Novel Complex of Copper and a Salicylaldehyde Pyrazole Hydrazone

Jul 22, 2009 - Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China, Institute of Organic Chemistry, S...
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Chem. Res. Toxicol. 2009, 22, 1517–1525

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Novel Complex of Copper and a Salicylaldehyde Pyrazole Hydrazone Derivative Induces Apoptosis through Up-Regulating Integrin β4 in Vascular Endothelial Cells Chuandong Fan,†,§,| Jing Zhao,†,§,| Baoxiang Zhao,*,‡ Shangli Zhang,†,§ and Junying Miao*,†,§ Institute of DeVelopmental Biology, School of Life Science, Shandong UniVersity, Jinan 250100, China, Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong UniVersity, Jinan 250100, China, and The Key Laboratory of CardioVascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong UniVersity, Qilu Hospital, Jinan 250012, China ReceiVed March 22, 2009

To determine apoptosis modulators of human umbilical vein endothelial cells (HUVECs), we prepared 9 novel complexes of copper (Cu) and salicylaldehyde pyrazole hydrazone (SPH) derivatives (Cu-SPHs). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay revealed that all of the SPHs and Cu-SPHs effectively inhibited cell growth. Six of the 9 Cu-SPHs induced apoptosis in HUVECs. Among the 9 Cu-SPHs, the complex of Cu and (E)-N′-(2-hydroxybenzylidene)-1-benzyl-3-phenyl-1H-pyrazole5-carbohydrazide, named Cu-15, was one of the most effective apoptosis inducers and inhibited angiogenesis on Matrigel and HUVEC migration in Vitro. We further studied the mechanism of Cu-15 action and found that the protein level of integrin β4 increased with 10 µM Cu-15 treatment for 12 or 24 h. Knockdown of integrin β4 by RNA interference significantly inhibited apoptosis induced by Cu-15 in HUVECs. Thus, high level of integrin β4 could promote apoptosis induced by Cu-15. Cu-15 might be a useful tool for further investigating the functions of integrin β4 in regulating angiogenesis and HUVEC apoptosis. Introduction Copper (Cu1) is a trace element that plays important roles in promoting angiogenesis (1-3). Angiogenesis is induced in many pathological states, such as wound healing, chronic inflammation, restenosis, atherosclerosis, and tumors. A large body of evidence indicated Cu chelation as an effective method to inhibit angiogenesis (1, 4-6). Cu chelators have become promising agents in the treatment of vascular hyperplasia diseases and inhibit angiogenesis through decreasing the level of Cu (7, 8), but the effect of a Cu-chelator complex on angiogenesis has been rarely investigated. Some Cu chelators, such as pyrrolidine dithiocarbamate (PDTC) (9, 10) or salicylaldehyde benzoylhydrazone (SBH) (11), acquire more effective or novel bioactivity after forming a Cu-chelator complex; therefore, a Cu-chelator complex might modulate angiogenesis. In recent years, the metallic complex of SBH has generated considerable attention for its biological activities. SBH appears * To whom correspondence should be addressed. Phone: 86-53188364929. Fax: 86-531-88565610. E-mail: [email protected] (J.M.); [email protected] (B.Z.). † Institute of Developmental Biology. ‡ Institute of Organic Chemistry. § The Key Laboratory of Cardiovascular Remodeling and Function Research. | These authors made equal contributions to this work. 1 Abbreviations: Cu, copper; SBH, salicylaldehyde benzoylhydrazone; SPH, salicylaldehyde pyrazole hydrazone; PDTC, pyrrolidine dithiocarbamate; VEC, vascular endothelial cell; HUVEC, human umbilical vein endothelial cell; Cu-15, the complex of Cu and (E)-N′-(2-hydroxybenzylidene)-1-benzyl-3-phenyl-1H-pyrazole-5-carbohydrazide; FGF-2, fibroblast growth factor 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; LDH, lactate dehydrogenase; DMSO, dimethyl sulfoxide; PARP, poly(ADP-ribose) polymerase; PBS, phosphate buffered saline; PVDF, polyvinylidene fluoride; RNAi, RNA interference.

to be an unusually potent inhibitor of DNA synthesis and cell growth in various cultured human and rodent cell lines (12, 13). SBH is one of the most important Schiff bases, which can react with a large variety of transition metal cations, including Cu2+, and thus can form a number of complexes (11, 14). Cu-SBH complexes were found to be significantly more cytotoxic than metal-free ligands and complexes of other transitional metals (Cu > Ni > Zn ) Mn > Fe ) Cr > Co) in MOLT-4 cells, an established human T-cell leukemia cell line (15). In our previous studies, we synthesized 9 novel salicylaldehyde pyrazole hydrazone (SPH) derivatives that inhibited the growth of A549 human lung cancer cells (16). SPH has identical ligands for Cu2+ as SBH. To date, no study has investigated the biological activity of SBH and Cu-SBH complexes in angiogenesis. Considering the wide biological activity of SBH, we deduced that SPH and Cu-SPH complexes might affect angiogenesis. Vascular endothelial cells (VECs) have important roles in angiogenesis. The regulation of endothelial cell apoptosis is a potential therapeutic method in blood vessel diseases (17, 18). To determine novel angiogenesis modulators, we investigated the effect of 9 newly synthesized SPH derivatives and their Cu-SPH complexes on human umbilical vein endothelial cells (HUVECs) and angiogenesis in Vitro. Furthermore, although many reports have described the molecular mechanisms of HUVEC apoptosis under different stimuli, the exact mechanisms have not been fully elucidated (19, 20). Integrin β4 plays an important role in signaling networks that drive pathological angiogenesis and tumor progression (21) and is involved in endothelial cell apoptosis (22, 23). To reveal the mechanism by which Cu-SPH complexes induce apoptosis in HUVECs, we investigated the role

10.1021/tx900111y CCC: $40.75  2009 American Chemical Society Published on Web 07/22/2009

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Table 1. Growth Inhibitory Properties IC50 (µM) for the Compounds at 48 h in HUVECs SPH

4

8

12

15

16

17

18

19

20

IC50 (µM)

1.78

1.54

1.14

1.52

2.23

2.26

4.85

1.58

2.19

Cu-SPH

Cu-4

Cu-8

Cu-12

Cu-15

Cu-16

Cu-17

Cu-18

Cu-19

Cu-20

IC50 (µM)

0.92

0.91

0.98

0.97

3.37

1.88

4.85

0.95

3.78

of integrin β4 in HUVEC apoptosis induced by the complex of Cu and (E)-N′-(2-hydroxybenzylidene)-1-benzyl-3-phenyl-1Hpyrazole-5-carbohydrazide (Cu-15), a representative of the Cu-SPH complexes.

Experimental Procedures Reagents, Chemicals, and the Preparation of Drugs. Fetal bovine serum (FBS) and M199 medium were obtained from Hycolon Co. (USA). Recombinant bovine fibroblast growth factor 2 (FGF-2) was from EssexBioGroup (China), and Hoechst 33258 was from Sigma (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium (MTT) was purchased from Amresco (Ohio, USA). Matrigel was purchased from BD Biosciences Co. (CA, USA) Rabbit polyclonal integrin β4 primary antibody (sc9090) and horseradish peroxidase-conjugated or FITC-conjugated goat antirabbit secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-poly(ADP-ribose) polymerase (PARP) antibody (AP-102) was purchased from Beyotime Co. (Jiangsu, China). Preparation of Cu-SPH complexes. SPH derivatives (compounds 4, 8, 12, 15, 16, 17, 18, 19, and 20) were previously synthesized in our laboratory (16). The 9 Cu-SPH complexes were prepared by the method described previously (24). Briefly, the SPH and CuCl2 were dissolved separately in DMSO at 20 mM as stock solutions. Then equal volumes of the SPH and CuCl2 were mixed to form Cu-SPH complexes at 10 mM as a stock solution for cell treatment. The final concentration of DMSO used in the culture medium was no more than 0.1% (v/v) and did not affect cell growth. Spectroscopy Study of Cu-SPH. The formation of the complexes was determined by spectroscopy as described (14). For the preparation of SPHs and CuCl2 stock solutions, SPHs were dissolved in DMSO as stock solutions at 20 mM, and CuCl2 was dissolved in methanol as a stock solution at 20 mM for spectroscopy. Subsequently, 3 mL of methanol was added in a spectroscopic quartz cuvette with a 1-cm optical path, then 3 µL of SPH stock solution (20 mM) was added at 20 µM in methanol. The UV-vis spectra were recorded by use of a V-550 UV-visible spectrometer (JASCO, Japan). Then, CuCl2 stock solution (20 mM) was added step by step at 5, 10, 20, and 30 µM in methanol, with the UV-vis spectra recorded after each addition. Cell Culture. HUVECs were obtained in our laboratory as described (25). Cells were cultured on gelatin-coated plastic dishes with M199 medium supplemented with 20% FBS and recombinant bovine FGF-2, 8.4 IU/mL, at 37 °C in 5% CO2 and 95% air. A population doubling level (PDL) for HUVECs of 10-20 was used for all experiments. The cells treated with DMSO were used as the controls in our experiment. MTT Assay. Cell growth was determined by the MTT assay as described (26). To determine the relationship between optical absorbance and the cell number, we seeded HUVECs into 96-well plates at 500 to 16,000 cells/well in 100 µL of culture medium. Then, we incubated the cells for 4 h at 37 °C and allowed them to attach. One hundred microliters of culture medium without HUVECs was added in a well, which was used as a blank control. Since 16,000 HUVECs per well (5 × 104/cm2) reached a 100% confluence, we took it as the highest density in the experiment. Then, 20 µL of MTT (5 mg/mL) in PBS was added into the well. After the cells were incubated for 4 h, the culture medium was removed gently, and 100 µL of DMSO was added. Subsequently, the plate was shaken on a rocker under dark conditions for 20 min. Light absorption was measured at 570 nm by use of a SpectraMAX 190 microplate spectrophotometer (GMI Co., USA).

For determination of the compounds’ effects on HUVEC growth, the cells were seeded in 96-well plates at 1.25 × 104/cm2 (4000 cells /well). After 24 h, the cells were treated with 0.1% DMSO (v/v, as control), the SPH or Cu-SPH at the indicated concentrations and times, and the cell growth was determined by MTT assay as described above. Lactate Dehydrogenase Assay. HUVEC culture medium was collected after treatment with 0.1% DMSO (v/v, as control), 10 µM CuCl2, or 10 µM Cu-SPH for 24 h. Lactate dehydrogenase (LDH) release was detected by use of a kit (Nanjing Jiancheng Co., China) according to the manufacturer’s instructions. Hoechst33258 Staining. Cells were plated onto 24-well plates at 1.25 × 104 cells/cm2 24 h before treatment with 0.1% DMSO (v/v, as control), CuCl2, SPH, or Cu-SPH. Then, 24 h after treatment, living cells were stained with 10 µg/mL Hoechst 33258 for 10 min. Cells were gently washed with PBS once. The percentage of apoptotic cells was determined by comparing cells containing normal DNA staining and those with condensed DNA staining of nuclei. A minimum of 500 cells was scored for each sample, and each experiment was performed in triplicate. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assay. TUNEL assay was used to detect in situ nuclear DNA fragmentation and measure apoptosis ratio (27). Briefly, after cells were treated in the presence or absence of 10 µM Cu-15 for 6, 12, 18, 24 h, DNA fragmentation was detected by DeadEndTM Fluorometric TUNEL System according to the manufacturer’s protocol. Cells were evaluated under a laser scanning confocal microscope (Leica, Germany). The apoptosis rate was quantified by the TUNEL-positive rate. Capillary-Like Tubule Formation. The formation of capillarylike structures by HUVECs on Matrigel was studied as previously described (28). Culture plates with 24 wells were coated with Matrigel according to the manufacturer’s instructions. HUVECs were seeded at 2.5 × 104 cells/cm2 in fresh M199 medium deprived of serum and FGF-2, and CuCl2 or compound 15 or Cu-15 was added. Images of tubule formation were captured by use of a microscope (Nikon, Japan) and quantified by use of Image-Pro Plus 6.0 (MD, USA). Cell Migration. Cells were seeded onto 24-well plates at 7.5 × 104 cells/cm2. With cells at a postconfluent state, strips of wounds were created by scraping cell monolayers with a sterile pipet tip. Images of cell migration were taken immediately after scraping and at 6, 12, and 24 h later. Cell migration was quantified by measuring the distance

Figure 1. Determination of UV-vis absorption spectra of 20 µM compound 15 in the presence of 0, 5, 10, 20, and 30 µM CuCl2. The figure presents only the spectra between 200 and 500 nm.

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Figure 2. Cu-SPHs triggered HUVECs to undergo apoptosis. (A) Effects of Cu-SPHs on nuclear fragmentation and condensation of HUVECs. Cells were treated with 10 µM CuCl2 or Cu-SPHs for 24 h and stained with Hoechst 33258. Magnification 400×. (B) Quantity of apoptotic cells calculated by Hoechst 33258 staining; at least 500 cells were counted. (C) Effect of Cu-SPHs on LDH release from HUVECs. (*P < 0.05, **P < 0.01 vs control, n ) 3.)

Figure 3. Effects of CuCl2, compound 15 and Cu-15 on the growth of HUVECs at 24 and 48 h. (A) MTT standard curve for HUVEC number showing relative changes in MTT absorbance readings as reflected in cell numbers. (B) Growth curves for HUVECs treated with CuCl2 and compound 15. (C) Growth curves for HUVECs treated with Cu-15. CuCl2 and Compound 15 added into the culture medium both at a final concentration of 10 µM in B inhibited the cell growth to the same extent as 10 µM Cu-15 in C. (*P < 0.05, **P < 0.01 vs control, n ) 4.)

between the wound edges before and after injury by use of the AnalySIS software (29). Data are the means of 3 independent experiments with three replications of platings each.

Western Blot Analysis. Cells were cultured in the absence or presence of 10 µM compound 15 or Cu-15 for 6, 12, and 24 h. After treatments, cells were washed twice with ice cold PBS and

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Figure 4. Cu-15 triggered HUVECs to undergo apoptosis in a dose- and time-dependent manner. (A) TUNEL assay of the effect of Cu-15 at 12, 18, and 24 h. (Bar ) 20 µm). (B) Effect of 10 µM compound 15 and 2.5, 5, 10 µM Cu-15 at 24 h detected by Hoechst 33258 staining. CuCl2 and compound 15 added into the culture medium at a final concentration of 10 µM induced apoptosis in HUVECs to the same extent as that with 10 µM Cu-15. Magnification 400×. (C) Western blot assay of PARP cleavage. (D) Quantity of apoptotic cells calculated by TUNEL assay in A. (E) Quantity of apoptotic cells calculated by Hoechst33258 staining in B, with at least 500 cells counted. (*P < 0.05, **P < 0.01 vs control, n ) 3.)

lysed in protein lysis buffer (1% SDS in 25 mM Tris-HCl, pH 7.5, 4 mM EDTA, 100 mM NaCl, 1 mM PMSF, 10 µg/mL leupeptin, and 10 µg/mL soybean trypsin inhibitor). The protein concentration of the cells was determined by the Bradford assay. An equal amount of protein was loaded in each lane on 7.5% SDS-polyacrylamide gel. After separation, the protein was electrophoretically transferred to a polyvinylidenefluoride membrane. The membrane was incubated in TBST (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat milk at

room temperature for 1 h. The membrane was probed with primary antibody overnight at 4 °C, then washed in TBST once, then in TBS twice, each for 5 min. The membrane was subsequently incubated with secondary antibody for 1 h at room temperature and washed with TBST and TBS. Then, the membrane was soaked in Ni-enhanced 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (18 mL of TBS, 24 mg of DAB, 2 mL of 0.3% CoCl2, 400 µL of 30% H2O2) until the protein strip could be visualized; the dyeing reaction was completed by the addition of double

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Figure 5. Effect of compound 15 and Cu-15 on tubule formation of HUVECs. (A) Photomicrographs of tubule formation of HUVECs on Matrigel at 6 h. (B) Relative quantity of tubule length. Treatment with Cu-15 inhibited tubule formation of HUVECs deprived of FGF-2 in a dose-dependent manner. CuCl2 and compound 15 added into the culture medium both at a final concentration of 10 µM inhibited tubule formation to the same extent as 10 µM Cu-15. Magnification 100×. (*P < 0.05, and **P < 0.01 vs control, n ) 4.)

distilled water. Intensity of the immunoreactive bands was quantified by use of Quantity-One software (Bio-Rad). RNA Interference (RNAi). RNAi involved the use of a specific integrin β4 small interfering RNA (siRNA), a pool of three targetspecific, 20- to 25-nt siRNAs (sc-35678; Santa Cruz Biotechnology, CA, USA). HUVECs were transfected with 40 nM integrin β4 siRNA with RNAi-Fect transfection reagent (Qiagen, Hilden, Germany) as described by the manufacturer. Cells were transfected with 40 nM scramble control siRNA (Santa Cruz Biotechnology) or 40 nM integrin β4 siRNA for 24 h and then incubated with M199 medium or 10 µM CuCl2, or 10 µM Cu-15 for another 24 h. We monitored the effect of gene silencing by immunofluorescence assay. Briefly, cells were fixed with 4% paraformaldehyde for 10 min. After washing with PBS, they were blocked with sheep serum for 20 min at room temperature. Then, primary antibodies for integrin β4 were added and incubated in a humid chamber overnight at 4 °C. After washing with PBS, cells were incubated with FITC-labeled secondary antibodies for 20 min at 37 °C. After washing with PBS, samples were evaluated by laser scanning confocal microscopy (Leica, Gemany). We randomly selected the region of interest (ROI) and zoomed in the same frames. The value of relative fluorescence intensity per cell was defined as the total value of fluorescence in the scan zoom divided by the total number of cells (at least 200 cells) in the same zoom. The effect of integrin β4 siRNA on HUVEC apoptosis was evaluated by Hoechst 33258 staining. Statistical Analysis. Data were presented as the means ( SE from at least 3 independent experiments and analyzed by Student’s t-test. Differences at p < 0.05 were considered statistically significant.

Results Determination of UV-Vis Spectra of SPH Derivatives in the Presence of Cu. The determination of the UV-vis spectra of compound 15 in the presence of CuCl2 is illustrated

in Figure 1. SPH in methanol gave three peaks of absorption at 224, 294, and 325 nm. With CuCl2 solution added, the SPH peaks at 294 and 325 nm disappeared, and new peaks at 254 and 387 nm appeared. The intensity of the new peaks increased with increasing CuCl2 concentration, and the peak at 387 nm reached a maximum when the molar of equivalence was reached. Beyond the molar of equivalence, the spectrum in the range from 300 to 700 nm did not change in the presence of 1.5 times of CuCl2 equivalence. The evolution of spectra indicated that the Cu-SPH complexes were formed during the addition of CuCl2, and the reaction was almost complete at equal molar quantity. The UV-vis spectra of the other 8 SPHs were similar to that of compound 15 (data not shown). DMSO at 0.1% (v/v) used in this study did not influence the spectral characteristics of SPH and Cu-SPH complexes (data not shown). Inhibitory Effects of SPHs and Cu-SPH Complexes on the Proliferation of HUVECs. MTT assay revealed that compound SPHs and Cu-SPHs inhibited the growth of HUVECs (Table 1), with Cu-SPH having a higher growth inhibitory effect than the compound SPHs, except for Cu-16, Cu-18, and Cu-20, each of which has a 4-tert-butylbenzyl group. Apoptosis Induced by Cu-SPH in HUVECs. To demonstrate that the inhibition of cell growth by Cu-SPH was due to apoptosis, we examined chromatin condensation of treated cells by Hoechst 33258 staining. Treatment with 10 µM Cu-4, Cu8, Cu-12, Cu-15, Cu-17, and Cu-19 for 24 h produced chromatin condensation and fragmentation (Figure 2A). However, none of the 9 SPHs triggered apoptosis in cells (data shown for compound 15 only, Figure 4). Release of LDH, a measure of cell necrosis, with Cu-SPH treatment for 24 h did not differ from that with control treatment

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Figure 6. Effect of compound 15 and Cu-15 on migration of HUVECs. (A) Photomicrographs of HUVEC migration at 0, 6, 12, and 24 h. (B) Quantification of migration distance. Cell migration of HUVECs was inhibited after the addition of 10 µM Cu-15 at 6, 12, and 24 h as measured by migration distance on 100× photomicrographs. (**P < 0.01 vs control, n ) 3.)

(Figure 2C), indicating that Cu-SPH did not induce necrosis in HUVECs. The results suggested that the compounds Cu-SPH inhibited HUVEC proliferation by inducing apoptosis. Among the apoptosis inducers, Cu-15 and Cu-19 were the most effective (Figure 2B). Compound 15 had a lower molecular weight and a simpler molecular structure than compound 19; therefore, we further investigated the effect of Cu-15 as a representative apoptosis inducer in HUVECs. Effects of Compound 15 and Cu-15 on HUVEC Growth. The MTT standard curve showed that the optical absorbance had a linear correlation with the HUVEC number in the range from 500 to 16,000 (Figure 3A). The MTT assay revealed both compound 15 and Cu-15 effectively inhibiting HUVEC growth (Figure 3B,C), with Cu-15 having a higher growth inhibitory effect than compound 15 (IC50 values 0.97 vs 1.52 µM at 48 h; Table 1). Ten micromolar CuCl2 did not inhibit HUVEC growth (Figure 3B). Interestingly, when CuCl2 and compound 15 were sequentially added into the culture medium, both at a final concentration of 10 µM, they inhibited HUVEC growth more effectively than compound 15 alone (Figure 3B). The results showed that CuCl2 and compound 15 might be able to form a complex in the culture medium and subsequently work as a whole. Cu-15 Induced HUVEC Apoptosis in a Dose- and TimeDependent Manner. To obtain more information about the apoptosis process induced by Cu-15, we examined the DNA rupture at different times and doses. The TUNEL assay revealed cells with DNA rupture on treatment with 10 µM Cu-15 for

Figure 7. Effects of 10 µM CuCl2, compound 15, and Cu-15 on the protein level of integrin β4 in HUVECs. Cells were cultured in the presence of 10 µM CuCl2, compound 15, or Cu-15 for 6, 12, or 24 h. (A) Western blot analysis of the protein level of integrin β4 and β-actin as a normalization control. (B) Quantificaton of integrin β4 protein level. (*P < 0.05, and **P < 0.01 vs control, n ) 3.)

12, 18, or 24 h (Figure 4A,D). Hoechst33258 staining showed cells treated with 2.5, 5, and 10 µM Cu-15 for 24 h with chromatin condensation and fragmentation characteristics (Figure 4B). PARP cleavage is an indicator of caspase activation and apoptosis. Western blot showed that PARP was cleaved in

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Figure 8. Knockdown of integrin β4 inhibited Cu-15-induced apoptosis in HUVECs. First column (control): cells were transfected with 40 nM scramble control siRNA for 24 h and incubated in culture medium for another 24 h. Second column (CuCl2): cells were transfected with 40 nM scramble control siRNA for 24 h and incubated with 10 µM CuCl2 for another 24 h. Third column (Cu-15): cells were transfected with 40 nM scramble control siRNA for 24 h and incubated with 10 µM Cu-15 for another 24 h. Fourth column (RNAi Cu-15): cells were transfected with 40 nM integrin β4 siRNA for 24 h and incubated with 10 µM Cu-15 for another 24 h. (A) Fluorescent micrographs showing the relative intensity and location of integrin β4 in HUVECs (bar ) 20 µm). (B) Hoechst33258 staining showing apoptotic cells. Magnification 400×. (C) Apoptosis rate in HUVECs determined by Hoechst33258 staining, with at least 500 cells counted. (*P < 0.05 vs control, n ) 4.)

HUVECs treated with 10 µM Cu-15 at 12 and 24 h, but not in HUVECs treated with 10 µM CuCl2 or compound 15 (Figure 4C). Interestingly, when CuCl2 and compound 15 were sequentially added into the culture medium, both at a final concentration of 10 µM, the HUVECs also underwent apoptosis determined by Hoechst33258 staining and PARP cleavage (Figure 4B,C) and exhibited an apoptosis rate similar to that in the cells treated with 10 µM Cu-15 (Figure 4B,E). This finding suggested that CuCl2 and compound 15 might be able to form a complex in the culture medium and subsequently work as a whole. Effect of Cu-15 on the Formation of Capillary-Like Structure of HUVECs. To demonstrate the effect of Cu-15 on HUVEC differentiation, we investigated the angiogenic property of HUVECs treated with CuCl2, compound 15, and Cu-15. Cu-15 at 1.25, 2.5, 5, and 10 µM inhibited the capillarylike structure formation of HUVECs (Figure 5). CuCl2 or compound 15 at 10 µM had no significant effect on the tubule formation of cells. Effect of Cu-15 on HUVEC Migration. To demonstrate how Cu-15 inhibited angiogenesis in Vitro, we investigated the migration of HUVECs treated with Cu-15. Cu-15 at 10 µM inhibited the migration ability of migration in HUVECs (Figure 6), but 10 µM CuCl2 and compound 15 and 5 µM Cu-15 had no significant effect on cell migration. Therefore, Cu-15 might not inhibit angiogenesis by cell migration at low concentrations such as 5 µM. Effect of Cu-15 on the Level of Integrin β4. To understand the mechanism by which Cu-15 induced apoptosis in HUVECs, we examined the changes in integrin β4 protein level after treatment with 10 µM Cu-15 for 6, 12, or 24 h. Cu-15 elevated the protein level of integrin β4 significantly at 12 and 24 h, but CuCl2 and compound 15 did not change the level of integrin β4 (Figure 7). Knockdown of Integrin β4 Inhibited Apoptosis Induced by Cu-15 in HUVECs. To further demonstrate the role of integrin β4 in HUVEC apoptosis induced by Cu-15, we knocked down integrin β4 using RNAi. The apoptosis rate of cells was significantly decreased, from 51% to 37%, with integrin β4 silencing and 10 µM Cu-15 treatment (Figure 8).

Discussion Cu plays an essential role in promoting angiogenesis. It might perform its function in angiogenesis through serving as a cofactor of angiogenic factors, including vascular endothelial growth factor (VEGF), acidic and basic FGFs, and angiotropin (1). Cu also appears to switch the normally quiescent endothelium into a proliferative state by activation of angiogenic growth factors. Recently, Cu was found to induce VEGF expression in primary and transformed human keratinocytes (3) and stimulate endothelial cells to release interleukin 8 (2). Increasing reports suggest that newly vascularized tissue sequesters extra Cu (1, 4, 30-34). In a rabbit cornea model, when prostaglandin E1 induced angiogenesis, Cu accumulated in the newly vascularized region (31). Furthermore, tumors in animal models can sequester extra Cu (1, 4). Consistently, high levels of copper have been found in several types of human cancers (30, 32-34). Accumulating evidence has shown that Cu chelation can effectively inhibit angiogenesis (1, 4-6). The current Cu chelators include tetrathiomolybdate, ATN-224, D-penicillamine, trientine, methotrexate, and PDTC. The mechanism of angiogenesis inhibition by Cu chelators through decreasing Cu level has been well studied, but the nonchelating effect of Cu chelators and Cu-chelator complexes on angiogenesis has been rarely investigated. A recent study discovered that Cu-chelator PDTC could induce VEC death and apoptosis in rat smooth muscle cells (rSMCs), and the addition of Cu2+ could enhance apoptosis induced by PDTC in rSMCs (9). The study did not report the effect of Cu2+ on PDTC-induced death in VECs. A recent study revealed that ATN-224 could selectively induce apoptosis in tumor cells but not in endothelial cells (35). In our study, the 9 SPH derivatives and their Cu-SPH complexes could effectively inhibit HUVEC growth. More importantly, 6 of the 9 Cu-SPH complexes but not their Cufree organics could effectively induce apoptosis in HUVECs. Cu-15 was the most effective of the apoptosis inducers. Cu-15 rather than compound 15 could inhibit angiogenesis and HUVEC migration in Vitro. Also, compound 15 and Cu could form a complex resembling Cu-15 in the medium that induced HUVEC apoptosis. Thus, compound 15 itself could not inhibit

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angiogenesisthroughdecreasingtheCulevel,buttheCu-compound 15 complex could inhibit angiogenesis and induce apoptosis in HUVECs. Considering the relatively high level of Cu in newly vascularized tissue, as a potential drug, compound 15 might form a complex with Cu at a relatively high concentration there, thus selectively inducing apoptosis of VECs regionally rather than in the whole body. More importantly, although compound 15 could effectively inhibit HUVEC growth, most of the somatic VECs are quiescent, and thus, compound 15 may be nontoxic to them. Therefore, compound 15 might be a potential selective therapeutic drug for hypervascularization disease and have minimal side effects. Also, from a chemical biology perspective, Cu-15 could be used as a chemical probe for understanding the functions of target proteins, to advance the functional research of genes and proteins in angiogenesis and vascular disease. Integrin β4 plays important roles in endothelial cell apoptosis. Our previous studies showed that the level of integrin β4 was increased in HUVEC apoptosis induced by rattlesnake venom (23) or by safrole oxide (36). Furthermore, the functional monoclonal antibody against integrin β4 inhibited the apoptosis of HUVEC induced by rattlesnake venom (22) and by deprivation of serum and growth factors (37). Our recent studies showed that apoptosis induced by deprivation of growth factors were inhibited by a novel butyrolactone derivative through depressing integrin β4 level in HUVECs (38, 39). The data suggested that integrin β4 might be an apoptosis promoter in HUVECs, and our present study showed that the protein level of integrin β4 was increased during HUVEC apoptosis induced by Cu-15; knockdown of integrin β4 by RNAi significantly inhibited apoptosis induced by Cu-15 in HUVECs, which thus supports integrin β4 as a promoter of apoptosis in HUVECs. In summary, we reveal Cu-SPH complexes inducing apoptosis in HUVECs and suggest that Cu-15 might be a useful tool for further investigating the functions of integrin β4 in the regulation of angiogenesis and HUVEC apoptosis. Acknowledgment. This study was supported by the National 973 Research Project (No. 2006CB503803), National Natural Science Foundation of China (No. 90813022), and Natural Science Foundation of Shandong Province (Z2008B10 and Z2008D04).

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(7)

(8) (9)

(10)

(11)

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(14)

(15)

(16)

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References (1) Brewer, G. J. (2001) Copper control as an antiangiogenic anticancer therapy: Lessons from treating Wilson’s disease. Exp. Biol. Med. 226, 665–673. (2) Bar-Or, D., Thomas, G. W., Yukl, R. L., Rael, L. T., Shimonkevitz, R. P., Curtis, C. G., and Winkler, J. V. (2003) Copper stimulates the synthesis and release of interleukin-8 in human endothelial cells: A possible early role in systemic inflammatory responses. Shock 20, 154– 158. (3) Sen, C. K., Khanna, S., Venojarvi, M., Trikha, P., Ellison, E. C., Hunt, T. K., and Roy, S. (2002) Copper-induced vascular endothelial growth factor expression and wound healing. Am. J. Physiol. Heart Circ. Physiol. 282, H1821-H1827. (4) Brem, S. S., Zagzag, D., Tsanaclis, A. M., Gately, S., Elkouby, M. P., and Brien, S. E. (1990) Inhibition of angiogenesis and tumor growth in the brain. Suppression of endothelial cell turnover by penicillamine and the depletion of copper, an angiogenic cofactor. Am. J. Pathol. 137, 1121–1142. (5) Pan, Q., Kleer, C. G., van Golen, K. L., Irani, J., Bottema, K. M., Bias, C., De Carvalho, M., Mesri, E. A., Robins, D. M., Dick, R. D., Brewer, G. J., and Merajver, S. D. (2002) Copper deficiency induced by tetrathiomolybdate suppresses tumor growth and angiogenesis. Cancer Res. 62, 4854–4859. (6) Yoshiji, H., Yoshii, J., Kuriyama, S., Ikenaka, Y., Noguchi, R., Yanase, K., Namisaki, T., Kitade, M., Yamazaki, M., and Fukui, H. (2005)

(21) (22) (23)

(24)

(25)

(26) (27)

Combination of copper-chelating agent, trientine, and methotrexate attenuates colorectal carcinoma development and angiogenesis in mice. Oncol. Rep. 14, 213–218. Brewer, G. J., Dick, R. D., Grover, D. K., LeClaire, V., Tseng, M., Wicha, M., Pienta, K., Redman, B. G., Jahan, T., Sondak, V. K., Strawderman, M., LeCarpentier, G., and Merajver, S. D. (2000) Treatment of metastatic cancer with tetrathiomolybdate, an anticopper, antiangiogenic agent: Phase I study. Clin. Cancer Res. 6 (1), 1–10. Brewer, G. J. (2005) Anticopper therapy against cancer and diseases of inflammation and fibrosis. Drug DiscoVery Today 10 (16), 1103– 1109. Erl, W., Weber, C., and Hansson, G. K. (2000) Pyrrolidine dithiocarbamate-induced apoptosis depends on cell type, density, and the presence of Cu2+ and Zn2+. Am. J. Physiol.: Cell Physiol. 278, C1116-C1125. Chen, J., Du, C. S., Kang, J. H., and Wang, J. M. (2008) Cu2+ is required for pyrrolidine dithiocarbamate to inhibit histone acetylation and induce human leukemia cell apoptosis. Chem.-Biol. Interact. 171, 26–36. Ainscough, E. W., Brodie, A. M., Denny, W. A., Finlay, G. J., Gothe, S. A., and Ranford, J. D. (1999) Cytotoxicity of salicylaldehyde benzoylhydrazone analogs and their transition metal complexes: quantitative structure-activity relationships. J. Inorg. Biochem. 77, 125– 133. Johnson, D. K., Murphy, T. B., Rose, N. J., Goodwin, W. H., and Pickart, L. (1982) Cytotoxic chelators and chelates 1. Inhibition of DNA synthesis in cultured rodent and human cells by aroylhydrazones and by a copper(II) complex of salicylaldehyde benzoyl hydrazone. Inorg. Chim. Acta 67, 159–165. Pickart, L., Goodwin, W. H., Burgua, W., Murphy, T. B., and Johnson, D. K. (1983) Inhibition of the growth of cultured cells and an implanted fibrosarcoma by aroylhydrazone analogs of the Gly-His-Lys-Cu(II) complex. Biochem. Pharmacol. 32, 3868–3871. Lu, Y. H., Lu, Y. W., Wu, C. L., Shao, Q., Chen, X. L., and Bimbong, R. N. B. (2006) UV-visible spectroscopic study of the salicyladehyde benzoylhydrazone and its cobalt complexes. Spectrochim. Acta [A] 65, 695–701. Koh, L. L., Kon, O. L., Loh, K. W., Long, Y. C., Ranford, J. D., Tan, A. L. C., and Tjan, Y. Y. (1998) Complexes of salicylaldehyde acylhydrazones: Cytotoxicity, QSAR and crystal structure of the sterically hindered t-butyl dimer. J. Inorg. Biochem. 72, 155–162. Xia, Y., Fan, C. D., Zhao, B. X., Zhao, J., Shin, D. S., and Miao, J. Y. (2008) Synthesis and structure-activity relationships of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives as potential agents against A549 lung cancer cells. Eur. J. Med. Chem. 43, 2347–2353. Dimmeler, S., and Zeiher, A. M. (2000) Endothelial cell apoptosis in angiogenesis and vessel regression. Circ. Res. 87, 434–439. Segura, I., Serrano, A., De Buitrago, G. G., Gonzalez, M. A., Abad, J. L., Claveria, C., Gomez, L., Bernad, A., Martinez, A., and Riese, H. H. (2002) Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis. FASEB J. 16, 833–841. Folkman, J. (2003) Angiogenesis and apoptosis. Semin. Cancer Biol. 13 (2), 159–167. Affara, M., Dunmore, B., Savoie, C, Imoto, S., Tamada, Y., Araki, H., Charnock-Jones, D. S., Miyano, S., and Print, C. (2007) Understanding endothelial cell apoptosis: what can the transcriptome, glycome and proteome reveal? Philos. Trans. R. Soc. London, Ser. B 362, 1469–1487. Giancotti, F. G. (2007) Targeting integrin beta 4 for cancer and antiangiogenic therapy. Trends Pharmacol. Sci. 28, 506–511. Zhao, Q. T., Wang, N., Jia, R., Zhang, S. L., and Miao, J. Y. (2004) Integrin beta(4) is a target of rattlesnake venom during inducing apoptosis of vascular endothelial cells. Vasc. Pharmacol. 41, 1–6. Zhao, Q. T., Araki, S., Zhang, S. L., and Miao, J. Y. (2004) Rattlesnake venom induces apoptosis by stimulating PC-PLC and upregulating the expression of integrin beta(4), P53 in vascular endothelial cells. Toxicon 44, 161–168. Daniel, K. G., Chen, D., Orlu, S., Cui, Q. C., Miller, F. R., and Dou, Q. P. (2005) Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells. Breast Cancer Res. 7, R897-R908. Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. InVest 52, 2745–2756. Price, P., and McMillan, T. J. (1990) Use of the tetrazolium assay in measuring the response of human tumor cells to ionizing radiation. Cancer Res. 50, 1392–1396. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501.

Up-Regulating Integrin β4 (28) Nagata, D., Mogi, M., and Walsh, K. (2003) AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress. J. Biol. Chem. 278, 31000– 31006. (29) Vasvari, G. P., Dyckhoff, G., Kashfi, F., Lemke, B., Lohr, J., Helmke, B. M., Schirrmacher, V., Plinkert, P. K., Beckhove, P., and HeroldMende, C. C. (2007) Combination of thalidomide and cisplatin in an head and neck squamous cell carcinomas model results in an enhanced antiangiogenic activity in vitro and in vivo. Int. J. Cancer 121, 1697– 1704. (30) Diez, M., Arroyo, M., Cerdan, F. J., Munoz, M., Martin, M. A., and Balibrea, J. L. (1989) Serum and tissue trace metal levels in lung cancer. Oncology 46, 230–234. (31) Ziche, M., Jones, J., and Gullino, P. M. (1982) Role of prostaglandin E1 and copper in angiogenesis. J. Natl. Cancer Inst. 69, 475–482. (32) Margalioth, E. J., Schenker, J. G., and Chevion, M. (1983) Copper and zinc levels in normal and malignant tissues. Cancer 52, 868– 872. (33) Kuo, H. W., Chen, S. F., Wu, C. C., Chen, D. R., and Lee, J. H. (2002) Serum and tissue trace elements in patients with breast cancer in Taiwan. Biol Trace Elem. Res 89, 1–11. (34) Sharma, K., Mittal, D. K., Kesarwani, R. C., Kamboj, V. P., and Chowdhery. (1994) Diagnostic and prognostic significance of serum and tissue trace elements in breast malignancy. Indian J. Med. Sci. 48, 227–232.

Chem. Res. Toxicol., Vol. 22, No. 9, 2009 1525 (35) Juarez, J. C., Betancourt, O., Pirie-Shepherd, S. R., Guan, X. J., Price, M. L., Shaw, D. E., Mazar, A. P., and Donate, F. (2006) Copper binding by tetrathiomolybdate attenuates angiogenesis and tumor cell proliferation through the inhibition of superoxide dismutase 1. Clin. Cancer Res. 12, 4974–4982. (36) Zhao, J., Miao, J. Y., Zhao, B. X., and Zhang, S. L. (2005) Upregulating of Fas, integrin beta 4 and P53 and depressing of PCPLC activity and ROS level in VEC apoptosis by safrole oxide. FEBS Lett. 579, 5809–5813. (37) Zhao, K. W., Zhao, Q. T., Zhang, S. L., and Miao, J. Y. (2004) Integrin beta 4 mAb inhibited apoptosis induced by deprivation of growth factors in vascular endothelial cells. Acta Pharmacol. Sin. 25, 733– 737. (38) Wang, W. W., Liu, X., Zhang, Y., Zhao, J., Zhao, B. X., Zhang, S. L., and Miao, J. Y. (2007) Both senescence and apoptosis induced by deprivation of growth factors were inhibited by a novel butyrolactone derivative through depressing integrin beta 4 in vascular endothelial cells. Endothelium. 14, 325–332. (39) Wang, W. W., Liu, X., Zhao, J., Zhao, B. X., Zhang, S. L., and Miao, J. Y. (2007) A novel butyrolactone derivative inhibited apoptosis and depressed integrin beta 4 expression in vascular endothelial cells. Bioorg. Med. Chem. Lett. 17, 482–485.

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