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Original article
2021
:14;
202107
doi:
10.1016/j.arabjc.2021.103241

Development of lactobionic acid conjugated-copper chelators as anticancer candidates for hepatocellular carcinoma

, , , , , , , ,
a National Local Joint Engineering Laboratory for Advanced Textile Processing and Clean Production, Wuhan Textile University, Wuhan 430073, Hubei, PR China
b College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 Beijing, PR China
c School of Chemistry, Central China Normal University, Wuhan 430079, Hubei, PR China

⁎Corresponding author. xwdong@wtu.edu.cn (Xiongwei Dong)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Hepatic copper deposition leads to metabolic disorders, rapid increase in reactive oxygen species (ROS) levels, and even the occurrence and metastasis of hepatocellular carcinoma. Copper chelation or copper transporter inhibition have already been developed into an effective method to control the canceration of hepatocytes and kill the hepatocarcinoma cells. Here, we designed three novel lactobionic acid conjugated copper chelators (GT1, 9 and 10), which have the potential to be recognized by asialoglycoprotein receptor (ASGPR), a high-capacity C-type lectin receptor selectively expressed in liver. Both GT1, 9 and 10 can selectively and efficiently coordinate with copper in solution and in the high-copper treated hepatocellular carcinomas model (HC HepG2 cells). The thiosemicarbazone-based chelator GT1 should more effectively eliminate copper and promote apoptosis of HC HepG2 cells, which might have application prospects in preventing cancerization and other pathological lesions caused by copper deposition of liver. Moreover, our results also revealed the potential of GT1 to be harnessed as preventive leading structures of the hepatocellular carcinomas.

Keywords

Copper chelation
Hepatocellular carcinoma
Copper deposition
Apoptosis
Hepatic targeting
1

1 Introduction

Copper, a redox active metal ion, is essential for most organisms, which serves as cofactor for various enzymes and regulates energy generation, oxygen transport, cellular metabolism, signal transduction, and other biochemical processes (Kim et al., 2008). In the organism, copper accumulation is associated with cellular and apoptotic injury (Johncilla and Mitchell, 2011), and copper deficiency will cause severe malnutrition, chronic diarrhea, and genetic diseases (Williams, 1983). Therefore, cellular copper concentrations must be maintained somewhere below toxicity and above nutrient deficiency (Kim et al., 2008; Harris, 2001). Disruption of copper homeostasis in the organisms is closely related to the occurrence and development of many diseases, such as Alzheimer’s disease, Wilson’s disease, prion diseases, carcinogenesis, and so on (Członkowska et al., 2018; Daniel et al., 2004; Stepien et al., 2017; Bandmann et al., 2015).

Liver is the key organ in the supply, storage, and excretion of copper, in which copper can be used for protein and energy production (Roberts and Sarkar, 2008). Catalytic copper, because of its redox activity and mobilization, introduce the formation of reactive oxygen species (ROS) through Haber-Weiss-like reactions, which gives rise to fast DNA damage and also leads to carcinogenesis (Zubair et al., 2013; Aruoma et al., 1991; Theophanides and Anastassopoulou, 2002). A large number of clinical and experimental evidences indicate that copper deposition in the liver should lead to a variety of serious diseases, especially the hepatocellular carcinoma (Theophanides and Anastassopoulou, 2002; Smallwood et al., 1968; Wooton-Kee et al., 2015; Danks, 1991). Copper deposition leads to the ROS production, which makes liver cells proliferate abnormally and activate oncogenes. On the other hand, liver metabolic disorders caused by copper deposition also further increase the copper level in the liver, causing a vicious circle (Hussain et al., 2007, 2003; Merlot et al., 2013; Paulsen and Carroll, 2013).

The rapid proliferation, invasion and metastasis of hepatocarcinoma cells always depend on high levels of copper (Chandan et al., 2018; Bruix et al., 2004; Ueda et al., 1993). Intracellular copper is required for oncogenic BRAF signaling, tumorigenesis and activation of the mitogen-activated protein kinase (MAPK) pathway (Turski et al., 2012; Brady et al., 2014). Because copper accumulation is important for the cancerization and deterioration of the liver, chelating copper ions or inhibiting the activity of copper transporters have already become effective intervention methods for hepatocellular carcinoma (Brady et al., 2014; Wang et al., 2015; Krasnovskaya et al., 2016; Kacar et al., 2020; Yoshii et al., 2001; Porchia et al., 2020; Gupte and Mumper, 2009; Santini et al., 2014; Safi et al., 2014). Previously, Peter J. Crouch and the collaborators had developed a highly effective copper chelator GTSM, glyoxalbis(N(4)-methylthiosemicarbazonato), which achieved intracellular copper chelation, transport and re-release, and increased intracellular copper bioavailability (Crouch et al., 2009). Referring to the coordination mechanism of GTSM with copper, we are expected to design novel chelators to eliminate copper deposition and biologically reuse the excessive copper in the liver, and ultimately achieve the intervention and treatment of copper-dependent hepatocellular carcinoma.

In order to develop copper chelators for hepatocyte-specific targeting, we can utilize asialoglycoprotein receptor (ASGPR), a high-capacity C-type lectin receptor selectively expressed on mammalian hepatocytes, which recognizes N-acetylgalactosamine and galactose-containing glycopeptide substrates (Rigopoulou et al., 2012). Hitherto, conjugating drugs with galactose or its glycomimetic have developed into an effective strategy for hepatocyte-targeted drug delivery via ASGPR (Mamidyala et al., 2012; Huang et al., 2017; Bernardes et al., 2010; Pujol et al., 2011; Sanhueza et al., 2017). Besides, the capacity of ASGPR to import molecules across the cellular plasma membrane also makes it an enticing target for delivery to hepatocellular carcinomas system via ASGPR-mediated endocytosis (Li et al., 2008).

Here, based on the copper coordination chemistry and the selective recognition mechanism of ASGPR, three novel hepatocyte-targeted copper chelators were designed by combining phenol, thiosemicarbazone or semicarbazone, and lactobionic acid. Comparing with semicarbazone-based chelators GT9 and GT10, the thiosemicarbazone-based chelator GT1 has stronger binding ability to copper, and can more effectively chelate copper ions in solution and in high-copper treated hepatocellular carcinomas model (HC HepG2 cells). The lethal concentration of GT1 to HC HepG2 cells approach micro-molar levels, and 5 μM GT1 can effectively promote apoptosis and kill HC HepG2 cells. Furthermore, our results also indicated that GT1 has the proper binding capacity to copper ions (log KCu2+ = 17.48 ± 0.11), and so will not interfere with the function of copper-containing proteins and enzymes, such as copper/zinc superoxide dismutase (SOD1). Thus, this study established the power of GT1 in the killing of copper-deposited hepatocellular carcinomas and in the removing of copper ions from liver cells.

2

2 Experimental section

2.1

2.1 Materials and instrumentation

Unless otherwise noted, materials were purchased from commercial suppliers and used without further purification. All the solvents were treated according to standard methods. DMEM, FBS, 0.25% trypsin-EDTA, penicillin, streptomycin and PBS were purchased from Gibco Inc (Gibco, USA). UV–vis spectra were recorded on an Analytik Jena Specord 210 spectrophotometer (Analytic Jena AG). 1H NMR spectra were recorded on a Varian Mercury 600 spectrometer. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet), coupling constants (Hz) and integration. The MS data were obtained using a Quattro II ESI mass spectrometer (Waters, USA). The data of flow cytometry were performed on BD flow cytometer (BD, USA). Potentiometric titrations were performed with a Metrohm 877 Titrino Plus automated titrator equipped with a Metrohm 6.0262.100 glass electrode calibrated against standard buffers. Elementary analysis was carried out on a PerkinElmer 2400 analyser (PerkinElmer, USA). FTIR spectra were recorded on a Bruker Vertex 70 device (Bruker, Germany) using KBr pellets.

2.2

2.2 Determination of acidity and stability constants

Acidity constants (pKa) of the chelators and stability constant (pK) of the M2+-complexes (M = Cu, Zn, Mn and Fe) were determined by potentiometric titrations. The water-jacketed titration vessel was maintained at 25.0 °C (±0.5 °C). To estimate pKa values, chelators were predissolved in an excess of HCl (1.0 mM) and diluted with water [10% (vol/vol) DMSO] in the titration vessel (25.0 mL), the final concentrations of HCl and chelators were 100 μM. All measurements were conducted in the presence of 100 mM KCl to maintain constant ionic strength. Solutions were titrated with 75.5 mM NaOH, which was prepared with degassed water and standardized with Potassium biphthalate prior to each titration. Data analysis was carried out with the program HyperQuad 2013 (Protonic Software, UK) (Gans et al., 1999, 1996). Stability constant of M2+-complex was determined by the titrations performed in the presence of 1.0 equiv of 100.0 μM metal nitrates using the pKa values determined above. Species distribution plots and titration simulations were built with the program HySS2009 (Protonic Software) (Thomas et al., 1979; Alderighi et al., 1999). The data represented here were the mean ± SD of the results obtained from at least three independent measurements.

2.3

2.3 Cell culture

HepG2, HeLa and MCF-7 cells were cultured in culture media (DMEM supplemented with 10% FBS, 50 unit/mL of penicillin, and 50 μg/mL of streptomycin). MCF-10A cells were cultured in CM-0525 culture media (Procell, China). All cells were cultured under standard conditions (37 °C, 5% CO2, 100% humidity) with weekly passaging. Culture media were replaced with fresh media every day.

2.4

2.4 Intracellular copper detection by flow cytometry

The cells were seeded in a 6-well plate at a density of 104 cells per mL in culture media. After 24 h, the cells were treated without or with Cu2+ in culture media for 48 h at 37 °C. After washing with PBS, the cells were further incubated without or with chelators in culture media for 24 h at 37 °C. Because DMSO was used as a co-solvent for chelator, the same amount of DMSO (5‰) was added as a control group. In other subsequent intracellular experiments, we also used DMSO (5‰) as a control group. Then, the cells were washed three times with PBS and incubated with 5 μM Cu2+ fluorescent probe (Rhodamine 6G hydrazide) for another 1 h. Finally, cells were washed by PBS, dissociated by 0.25% trypsin-EDTA and re-suspended in PBS. Fluorescence data was collected and analyzed on the BD flow cytometry.

2.5

2.5 Cell apoptosis assay

FITC-conjugated annexin-V assays were used to assess cell apoptosis. Following the manufacturer’s instructions of Annexin V-FITC Apoptosis Detection Kit (Abcam, ab14085), cells were washed by cold PBS and re-suspended in 500 μL of 1 × Binding Buffer. Then, 5 μL of Annexin V-FITC and 5 μL of propidium iodide were added into the Binding Buffer and incubated at room temperature for 5 min in the dark, and the stained cells were analyzed by flow cytometry.

2.6

2.6 Cell viability assay

Cytotoxicity of Cu2+ and chelators was evaluated by MTS assay. Briefly, after cells reached 80% confluence in 96-well plates, the cells were treated with Cu2+ or chelators for a suitable time. Then, 20 µL of CellTiter 96® AQueous One Solution Reagent (Promega, G3580) was added into each well of the 96-well assay plate containing the samples in 200 µL of culture medium. The MTS reagent was incubated with cells at 37 °C for 2 h, and then the absorbance at 490 nm was recorded by a SpectraMax M5 Microplate Reader. At the same time, every experimental groups subtracted the background absorbance of CellTiter 96® AQueous One Solution Reagent.

2.7

2.7 SOD1 activity assay

SOD1 activity was determined by measuring the inhibition of the reduction of the water-soluble tetrazolium salt, WST-1, which produced a water-soluble formazan dye upon reduction with O2−. The determinations were performed with a HT Superoxide Dismutase Assay Kit (Trevigen). Bovine SOD1 (Sigma), which has been proved to be equivalent to human SOD1, was used to generate a standard curve. After incubating 1 μM SOD1 with 0, 1, 5, 10, 30, 50 μM GT1 for 24 h at 37 °C, the activity of SOD1 was measured according to the manual.

3

3 Results and discussion

3.1

3.1 Design of hepatocyte-targeted copper chelators

In order to rationally design hepatocyte-targeted copper chelators based on the copper coordination chemistry and the recognition mechanism of ASGPR for galactose, several pivotal factors to be considered include: (1) the appropriate stability constants of the copper complexes, which cannot be much higher than the affinity constant of copper binding protein for copper ions, and so the logKCu2+ value of our chelators should be within the range of 11–17 (Banci et al., 2010); (2) the N-acetylgalactosamine or galactose-containing structure, which could be recognized by hepatocellular ASGPR; (3) the strong transmembrane ability, which allows both the chelators and the copper complexes in and out of the cells to increase the copper bioavailability.

Based on our experience in designing specific SOD1 inhibitors (Dong et al., 2016; Li et al., 2019), three Cu2+-chelating agents were designed via a combination of phenol, thiosemicarbazone or semicarbazone, and lactobionic acid (Fig. 1, GT1, 9 and 10). The thiosemicarbazone and semicarbazone contain strongly copper-chelating and a H-bonding moiety —C(SH)—NH— and —C(OH)—NH—, respectively. The phenolic hydroxyl would modulate and facilitate the coordinating ability of thiosemicarbazone and semicarbazone to copper (Paterson and Donnelly, 2011). Finally, in order to achieve hepatocyte-targeted chelating agent delivery, lactobionic acid was covalently linked to the chelators, because galactose structure of lactobionic acid could be recognized by hepatocellular ASGPR.

The rational design of hepatocyte-targeted copper chelators. The design strategy of hepatocyte-targeted copper chelators through lactobionic acid conjugating.
Fig. 1
The rational design of hepatocyte-targeted copper chelators. The design strategy of hepatocyte-targeted copper chelators through lactobionic acid conjugating.

The metal-coordinating moieties (TH1, 9 and 10) were synthesized by our previous procedures (Dong et al., 2016), and then the hepatocyte-targeted copper chelators containing lactobionic acid (GT1, 9 and 10) were synthesized according to Scheme S1. All compounds were fully characterized by NMR (1H and 13C) and MS in our ESI (Figs. S1–S18), which adequately demonstrated the accuracy of these compounds. As shown in Figs. S19a and S19c, lactobionic acid conjugation leaded to the disappearance of ν(—NH2) characteristic peaks (TH1: 3289 cm−1, TH9: 3344 cm−1, TH10: 3352 cm−1) in GT1, GT9, and GT10, which verified that lactobionic acid was successfully covalently coupled to respective metal-coordinating moieties (Seleem et al., 2005; Shebl et al., 2010).

3.2

3.2 Coordination of chelators to copper

For the metal-coordinating moieties (TH1, 9 and 10) and copper chelators (GT1, 9 and 10), the stability constants of the complexes formed respectively with Cu2+, Zn2+, Mn2+ and Fe2+ were determined via potentiometric titrations at 25 °C. Considering the physical and chemical properties of trace elements similar to Cu2+ in human cells, Fe2+, Zn2+ and Mn2+ are used for comparison in this work. As shown in Table 1, the binding capacities of each copper chelator and respective metal-coordinating moiety to copper are basically equal, such as TH1 (logKCu2+ = 17.82 ± 0.07) and GT1 (logKCu2+ = 17.48 ± 0.11), which indicated that the covalent linkage of lactobionic acid almost did not affect the binding of metal-coordinating moiety to copper. Compared with GT9 and 10, the chelator GT1, designed on the basis of thiosemicarbazone, has a much stronger binding ability with copper (Table 1). The higher ability of thiosemicarbazone to bind metal is also confirmed by our previous work, in which the thiosemicarbazone based chelator LD18 have higher ability than semicarbazone based chelator LD27 to bind metal (Dong et al., 2016). Under our testing conditions, both GT1, 9 and 10 have stronger binding ability to Cu2+ than Fe2+, Zn2+ and Mn2+, which implies that the chelators have notable selectivity for Cu2+ over the other divalent metal ions tested (Table 1). On the other hand, Peep Palumaa and the collaborators had indicated that the stability constants (logKCu) between copper and common copper-containing proteins are in the range of 14–17 (Banci et al., 2010). Therefore, our chelators would not interfere with the normal function of copper proteins in the cell, because the stability constants of copper complexes formed by GT1, 9 and 10 are not significantly higher than the intracellular copper-containing proteins (Table 1).

Table 1 The data for coordination with different divalent metal ions of the metal-coordinating moieties and chelators.
Ligand (KM2+ value1) TH1 TH9 TH10 GT1 GT9 GT10
logKCu2+, n = 1, M−1 17.82 ± 0.07 16.17 ± 0.03 15.91 ± 0.02 17.48 ± 0.11 15.59 ± 0.18 14.52 ± 0.20
logKZn2+, n = 1, M−1 17.13 ± 0.05 15.28 ± 0.04 14.74 ± 0.09 15.90 ± 0.23 13.41 ± 0.18 12.83 ± 0.19
logKMn2+, n = 1, M−1 14.11 ± 0.03 14.86 ± 0.11 14.29 ± 0.07 14.85 ± 0.04 12.45 ± 0.14 11.83 ± 0.18
logKFe2+, n = 1, M−1 16.45 ± 0.05 14.34 ± 0.03 14.07 ± 0.06 15.10 ± 0.26 12.99 ± 0.41 12.32 ± 0.43
KCu2+/KZn2+ 4.90 7.76 1.48 × 101 3.80 × 101 1.51 × 102 4.90 × 101
KCu2+/KMn2+ 5.13 × 103 2.04 × 101 4.17 × 101 4.27 × 102 1.38 × 103 4.90 × 102
KCu2+/KFe2+ 2.34 × 101 6.76 × 101 6.92 × 101 2.40 × 102 3.98 × 102 1.58 × 102
pH range2 3–9 3–9 3–9 3–9 3–9 3–9
1 KM2+ is the stability constant of metal ion and chelator, which is calculated from ML (metal–ligand).
2 pH range indicates KM2+ was calculated in pH 3–9.

Next, solution speciation diagrams were calculated for the coordination of the metal-coordinating moieties and chelators to Cu2+, Fe2+, Zn2+ and Mn2+ (Fig. 2a and S20a) using the stability constants in Table 1. All of these diagrams indicated that the Cu2+-chelator complexes are the predominant species formed in the range of pH 3–9 (Table 1). This pH range requisite for the formation of stable complexes completely covers the pH range of intracellular environment. In our previous work, the complexes had been confirmed by the crystal structures of the Cu2+ complexes formed with thiosemicarbazone and semicarbazone based chelators (Dong et al., 2016), whose metal-coordinating moieties are similar to GT1, 9 and 10.

The metal binding characterization of hepatocyte-targeted copper chelators. (a) Species distribution plots measured using potentiometric titrations for the Cu2+, Zn2+, Mn2+ and Fe2+-chelator systems. The sources of Cu2+, Zn2+, Mn2+, and Fe2+ are CuCl2, ZnCl2, MnCl2 and FeCl2, respectively. Potentiometric titrations were performed for the solutions containing the chelators and equimolar amounts of metal ions at 25 °C (I = 0.1 M KCl). (b) Spectrophotometric titrations of chelators (10 mM) with Cu2+ (0–20 mM) at 25 °C.
Fig. 2
The metal binding characterization of hepatocyte-targeted copper chelators. (a) Species distribution plots measured using potentiometric titrations for the Cu2+, Zn2+, Mn2+ and Fe2+-chelator systems. The sources of Cu2+, Zn2+, Mn2+, and Fe2+ are CuCl2, ZnCl2, MnCl2 and FeCl2, respectively. Potentiometric titrations were performed for the solutions containing the chelators and equimolar amounts of metal ions at 25 °C (I = 0.1 M KCl). (b) Spectrophotometric titrations of chelators (10 mM) with Cu2+ (0–20 mM) at 25 °C.

In order to further demonstrate the binding properties of the hepatocyte-targeted copper chelators toward copper ions, we also evaluated the interactions between Cu2+ and these chelators by spectrophotometric titrations. As shown in Fig. 2b, upon titration of Cu2+, the intense absorption band at 345 nm (for GT1), 326 nm (for GT9) and 335 nm (for GT10) decreased gradually along with the increase of a new band at 395 nm (for GT1), 380 nm (for GT9) and 393 nm (for GT10), respectively. Furthermore, the well-defined isosbestic points at 370 nm (for GT1), 348 nm (for GT9) and 352 nm (for GT10) distinguished the free chelators and the respective copper complexes clearly (Fig. 2b). In physiology-like conditions (10% FBS), the characteristic absorption peaks of respective copper complexes at 395 nm (for GT1), 380 nm (for GT9) and 393 nm (for GT10) were also obtained upon titration of Cu2+ (Fig. S20b), which suggested that both GT1, 9 and 10 might coordinate with Cu2+ in physiology conditions. According to the results of potentiometric and spectrophotometric titrations, both GT1, 9 and 10 can preferentially bind to Cu2+ in vitro, and the binding capacity of GT1 is slightly stronger than GT9 and 10. Furthermore, the results of infrared spectrum also confirmed the coordinating of cooper with chelators. The strong bands at 1618, 1630, and 1626 cm−1 assigned to ν(C⚌N) for GT1, GT9 and GT10, respectively, were shifted to lower wave numbers (19–34 cm−1) in copper complexes (Figs. S19b and S19c). These indicated the participation of the azomethine nitrogen in the chelation (Shebl et al., 2010; Chandra and Kumar, 2007; Sen et al., 2008). In copper complexes of GT1; GT9 and GT10, the disappearance of ν(C⚌S) or ν(C⚌O) bands provided an evidence that the thiolic S or enolic O participate in the chelation after deprotonation leading to a covalent linkage (Singh and Singh, 2003). Therefore, our IR spectra of copper complexes are consistent with the IR results of thiosemicarbazone- and semicarbazone-based copper complexes in the literature (Seleem et al., 2005; Shebl et al., 2010; Chandra and Kumar, 2007; Sen et al., 2008; Singh and Singh, 2003), and are also consistent with the single crystal structure of copper complexes in our previous work (Dong et al., 2016).

3.3

3.3 Construction of copper deposition cell model

Our results indicated that the chelators (GT1, 9 and 10) could preferentially coordinate with copper, and the binding strength was also moderate. In order to systematically evaluate the removal of hepatocellular copper deposition by our chelators, we should first construct an appropriate cellular model of copper deposition. Thus, we detected the effects of incubation time on the survival of Cu2+ treated liver hepatocellular cells (HepG2). As the treatment time increases (0, 24, 48, 72 and 96 h), 100 μM Cu2+ could gradually reduce the cell viability of HepG2, and the treatment time over 48 h was more detrimental to the cell survival (Fig. 3a). Next, we further evaluated the dependence of HepG2 cytotoxicity on Cu2+ concentration. The cytotoxicity of HepG2 increased with the increase of Cu2+ concentration, and the cell survival rate was close to 85% when treated with 50 μM Cu2+ for 48 h (Fig. 3b). Finally, we evaluated the relative concentration of intracellular copper ions using commercial Cu2+ fluorescent probe, Rhodamine 6G hydrazide (CAS: 932013-08-6) (Dujols et al., 1997). After treating HepG2 cells with 50 μM Cu2+ for 48 h, the cells were washed with PBS, and then incubated with a 5 μM fluorescent probe for 1 h to characterize the intracellular Cu2+ contents. The results indicated that the fluorescence intensity of intracellular Cu2+ increased by approximate 11.5 times, which revealed the successful construction of high-copper cell model (Fig. 3c). In order to balance the copper concentration and survival rate, we treated HepG2 cells with 50 μM Cu2+ for 48 h to construct the high-copper treated hepatocellular carcinomas model (HC HepG2), which was used as copper deposition model for our subsequent studies.

Construction of high-copper treated hepatocellular carcinomas model (HC HepG2 cells). (a) After incubating HepG2 cells with 100 μM Cu2+ for 0, 24, 48, 72 and 96 h, the cell viability was evaluated using commercial MTS kit. (b) After incubating HepG2 cells with 0, 5, 10 50 and 100 μM Cu2+ for 48 h, the cell viability was evaluated using commercial MTS kit. (c) The HepG2 cells were incubated with 50 μM Cu2+ for 48 h, washed with PBS three times, and then treated with 5 μM Cu2+ fluorescent probe (Rhodamine 6G hydrazide) for another 1 h to characterize the intracellular Cu2+ contents. All experiments were normalized by respective control data. Data are mean of triplicate samples ± SD (*P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t test), and all error bars are SD.
Fig. 3
Construction of high-copper treated hepatocellular carcinomas model (HC HepG2 cells). (a) After incubating HepG2 cells with 100 μM Cu2+ for 0, 24, 48, 72 and 96 h, the cell viability was evaluated using commercial MTS kit. (b) After incubating HepG2 cells with 0, 5, 10 50 and 100 μM Cu2+ for 48 h, the cell viability was evaluated using commercial MTS kit. (c) The HepG2 cells were incubated with 50 μM Cu2+ for 48 h, washed with PBS three times, and then treated with 5 μM Cu2+ fluorescent probe (Rhodamine 6G hydrazide) for another 1 h to characterize the intracellular Cu2+ contents. All experiments were normalized by respective control data. Data are mean of triplicate samples ± SD (*P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t test), and all error bars are SD.

3.4

3.4 Elimination of hepatocellular copper deposition

In order to determine the concentration of chelators used in intracellular experiments, we first measured the cytotoxicity of GT1, 9 and 10 in normal and HC HepG2 cells. After constructing the high-copper cell model, the HC HepG2 cells were incubated with chelators for another 24 h, and the relative viability was recorded using MTS tetrazolium compound, which could be bioreduced by cells into a colored formazan product (Mosmann, 1983). The results indicated that all chelators killed HC HepG2 cells at low doses (5–50 μM), and GT1 could cause cell death more efficiently. The treatment with 5 μM GT1 killed ∼65% of HC HepG2 cells, whereas the cells treated with 5 μM GT9 or 10 maintained >90% cell viability (Fig. 4a). Besides, both GT1, 9 and 10 did not significantly affect normal HepG2 cells survival, in the chelator concentration range of 0–50 μM (Figs. S20c). Obviously, all copper chelators designed here can kill high-copper treated hepatocellular carcinomas with high efficiency, and the lethality decreased in the order of GT1 > GT9 ∼ GT10, which may be due to the stronger binding capacity of thiosemicarbazone contained GT1 to copper. It is well known that thiosemicarbazone- and semicarbazone-based chelators have already been developed into important anti-cancer agents, which can kill a variety of cancer cells (Bai et al., 2021; Gan et al., 2014; Patole et al., 2004; Kalinowski et al., 2009). Our results that thiosemicarbazone-based chelator GT1 can kill cancer cells more effectively is also consistent with many reported studies (Bai et al., 2021; Gan et al., 2014; Patole et al., 2004). For example, Bai et al. showed that thiosemicarbazone-based chelators (IC50 ≈ 2–11 μM) could kill triple negative breast cancer MDA-MB-231 cells more effectively than semicarbazone-based drugs (Bai et al., 2021), which also confirmed the reliability of our results. Compared with TH1, 9 and 10, chelators with lactobionic acid conjugation (GT1, 9 and 10) could kill HC HepG2 cells more effectively (Fig. S21a), which suggests that lactobionic acid conjugation can improve the targeting effect of chelators on hepatocytes.

Elimination of the hepatocellular copper deposition by chelators. After constructing the high-copper cell model, the HC HepG2 cells were incubated with 0, 1, 5, 10, 30 and 50 μM chelators (GT1, 9 and 10) for another 24 h. Then, the cell viability and intracellular Cu2+ contents were evaluated using commercial MTS kit (a) and Cu2+ fluorescent probe (b), respectively. SOD1 (1 μM) was incubated with 0, 1, 5, 10, 30, 50 μM GT1 for 24 h at 37 °C, and then the activity of SOD1 was measured by commercial kit (c). For chelator treatment, the same amount of DMSO (5‰) was added as a control group, because DMSO was used as a co-solvent. All experiments were normalized by respective control data. Data are mean of triplicate samples ± SD (*P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t test), and all error bars are SD.
Fig. 4
Elimination of the hepatocellular copper deposition by chelators. After constructing the high-copper cell model, the HC HepG2 cells were incubated with 0, 1, 5, 10, 30 and 50 μM chelators (GT1, 9 and 10) for another 24 h. Then, the cell viability and intracellular Cu2+ contents were evaluated using commercial MTS kit (a) and Cu2+ fluorescent probe (b), respectively. SOD1 (1 μM) was incubated with 0, 1, 5, 10, 30, 50 μM GT1 for 24 h at 37 °C, and then the activity of SOD1 was measured by commercial kit (c). For chelator treatment, the same amount of DMSO (5‰) was added as a control group, because DMSO was used as a co-solvent. All experiments were normalized by respective control data. Data are mean of triplicate samples ± SD (*P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t test), and all error bars are SD.

Then, we systematically evaluated the effects of GT1, 9 and 10 on copper elimination in HC HepG2 cells. The cells were incubated with different concentrations of GT1, 9 and 10 for 24 h, and then the relative copper content was detected using Rhodamine 6G hydrazide. For all chelators, the intracellular copper content of HC HepG2 cells gradually decreased as the concentration increases of GT1, 9 and 10. Consistent with the results in vitro, GT1 also has better elimination effects on copper in HC HepG2 cells (Fig. 4b). Respectively, 5 and 50 μM GT1 drastically eliminated ∼45% and ∼80% intracellular copper. For GT9 and 10, 50 μM chelator eliminated only ∼50% intracellular copper. However, 50 μM GT1 only slightly eliminated ∼15% and ∼20% intracellular copper in high-copper treated HeLa and MCF-7 cells, which suggested that GT1 could selectively eliminate copper for hepatocytes (Fig. S21b). Overall, our results suggested that GT1 can eliminate copper deposition in hepatocellular carcinomas more efficiently, which might help us control the deterioration of hepatocytes or kill the copper-deposited hepatocellular carcinomas.

Because the stability constants (logKCu) between copper and common copper-containing proteins are in the range of 14 to 17 (Banci et al., 2010); GT1, 9 and 10 should not interfere with the normal function of copper proteins (Table 1). To verify this hypothesis, we determined whether the chelator would inhibit the activity of copper/zinc superoxide dismutase (SOD1) using commercial kit. Our results reliably indicated that GT1 will not interfere with the catalytic activity of SOD1 (Fig. 4c). In general, the chelators we designed can effectively eliminate copper deposition in hepatocellular carcinomas, and will not affect the function of copper proteins. Among them, GT1 has the best performance, which can strongly bind with Cu2+ and successfully kill HC HepG2 cells at low doses.

3.5

3.5 Promotion of copper-deposited hepatocellular carcinomas apoptosis

The effect of GT1 on copper-deposited hepatocellular carcinomas apoptosis was observed via flow cytometry, because the elimination of copper is not conducive to the survival of HC HepG2 cells. The data showed that the HC HepG2 cells display a drastically elevation in apoptosis following exposure to GT1 for 24 h (Fig. 5). In detail, 5 μM GT1 promoted 33.9% of HC HepG2 cells to undergo apoptosis, and 10–50 μM GT1 increased the proportion of apoptotic cells to even more than 50%. As shown in Fig. S22a, 50 μM GT1 only promoted 15.9% of normal HepG2 cells to undergo apoptosis. These results indicated that eliminating the deposited copper in liver cancer cells can more effectively promote apoptosis, thereby killing such hepatocarcinoma cells. Earlier literature data also indicated that thiosemicarbazone-based metal chelator could cause a significant increasing in the population of apoptotic and dead cells (Bai et al., 2021). Therefore, both our results and literature data have shown that thiosemicarbazone-based metal chelator can kill cancer cells by promoting apoptosis. Furthermore, 50 μM GT1 only caused both apoptosis and death of 16.5% MCF-10A cells (human normal mammary epithelial cells), which indicated that GT1 has relatively small effects on non-cancerous cells (Fig. S22b).

Promotion of copper-deposited hepatocellular carcinomas apoptosis by GT1. After constructing the high-copper cell model, the HC HepG2 cells were incubated with 0, 5, 10 and 50 μM GT1 for another 24 h. For chelator treatment, the same amount of DMSO (5‰) was added as a control group, because DMSO was used as a co-solvent. Flow cytometric analysis of cell apoptosis was carried out with Annexin V and propidium iodide staining.
Fig. 5
Promotion of copper-deposited hepatocellular carcinomas apoptosis by GT1. After constructing the high-copper cell model, the HC HepG2 cells were incubated with 0, 5, 10 and 50 μM GT1 for another 24 h. For chelator treatment, the same amount of DMSO (5‰) was added as a control group, because DMSO was used as a co-solvent. Flow cytometric analysis of cell apoptosis was carried out with Annexin V and propidium iodide staining.

The chelator GT1 was designed via a combination of phenol, thiosemicarbazone, and lactobionic acid (Fig. 1). The use of thiosemicarbazone group enables GT1 to have suitable binding capacity with copper (log KCu2+ = 17.48 ± 0.11). While effectively removing the copper deposits of hepatocarcinoma cells, it will not interfere with the functions of other copper proteins (Fig. 4b and 4c). Besides, lactobionic acid group of GT1 can theoretically be recognized by hepatocellular ASGPR, which is expected to achieve hepatocyte-targeted chelating agent delivery. Because copper deposition is an important induction factor of hepatic carcinoma, GT1 may have application prospects in intervening copper deposition and cancerization of liver. For the copper-deposited cells that have already undergone cancerous transformation, GT1 can also promote cell apoptosis by chelating copper, thereby killing the cells (Figs. 4 and 5).

4

4 Conclusions

In this study, three specific and efficient copper chelators (GT1, 9 and 10) were designed through combining copper-coordinating group, phenol, and lactobionic acid. Among them, GT1 has the strongest binding capacity with copper, and can effectively chelate Cu2+ in the solution and in the cell. Through eliminating the intracellular copper, GT1 facilitates the apoptosis and death of HC HepG2 cells, a model of copper-deposited hepatocarcinoma cells. Due to its moderate copper-coordinating ability, GT1 will not interfere with the normal function of intracellular copper proteins, such as SOD1. Besides, the conjugation of lactobionic acid has the potential to achieve hepatic targeting delivery of chelators through ASGPR. Therefore, GT1 may have important application prospects in hepatic targeting copper elimination, which might be a potential method to inhibit copper-related cancerization of liver.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [21701128]; Hubei Provincial Natural Science Foundation of China [2017CFB206]; Educational Commission of Hubei Province of China [Q20171601].

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103241.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1


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