit cancer cell growth by targeting TrxR.42 Au complexes have found to suppress TrxR expression , leading to inhibiting tumor growth and angiogenesis.43 Our previous studies revealed that phenanthroline Ru(II) compounds could target TrxR and induce reactive oxygen species (ROS) overproduction which accounted for the DNA damage and apoptosis.22 7 ACS Paragon Plus Environment


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Therefore, we are motivated to investigate whether those Fe(II) complexes could also act as TrxR inhibitors. It was reported that the interaction between gold complexes with the active site of TrxR was highly related to ligand-exchange processes between cysteine/selenocysteine residues of enzyme and the coordinated ligands at gold center.23 Thus, the binding mode between complex 3 and TrxR peptide (AGUVGAGLIK) was determined by MALDI-TOF-MS experiment to monitor the chemical reaction on molecular level. As indicated in Figure. 3, the molecular weight of the individual TrxR peptide is m/z 1056.377, which protected with 1-methoxy-4-methylbenzyl substituent ground. The signal of TrxR containing selenopeptide nearly vanished after incubated with complex 3 for 12 h and a new peak at m/z 535.04 was found in the MALDI-TOF-MS spectra (Figure. 3c). The m/z value of this new adduct is the same as a naked iron atom attached to the model peptide. These results indicate complex 3 could interact with TrxR (as a ligand) in ligand-exchange pathway then inhibit cancer cells proliferation. Generally, inhibition of TrxR activity requires a moderate ligand-exchange reaction with thiols. The high thiol binding affinity of Fe(II) complexes may result in binding interactions with blood thiols including BSA and GSH, which limit the bioavailability to tumor tissues. Therefore, we examined the stability of complex 3 towards GSH and BSA. UV-vis spectrum was used to detect the interaction between complex 3 and biomolecules according to recent study.16 As indicated in Figure. 4B, there was no obvious change in the characteristic absorption of complex 3 after incubated with excessive GSH or BSA for 48 h, which strongly supported that complex 3 was inert to the blood thiols. Furthermore, analysis of the aqueous solutions of complex 3 and excessive GSH by ESI-MS, the signals could be assigned to the adduct for GSH and iron atom was not found (Figure. 4A and 4C). Complex 3 did not react with BSA either under the same 8 ACS Paragon Plus Environment

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condition, since the adduct signals did not show on Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) (Figure. 5A and 5B,). Encouraged by these results, the inhibition of complex 3 on TrxR activities of different cervical cancer cells was further examined through TrxR assay kit. As shown in Figure. 6A, the TrxR activity of Caski, SiHa and HeLa cells decreased to 48.92 %, 84.51% and 86.01% respectively after its treatment with complex 3, which was consistent with the cytotoxic effects of complex 3 to these cervical cancer cells. The expressions of Trx and TrxR in three cervical cancer cell lines treated with complex 3 were also examined by Western blotting. As shown in Figure. 6B, complex 3 downregulated the expressions of TrxR in Caski cells significantly, but the expressions of TrxR in the SiHa cells was upregulated dramatically and increased slightly in HeLa cells. For treatment with complex 3, the expression level of Trx was slightly decreased in SiHa cells, in contrast with the significant increase in Caski and HeLa cells. These results revealed that the effective regulation on the expressions of TrxR and Trx induced by complex 3 may contribute to highest anticancer activity in Caski cells. . The normal physiological function of TrxR is mainly due to the active sites of cysteine on the 58/63 sites from N-terminal, and the cysteine and selenocysteine on the 497/498 sites from C-terminal. The cysteine and selenocysteine on the end of C-terminal arm can flexibly swings are to work as electron carriers and can be recognized as target sites to develop anticancer drugs. Meanwhile, the active site of Trx is an open and highly preserved with two cysteine residues.20 Owing to the difference of active site between the TrxR and Trx, complex 3 may exhibit different inhibitory activity toward the TrxR and Trx. Therefore, the obtained results in this study demonstrate the potency of Fe(II) complexes as TrxR inhibitors. Taken together, it could be concluded that complex 3 could selectively inhibit the activity of TrxR. 9 ACS Paragon Plus Environment


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Down-regulation of intracellular ROS generation by complex 3. The imbalance of redox system has been considered as the principal action mechanism for the chemotherapeutic drugs to initiate apoptosis in cancer cells.10,44-50. We set out to determine the ROS generation in cervical cancer cells after incubation with complex 3 by using dihydroethidium (DHE) as fluorescent probe. As shown in the Figure. 7A, the treatment of complex 3 dramatically down-regulated the generation of ROS in three cervical cancer cells. To further verify this result, fluorescence confocal microscopy imaging was carried out to visualize the ROS level in Caski cells. As demonstrated in Figure. 7C, the fluorescent intensity of DHE was decreased in 10 min after treatment with complex 3, and there was no obvious change observed by 120 min of treatment, which indicated that complex 3 could be induced imbalance of redox system in Caski cells. Furthermore, the scavenging capability of complex 3 was evaluated by ABTS+ free radical scavenging assay. As shown in Figure. 7B, the treatment of complex 3 decreased the absorbance of ABTS with the wavelength at 734 nm, indicating complex 3 has the strong scavenging capability of free radicals. This result revealed that, the down-regulation of intracellular ROS in Caski cells was caused by the strong scavenging capability of complex 3.

Cancer cell apoptosis induced by Fe(II) complexes. Apoptosis and cell cycle arrest in the cells are two main reasons accounting for the inhibition of cell growth.50 Our previous study indicated Fe(II) complexes containing phenantdihroline 10 ACS Paragon Plus Environment

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effectively inhibited the proliferation of cancer cells through induction of apoptosis.41 Herein, flow cytometric analysis was performed to understand the death mechanisms of the as-synthesized Fe(II) complexes in three cervical cancer cells. The results showed that, the treatment of different complexes caused apoptosis at different levels, as evidenced by the different percentages of sub-G1 population and the morphological changes in Caski cells (Figure. 8 and Figure. S2). These results suggested that Fe(II) complexes inhibited the growth of Caski cells by the induction of apoptosis. Meanwhile, the cell cycle distribution in SiHa, Caski and HeLa cells was determined after treatment of complex 3. As demonstrated in Figure. 9A and 9B, treatment of complex 3 significantly increased the apoptotic level in Caski cells in a dose-dependent manner, as we can see the increased percentage of sub-G1 peaks. Compared to the Caski cells, no obvious variations on cell cycle distribution and cellular morphology were observed in SiHa and HeLa cells after treatment of complex 3 (Figure. S3). Furthermore, the expression level of p-Histone that was associated with DNA damage was examined by Western blotting after treatment of complex 3. As shown in Figure. 9C, treatment of complex 3 significantly increased the expression level of p-Histone in Caski and SiHa cells, but decreased in HeLa cells at the same concentration. These results indicate that, DNA damage was involved in the complex 3-induced apoptosis in Caski and SiHa cells, but not in HeLa cells.

Fe(II) complexes inhibit cell migration, invasion and angiogenesis. Metastasis and invasion are important incidences in later period of cancer progression. Therefore, 11 ACS Paragon Plus Environment


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the inhibition of metastasis and invasion is critical for efficient cancer treatment. Herein, wound healing assay and transwell assay were introduced to evaluate the inhibition effect of complex 3 on cell migration and invasion. As shown in Figure. 10, complex 3 at sub-toxic concentrations strongly suppressed the migration and invasion of HUVECs after 24 h. For instance, the migration and invasion extent of HUVEC cells was reduced to 8.33% and 29.6% compared to control. Furthermore, the antiangiogenic effect of complex 3 was evaluated in this study. Chick embryo chorioallantoic membranes (CAM), which have plenty of branching of vessels and fairly sensitive to antiangiogenesis drugs, was suitable to investigate drug’s antiangiogenesis capability. As illustrated in Figure. 11A, the CAM neovascularization was significantly suppressed by complex 3 as the concentration was raised up to 10 µM. However, CAM exposed to cisplatin exerted inconspicuous antiangiogenic effect. VEGF is vascular endothelial growth factor, promoting the angiogenesis process.51 In this study, we demonstrated that the expression level of VEGF in HUVEC cells decreased with treatment of complex 3 in a dose-dependent manner (Figure. 11B), which suggested that Fe(II) complexes inhibited the angiogenesis by suppressing the expression of VEGF. Besides, numerous studies have reported that the antiangiogenic activity is highly relate to the expression of TrxR,52,53 and we have demonstrated that complex 3 effectively targeted TrxR and triggered apoptosis in Caski cells. Therefore, we supposed that complex 3 exerted strong antiangiogenic ability through inhibiting the expression of VEGF and TrxR. Next the hematological analysis was performed to evaluate the systemic 12 ACS Paragon Plus Environment

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cytotoxicity of complex 3 in vivo. As shown in Figure. 12, the results of hematological analysis revealed that the mice treated with complex 3 showed no significant impact on the function of liver, kidney and blood lipid, as reflected by the biochemical indexes of albumin (ALB), creatinine (CREA), glutamic acid (GLU), triglycerides (TG), low density lipoprotein cholesterol (LDLC) and cholesterol (CHOL). Furthermore, the results of H&E staining showed that no pathological changes were observed in the heart, liver, spleen, lungs and kidney in mice after treated with complex 3 for 72 h (Figure. 13), suggesting the negligible toxicity of complex 3 in vivo.

CONCLUSIONS In summary, we have rationally designed and synthesized a series of Fe(II) complexes with phenanthroline derivatives as ligand. The mechanism of cell death induced by complex 3 revealed that complex 3 suppressed the growth of cancer cells by apoptosis and specifically inhibited the activities of TrxR. Furthermore, complex 3 exhibited antiangiogenic activity and inhibited the cell migration and invasion against HUVEC cells. Moreover, results of hematological analysis and H&E staining demonstrated that complex 3 has negligible toxicity on function of the major organs of mice. Therefore, this study provides a strategy for drug design to exploit Fe-based phenanthroline derivative as chemotherapeutic agent in cancer treatment.

EXPERIMENTAL SECTION Chemicals and Reagents. (NH4)2Fe(SO4)2 , cisplatin, propidium iodide (PI), 13 ACS Paragon Plus Environment


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Dihydroethidium (DHE), Thiazolyl blue tetrazolium bromide (MTT) and BCA assay kit were purchased from Sigma-Aldrich. TrxR Activity Kit was purchased from Cayman Chemical. All used antibodies were brought from Cell Signaling Technology (Beverly, MA, USA). Fe(II) complexes used in this study were dissolved in DMSO. All organic solvents were analytical grade except otherwise stated. Elemental analysis were obtained on an EA2400II CHNS/O elemental analysis (USA), mass spectra were obtained on an ABI4000 Q TRAP liquid chromatography-mass spectrometer (ABI, USA). Column chromatography was performed by using alumina column chromatography. 1H NMR spectra were recorded on a Varian Unity Inova (400 MHz). The purity of the final products was >95% as determined and confirmed by HPLC, elemental analysis and 1H NMR. Synthesis of the ligands. The ligands 2-phenylimidazo[4,5-f][1,10]phenanthroline (pip), 2-(4-methylphenyl)imidazo[4,5-f][1,10]phenanthroline (pip-CH3), 2-(4-methoxyphenyl)imidazo[4,5-f][1,10]phenanthroline

(pip-OCH3),

2-(4-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline (pip-NO2), 2-(4- carboxyphenyl) imidazo[4,5-f][1,10]phenanthroline (pip-COOH) and 2-(4-hydroxyphenyl) imidazo[4,5-f][1,10]phenanthroline (pip-OH) were synthesized according to the reported procedures.54 Synthesis of the Fe(II) complexes. Complex 1 has been reported by our previous study, 17and complexes 2-6 were synthesized by using the following general procedure. (NH4)2Fe(SO4)2 (0.392g, 0.1 mM) aqueous solution (1 mL)was added dropwise to the 14 ACS Paragon Plus Environment

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ethanol solution (40 mL) of the ligand . (L1, 0.89 g; L2, 0.93 g; L3, 0.98 g; L4, 1.02 g; L5, 0.94 g; L6, 1.02 g; 3mM). The mixture was stirred for 3 h, until the color of mixture didn’t change. The solution was filtered and dried in vacuum. Finally, the crude products were further purified through alumina column chromatography with methanol and acetonitrile as eluent. [Fe(II)(pip-CH3)3]SO4 (2).

Yield: 42.6%. ESI-MS: m/z 493.4 [M-SO42-]2+.

Elemental analysis calc (%) for C60H42N12O4SFe: C, 66.77; H, 3.91; N, 15.52; found (%): C, 66.70; H, 3.81; N, 15.48. UV-vis ((nm), ε/104 (M-1 cm-1): 288 (0.80), 534 (0.16). IR (KBr): 3074 (N-H), 1610, 1460 (C = Carom) cm-1. 1H NMR (DMSO-d6,δ): 8.98 (d, 6H), 8.26 (s, 6H), 7.96 (s, 3H), 7.66 (m, 6H), 7.33 (d, 6H), 2.38 (s, 9H). [Fe(II)(pip-OCH3)3]SO4 (3). Yield 50.2%. ESI-MS: m/z 517.6 [M-SO42-)]2+. Elemental analysis calc (%) for C60H42N12O7SFe: C, 66.54; H, 3.91; N, 15.52; found (%): C, 66.43; H, 3.98; N, 15.39. UV-vis ((nm), ε/104(M-1 cm-1): 292 (0.76), 533(0.17). IR (KBr): 3453 (N-H), 1465, 1376 (C = Carom) cm-1. 1H NMR (DMSO-d6, δ): 9.32 (s, 3H), 9.08 (d, 3H), 8.41 (d, 6H), 7.75 (m, 12H), 7.25 (m, 12H), 3.88 (s, 9H). [Fe(II)(pip-NO2)3]SO4 (4). Yield 37.5%. ESI-MS: m/z 540.0 [M-SO42-)]2+. Elemental analysis calc (%) for C57H33N15O10SFe: C, 58.22; H, 2.83; N, 17.87; found (%): C, 58.20; H, 2.87; N, 17.85. UV-vis ((nm), ε/104(M-1 cm-1): 291 (0.76), 541 (0.29). IR (KBr): 3440 (N-H), 1545, 1386 (C = Carom) cm-1. 1H NMR (DMSO-d6, δ): 9.20(s, 6H), 8.68 (d, 6H), 8.45 (d, 6H), 7.75 (d, 12H). [Fe(II)(pip-COOH)3]SO4 (5). Yield 39.2%. ESI-MS: m/z 538.5 [M-SO42-)]2+. Elemental analysis calc (%) for C60H36N12O10SFe: C, 61.44; H, 3.09; N, 14.33; found 15 ACS Paragon Plus Environment


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(%): C, 61.36; H, 3.19; N, 14.31. UV-vis ((nm), ε/104(M-1 cm-1): 287 (0.98), 534 (0.06). IR (KBr): 3440 (N-H), 1545, 1386 (C = Carom) cm-1. 1H NMR (DMSO-d6, δ): 9.16 (s, 6H), 8.48 (d, 6H), 8.08 (d, 6H), 7.48 (m, 12H), 7.72 (s, 6H). [Fe(II)(pip-OH)3]SO4 (6). Yield 36.5%. ESI-MS: m/z 96.6[M-SO42-)]2+. Elemental analysis calc (%) for C57H36N12O7SFe: C, 62.87; H, 3.33; N, 15.44; found (%): C, 62.90; H, 3.35; N, 15.45. UV-vis ((nm), ε/104(M-1 cm-1): 296 (0.93), 540 (0.15). IR (KBr): 3409 (N-H), 1576, 1384 (C = Carom) cm-1. 1H NMR (DMSO-d6, δ): 11.72 (s, 3H), 9.02 (s, 6H), 8.25 (s, 6H), 7.75 (m, 12H), 7.02 (s, 6H). Measurement of lipophilicity. Log P was used to evaluate the concentrations distributed in the n-octanol phase and aqueous phases. Cell culture and MTT assay. Cancer cell lines used in this study were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The normal human liver cell lines (L02) was purchased from Nanjing Key GEN Biotech. All the cell lines in this work were maintained in DMEM medium supplemented streptomycin (50 units/ml), penicillin (100 units/ml) and fetal bovine serum (10%) at 37℃ in humidified incubator with 5% CO2 atmosphere. The anticancer activity induced by Fe(II) complexes (1-6) was analyzed by MTT assay after 72 h. Intracellular uptake of complex 3. Caski, SiHa and HeLa cells (cells/ml, 6 ml) were planted in 6 cm dishes and adhered for a period of times. The cells were then administrated with complexes 3 at 20 µM. Then shook up the medium with complex 3 and got supernatant 100 µl accurately in 96 plate for 0, 1, 2, 4, 6, 8, 10 h respectively. The content of unabsorbed Fe(II) complex was determined by UV-vis analysis at 16 ACS Paragon Plus Environment

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maximum absorption wavelength respectively. Transferrin competing assay. Intracellular uptake of complex 3 and the transferrin competing assay were performed as previous reported.55 Briefly, three cervical cancer cells were plated in 96-well plates and adhered for 24 h. The medium were removed and the cells incubated with medium with excess transferrin for 2 h. After that the cells were incubated with complex 3 with concentration of 20 µM. After that, the medium were removed and the cells were washed three times with PBS. 100 µL of 0.1% Triton X-100 in 0.1 N NaOH solution was added to lyse the cells and read the UV-vis absorption of complex 3. The cellular uptake was calculated followed the standard curve. ABTS assay. The ABTS liquid was prepared and have the absorption peak at 734 nm followed the instructions. Different concentrations of complex 3 were added to the ABTS liquid and the mixture were determined the absorption intensity at different times. The radical scavenging capability of complex 3 was expressed as the percentage of the absorption intensity of the tested wells to the absorption intensity of the positive control wells. Evaluation of TrxR inhibition. The cellular proteins of Caski, SiHa and HeLa treated with complex 3 were extracted and conducted the BCA assay to determine the concentration. The inhibition on TrxR activities in three cervical cancer cells lines induced by complex 3 were examined by TrxR assay kit (Cayman) referred to the program of manufacturer's instruction. Binding studies between complexes 3 and TrxR model peptide. Complex 3 (1 17 ACS Paragon Plus Environment


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µM) was added to the solution containing TrxR model peptide (AGUVGAGLIK) with the ratio of 1:1. Then MALDI-TOF-MS assay was conducted to examine the reaction between complex 3 and TrxR peptide after 12 h. The stability analysis of complex 3 toward thiols in blood. ESI-MS was used to investigate the reaction between complex 3 and GSH. Mixtures of 10 µM of complex 3 with 50-fold excess of GSH (0.5 mM) in freshly prepared aqueous (VCH3OH:VH2O=3:7) was analyzed by ESI-MS after 48 h. Similarly, the interaction between complex 3 and BSA was investigated by ESI-QTOF-MS and the ions intensity was analyzed after 48 h. Besides, UV-vis was also used to detect the reaction between the complex 3 and blood thiols and the experiment condition was same as above. Western blotting. The expressions of TrxR, Trx and TfR in three cervical cancer cells treated with or without complex 3 were determined by western blotting. The expressions of VEGF in HUVEC treated with different concentration complex 3 were determined by western blotting. Determination of intracellular ROS generation. The ROS generation levels in cervical cancer carcinoma cells induced by complex 3 were determined by DHE assay. ROS level was determined by fluorescence intensity through Tecan SAFIRE fluorescence reader. Relative DHE fluorescence intensity of cells treated with complex 3 was indicated as percentage of control. DHE assay was conducted as follow. Three cervical cancer cells were planted in plate which pre-incubated with DHE at 10 µM and then treated with complex 3, the fluorescence intensity was tested 18 ACS Paragon Plus Environment

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under a fluorescence microscope. Flow Cytometric Analysis. Flow cytometry assay was used to analyze the cell cycle distribution resulted in Fe(II) complexes. Cell cycle distribution of three cervical cancer cells was determined by Multi Cycle software (Phoenix Flow Systems, San Diego, CA) and the contents of apoptotic cells underwent hypodiploid DNA were measured by calculating the sub-G1 peak in the cell cycle distribution pattern. 10,000 events per sample for each experiment were recorded. Migration and Invasion assay. The migration and invasion effects of complex 3 on HUVECs were detected by wound-heal assay and Tranwell assay. The extent of wound heal was observed after 24 h by imaging with fluorescence microscope. In the invasion study, the non-invaded cells from the upper face of the Transwell membrane were removed and invaded cells were fixed and then stained with Giemsa solution. The invaded cells were calculated by manual counting in three experiments. Chorioallantoic membrane assay. The anti-angiogenesis effect of complex 3 was tested by CAM assay. Fertilized chicken eggs were incubated at 37 ℃ with humid atmosphere. Eggs were cracked open after embryonic day-5 and then various concentrations of complex 3 and cisplatin were slightly dropwise added to top of chicken CAM. The embryos were incubated for another 48 h. The CAM was observed under a camera and photographed. Acute toxicity experiments. Mice used in this study were purchased by Shanghai Super-B&K Laboratory Animal Corp. Ltd. The mice (body weight 35-40 g) were injected with saline, 2 mg/kg and 4 mg/kg of complex 3. The heart, liver, spleen, lung 19 ACS Paragon Plus Environment


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and kidney of the mice were taken out for H&E staining after 72 h. All animal experiments were approved by the Animal Experimentation Ethics Committee of Jinan University.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the Internet at http://pubs.acs.org. Safer index of Fe(II) complexes. Changes in the morphology of Caski cells exposed to different complexes for 72 h. Changes in the morphology of Caski, SiHa and HeLa cells exposed to different concentrations of complex 3 for 72 h. FT-IR spectra in KBr of complexes 2-6. UV-vis absorption spectra of complexes 2-6 in Tris-HCl (pH=7.2). ESI-MS spectra of complexes 2-6. 1H NMR spectra of complexes 2-6.

AUTHOR INFORMATION Corresponding Author

*E-mail: Tianfeng Chen, [email protected]. Phone: +86 20-85225962.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGENT This work was supported by Natural Science Foundation of China (21271002, 21371076), National High-level personnel of special support program, National High Technology Research and Development Program of China (SS2014AA020538), Science Foundation for Distinguished Young Scholars of Guangdong Province (S2013050014667), YangFan Innovative & Entepreneurial Research Team Project (201312H05), Guangdong Special Support Program and Guangdong Frontier Key Technological Innovation Special Funds (2014B050505012) and Fundamental Research Funds for the Central Universities.

ABBREVIATIONS USED TrxR, thioredoxin Reductase; Trx, thioredoxin; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; TfR, transferrin receptor; BSA, bovine serum albumin; GSH, glutathione; MTT, blue tetrazoline; DHE, dihydroethidium; PI, Propidium Iodide; ALB, albumin; CREA, creatinine; TG, triglyceride; LDLC, low density lipoprotein; GLU, glucose; CHOL, total cholesterol.

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Scheme 1. Structure of Fe(II) complexes in this study.


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Figure 1. Lipo-hydro partition coefficients and cytotoxicities of Fe(II) complexes. (A) The lipo-hydro distributions of as-synthesized Fe(II) complexes in H2O and n-octanol. (B) The cytotoxicities of Fe(II) complexes against Caski cells with different compounds’ lipophilicity. Values expressed are means ± SD of triplicates.



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Figure 2. Cellular uptake of complex 3 in different cervical cancer cells. (A) Scheme of cellular uptake of complex 3 in different cervical cancer cells. (B) The cellular uptake of complex 3 in cervical cancer cells. The cells were treated with 20 µM of Fe(II) complexes respectively. (C) The expression level of TfR in different cervical cancer cells. β-actin was used as loading control. (D) The cellular uptake of complex 3 pre-treated with anti-TfR in different cervical cancer cells. Significant difference between treatment and control groups is indicated at P< 0.05 (*) or P < 0.01(**) levels.


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a TrxR

1400

a

1200 1000 800 600 400 200 0

1052 200

1054

1056

1058

525

526

527

1060

b

150

b

Complex 3

100 50 0 524 40

528

c

30

3+TrxR

20 10

c 600

750

900

1050

0 533 1200

534

535

536

537

538

Figure 3. Interaction between TrxR and [Fe(II)(pip-OCH3)3]SO4. (a) The MALDI-TOF-MS analysis of TrxR in Milli-Q water. (b) The MALDI-TOF-MS analysis of complex 3 in Milii-Q water. (c) The MALDI-TOF-MS analysis of TrxR treated with complex 3 after 12 h.



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A B

120 110

2.4

GSH BSA Complex 3 GSH+Complex 3 BSA+Complex 3

1.8

308.2

100

1.2

90 0.6

80

517.4

0.0

70

330

C

440

550

660

60

Belonging to

50

[GSH+H]+ [GSH+Na]+ [GSH+2Na-H]+ [GSH+3Na-2H]+ [Fe(pip-OCH3)3 ]2+ [2GSH+H]+ [2GSH+Na]+ [2GSH+K]+ [2GSH+3Na-2H]+ [Fe(pip-OCH3)2 –H]+ [Fe(pip-OCH3 )2+K+CH3OH+H2O-2H]+ [Fe(pip-OCH3)2 –H]+

330.2

40 30

614.9

636.9

20

354.1

374.4 653.9

707.3 681.1

10

1033.0 793.1

0

200

400

600

800

1000

1200

1400

770 Measured Value(m/z) 308.2 330.2 354.1 374.4 517.4 614.9 636.9 653.9 681.1 707.3 793.1 1033.0

Figure 4. Interaction between complex 3 and substance containing thiol. (A) ESI-MS analysis of complex 3 (10 µM) treated with GSH (0.5 mM) at 25℃ for 48 h. (B) UV-vis absorption spectra of complex 3 (10 µM) in aqueous medium (VCH3OH/VH2O=1:1) treated with GSH (0.5 mM) and BSA (0.5 mM) at 25℃ for 48 h. (C) Peaks in Figure 5A attributed to corresponding substrates.


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Figure 5. The interaction between complex 3 and BSA. (A) QTOF analysis of complex 3 in aqueous solution (VCH3OH/VH2O= 3:7) with BSA. (B) QTOF analysis of complex 3 (10 µM) with 50 eq. excess of BSA (0.5 mM) in aqueous medium (VCH3OH/VH2O= 3:7) at 25℃ for 48 h.



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Figure 6. Complex 3-induced TrxR inhibition in different cervical cancer cancer cells. (A) Inhibition of complex 3 on TrxR activity in different cervical cancer cells. Significant difference between treatment and control groups is indicated at P< 0.05 (*) or P < 0.01(**) levels. (B) The expression levels of Trx and TrxR induced by complex 3 in different cervical cancer cells. β-actin was used as loading control.


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Figure 7. Decrease of intracellular ROS level induced by complex 3. (A) Intracellular ROS generation after treatment with complex 3 in different cells. (B) The free radical scavenging capacity of complex 3 was determined by ABTS assay. Values expressed are means ± SD of triplicates. (C) The detection of ROS in Caski cell treated with complex 3 in 120 min by fluorescence confocal microscopy, as detected by DHE staining. Scale bar = 50 µm.



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Figure 8. Flow cytometric analysis of Caski cells with the treatment of different complexes. (A) Cell cycle distribution of Caski cells after the treatment of different complexes by PI-flow cytometric analysis. (B) Quantitative analysis of Sub G1 proportion induced by complex 3 in Caski cells. Values expressed were means ± SD of triplicates. Different characters indicated the significant difference between treatment and control groups at P< 0.05 level.


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Figure 9. Complex 3 induced cancer cell apoptosis. (A) Cell cycle distribution of different cervical cancer cells induced by complex 3 by PI-flow cytometric analysis. (B) Quantitative analysis of the Sub-G1 proportion induced by complex 3 in different cervical cancer cells. Values expressed were means ± SD of triplicates. (C) The expression levels of P-Histone in different cells after treatment of complex 3. β-actin was used as loading control.



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Figure 10. Complex 3 inhibited the migration and invasion of HUVEC cell in vitro. (A) Different concentrations of complex 3 suppressed HUVEC cells migration. Scale bar = 0.5 mm (B) Different concentration of complex 3 suppressed HUVEC cells invasion. Scale bar = 0.1 mm (C) Quantitative analysis of the migration cells after incubation with 1, 2, 4 µM complex 3. (D) Quantitative analysis of the invasion cells after incubation with 1, 2, 4 µM complex 3. The quantitative data were analyzed by manual counting (% of control). Values expressed were means ± SD of triplicates. Different characters indicated the significant difference between treatment and control groups at P< 0.05 level.


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Figure 11. Antiangiogenic activity of complex 3 in CAM model. (A) Representative images of angiogenesis inhibition caused by complex 3 in CAM assay. Scale bar = 0.5 cm. (B) The expression level of VEGF in HUVEC cells. β-actin was used as loading control.



CREA (µmol/L)

14 7

0 2.8

CREA

12

4

2

0

TG

2.1 1.4 0.7 0.0

0.4 0.3 0.2 0.1 0.0

GLU

9 6 3 0 4

LDLC CHOL (mmol/L)

ALB (g/L)

21

6

GLU (mmol/L)

ALB

LDLC (mmol/L)

28

TG (mmol/L)

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CHOL

3 2 1 0

Figure 12. Hematology analysis of mice treated with complex 3 after 72 h. ALB represented for serum albumin, CREA represented for creatinine, GLU represented for blood glucose, TG represented for triglyceride, LDLC represented for low density lipoprotein cholesterol, CHOL represented for cholesterol. Each value represents means ± SD of triplicates.


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Figure 13. H&E staining of heart, liver, spleen, lung and kidney in mice after the treatment of complex 3 for 72 h. Scale bar = 200 µm.

Table 1. Cytotoxic effects of Fe complexes on human cancer and normal cell lines (IC50) IC50 (µM)

Complex

a b

a

b

Caski

SiHa

HeLa

U87

C6

Chem-5

L02

SI

SI

1

1.72

2.91

4.21

12.41

3.87

2.15

3.12

1.25

1.81

2

0.38

2.16

3.87

3.25

1.15

1.68

2.71

4.42

7.13

3

0.75

6.73

7.32

21.63

19.38

14.24

23.71

18.98

31.61

4

0.25

1.32

9.31

9.42

0.88

1.15

2.27

4.60

9.08

5

56.12

65.12

﹥80

﹥80

70.4

53.63

31.93

0.96

0.57

6

35.61

19.12

﹥80

﹥80

50.21

27.52

12.73

0.77

0.36

Auranofin

0.31

1.69

3.88

2.31

0.98

2.14

3.14

6.90

10.13

Cisplatin

0.59

17.04

11.81

9.33

8.78

9.67

3.32

16.39

5.63

: Safer Index = IC50(Chem-5) / IC50(Caski) : Safer Index = IC50(L02) / IC50(Caski)



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Table of contents

Herein, we designed a series of Fe(II) complexes act as TrxR inhibitors. The results indicated that complex 3 could inhibit the activity of TrxR and angiogenesis. Taken together, this study provides a strategy for drug design to exploit Fe-based phenanthroline derivative as chemotherapeutic agent in cancer treatment.


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Anticancer and Antiangiogenic Iron(II) Complexes That Target

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