We not only modified the types and numbers of coordinated ligands in a metal agent to enhance its anticancer activity, but we also designed a metal prodrug based on the N-donor residues of the human serum albumin (HSA) IIA subdomain to improve its delivery efficiency and selectivity in vivo. However, there may be a conflict in simultaneously achieving the two goals because Lys199 and His242 in the IIA subdomain of HSA can replace its two coordinated ligands, which will decrease its anticancer activity relative to the original metal agent. Thus, to improve the delivery efficiency of the metal agent and simultaneously avoid decreasing its anticancer activity in vivo, we decided to develop an anticancer metal prodrug by regulating its pharmacophore ligand so that it would not be displaced by the Lys199 residue of the folic acid (FA)-functionalized HSA nanoparticle (NP) carrier. To this end, we first synthesized two (E)-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide Schiff base (HL) Cu(II) compounds by designing a second ligand with a different coordinating atom with Cu2+/Cu(L)(QL)(Br) [C1, QL = quinolone] and Cu(L)(DMF)(Br) [C2, DMF = N,N-dimethylformamide]. As revealed by the structures of the two HSA complexes, the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. The QL ligand of C1 is replaced by Lys199, which coordinates with Cu2+, whereas the DMF ligand of C2 is kept intact and His242 is replaced with Br– of C2 and coordinates with Cu2+. The cytotoxicity of the Cu compounds was enhanced by the FA-HSA NPs in the Bel-7402 cells approximately 2–4-fold; however, they raise the cytotoxicity levels in the normal cells in vitro, and the FA-HSA NPs did not. Importantly, the in vivo data showed that FA-HSA-C2 NPs increased selectivity and the capacity to inhibit tumor growth and were less toxic than HSA-C2 NPs and C2. Moreover, C2/HSA-C2 NPs/FA-HSA-C2 NPs induced Bel-7402 cell death by potentially multiple mechanisms.Keywords: folic acid; human serum albumin; nanoparticles; prodrug; targeting;

•The processes of tautomeric change of ligands were calculated using DFT calculations.•The binding behavior of ligands with Cu(II) in aqueous medium containing 10% DMSO was examined.•The anticancer activity and anti-metastatic activity of these Cu(II) complexes were evaluated in vitro.•We revealed the anticancer mechanism of the Cu(II) complex.We synthesized two mixed-ligand Cu(II) complexes containing different aroylhydrazone ligands and a pyridine co-ligand, namely, [Cu(L1)(Py)] (C1) and [Cu(L2)(Py)(Br)] (C2) (L1 = (E)-2-hydroxy-N′-((2-hydroxynaphthalen-1-yl)methylene)benzohydrazide, Py = pyridine, L2 = (E)-2-hydroxy-N′-(phenyl(pyridin-2-yl)methylene)benzohydrazide), and assessed their chemical and biological properties to understand their marked activity. C2 showed better anticancer activity than C1 in various human cancer cell lines, including the cisplatin-resistant lung cancer cell line A549cisR. Both Cu(II) complexes, especially C2, displayed promising anti-metastatic activity against HepG2 cells. Spectroscopic titration and agarose gel electrophoresis experiments indicated that C2 exhibited binding affinity toward calf-thymus DNA and efficient pBR322 DNA-cleaving ability. Further mechanistic studies showed that C2 effectively induced DNA damage and thus led to cell cycle arrest at the G2/M phase, and also stimulated mitochondrial dysfunction mediated by reactive oxygen species and caspase-dependent apoptosis.Download high-res image (198KB)Download full-size image

To improve the selectivity, delivery, and activity of ferric (Fe) anticancer agents, we design prodrugs based on N-donor residues of the human serum albumin (HSA) carrier IIA subdomain. We synthesized six Fe(III) compounds derived from 2-hydroxy-1-naphthaldehyde thiosemicarbazone (7–12). HSA complex structure revealed that Fe compound binds to the hydrophobic cavity in the HSA IIA subdomain. Lys199 and His242 of HSA replace the two Cl atoms of Fe compound, coordinating with Fe3+. In vivo data revealed that compound 12 and HSA-12 complex inhibit the growth of the liver tumor and that the HSA-12 complex has stronger targeting ability and therapeutic efficacy than compound 12 alone. In addition, our results have shown that compound 12 and HSA-12 complex induce Bel-7402 cell death possible by several mechanisms.

To increase delivery efficiency, anticancer activity, and selectivity of anticancer metal agents in vivo, we proposed to develop the anticancer metal pro-drug based on His242 residue of the human serum albumin (HSA) carrier IIA subdomain. To confirm our hypothesis, we prepared two Cu(II) compounds [Cu(P4 mT)Cl and Cu(Bp44 mT)Cl] by modifying Cu(II) compound ligand structure. Studies with two HSA complex structures revealed that Cu(P4 mT)Cl bound to the HSA subdomain IIA via hydrophobic interactions, but Cu(Bp44 mT)Cl bound to the HSA subdomain IIA via His242 replacement of a Cl atom of Cu(Bp44 mT)Cl, and a coordination to Cu2+. Furthermore, Cu(II) compounds released from HSA could be regulated at different pHs. In vivo data revealed that the HSA-Cu(Bp44 mT) complex increased copper’s selectivity and capacity of inhibiting tumor growth compared to Cu(Bp44 mT)Cl alone.

•The DNA binding and cleavage properties of the ligand and its Fe complex have been investigated.•The proliferation inhibition of the ligand and its Fe complex was evaluated against various cancer cells.•The redox property of the Fe complex was examined by electrochemistry and biochemistry methods.•We revealed anticancer mechanism of the Fe(III) complex.We synthesized an α−N−heterocyclic thiosemicarbazone ligand (L) and its Fe complex (C1) and assessed their chemical and biological properties in order to understand their marked activity. Electrochemical studies and ascorbate oxidation studies demonstrated that C1 shows considerable redox activity, and FeIII/II redox potentials was within the range accessible to cellular oxidants and reductants. Absorption spectral, emission spectral and viscosity analysis reveal that L and C1 interacted with DNA through intercalation and C1 exhibited a higher DNA binding ability. Agarose gel electrophoresis experiments indicated that C1 exhibited the highest pBR322 DNA cleaving ability. In vitro, C1 showed significantly more anticancer activity than the ligand alone. Moreover, C1 induces production of reactive oxygen species (ROS) and DNA damage, resulting in activation of the p53 pathway, cell cycle arrest at the S phase, and mitochondria-mediated apoptosis by regulating the expression of Bcl−2 family proteins.

•Four Cu(II)-Schiff base complexes were synthesized through anion regulation.•Cu compounds possess high anticancer activity.•Cu compounds induce cancer cells apoptosis possible via mitochondrial pathway.•Cu compounds regulate the proteins of Bcl-2 family.Copper (Cu) compounds are a promising candidate for next generation metal anticancer drugs. Therefore, we regulated anions to synthesize four mononuclear and binuclear Cu(II) compounds derived from thiosemicarbazone Schiff base ligands and characterized them. Four of these compounds showed very high cytotoxicity to cancer cell lines in vitro. These Cu(II) compounds strongly promoted the apoptosis of BEL-7404 cells and had a capacity to arrest the cell cycle at S phase of those cells. Furthermore, reactive oxygen species (ROS), mitochondrial membrane potential and Western blot analyses revealed that these Cu(II) compounds exert their cytotoxicity through an ROS-mediated intrinsic mitochondrial pathway accompanied by the regulation of Bcl-2 family proteins.

When administering several anticancer drugs within a single carrier, it is important to regulate their spatial distribution so as to avoid possible mutual interference and to thus enhance the drugs’ selectivity and efficiency. To achieve this, we proposed to develop human serum albumin (HSA)-based multidrug delivery systems for combination anticancer therapy. We used three anticancer agents (an organic drug [5-fluorouracil, or 5FU], a metallic agent [2-benzoylpyridine thiosemicarbazide copper II, or BpT], and a gene agent [AS1411]) to treat liver cancer and confirm our hypothesis. The structure of the HSA-palmitic acid (PA)-5FU-BpT complex revealed that 5FU and BpT, respectively, bind to the IB and IIA subdomains of HSA. Our MALDI-TOF-MS spectral data show that one AS1411 molecule is conjugated to Cys-34 of the HSA-5FU-BpT complex via a linker. Compared with unregulated three-drug combination therapy, the HSA-5FU-BpT-AS1411 complex enhances cytotoxicity in Bel-7402 cells approximately 7-fold in vitro; however, in normal cells it does not raise cytotoxicity levels. Importantly, our in vivo results demonstrate that the HSA-5FU-BpT-AS1411 complex is superior to the unregulated three-drug combination in enhancing targeting ability, inhibiting liver tumor growth, and causing fewer side effects.Keywords: anticancer drug; combination therapy; drug delivery systems; human serum albumin; protein structure;

To synergistically enhance the selectivity and efficiency of anticancer copper drugs, we proposed and built a model to develop anticancer copper pro-drugs based on the nature of human serum albumin (HSA) IIA subdomain and cancer cells. Three copper(II) compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine, imidazole, or indazole ligands were synthesized (C1–C3). The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. Among them, the pyridine and imidazole ligands of C1 and C2 are replaced by Lys199, and His242 directly coordinates with Cu(II). The indazole and Br ligands of C3 are replaced by Lys199 and His242, respectively. Compared with the Cu(II) compounds alone, the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3–5-fold, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent. We find that the HSA complex has a stronger capacity for cell cycle arrest in the G2/M phase of MCF-7 by targeting cyclin-dependent kinase 1 (CDK1) and down-regulating the expression of CDK1 and cyclin B1. Moreover, the HSA complex promotes MCF-7 cell apoptosis possibly through the intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

•Four binuclear Cu(II)-Schiff base complexes were synthesized and characterized.•Cu complexes have high anticancer activity.•Cu complexes induce cells apoptosis possible via intrinsic ROS-mediated mitochondrial pathway.•Cu complexes regulate Bcl-2 family proteins.Copper (Cu) compounds are a promising candidate for next generation metal anticancer drugs and have been extensively studied. Therefore, four binuclear copper(II) compounds derived from Schiff base thiosemicarbazones (L1–L4), namely [CuCl(L1)]2 (C1), [CuNO3(L2)]2 (C2), [Cu(NCS) (L3)]2 (C3) and [Cu(CH3COO) (L4)]2 (C4) were synthesized and characterized. Four of these compounds showed very high cytotoxicity to cancer cell lines in vitro. These Cu(II) compounds strongly promoted the apoptosis of BEL-7404 cells. The formation of reactive oxygen species (ROS), change in mitochondrial membrane potential and western blot analysis revealed that Cu compounds could induce cancer cell apoptosis through the intrinsic ROS-mediated mitochondrial pathway accompanied by the regulation of Bcl-2 family proteins.

•Cu(II) compounds derived from plumbagin were synthesized and characterized.•Folic acid-human serum albumin (FA-HSA) has been used as a carrier for copper complexes.•Anticancer activities and targeting of the Cu compound is improved when bound to FA-HSA.•FA-HSA-metallodrug regulates expression of CDK1, cyclin B1 and proteins of Bcl-2 family.The folate (FA)-functionalized human serum albumin (HSA) carrier (FA-HSA) is promising for improving the target and efficiency of anticancer drugs. To develop FA-HSA carrier for metal anticancer drugs, we investigated anticancer properties and mechanism of FA-HSA carrier for Cu(II) complexes derived from plumbagin. The fluorescence spectroscopy and molecular docking revealed that Cu(II) complexes bind to IIA subdomain of HSA. Compared with Cu(II) complex alone, FA-HSA-metallodrug complex enhances cytotoxicity to FA-positive cancer cells (HeLa) but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent; FA-HSA-metallodrug complex has a stronger capacity for cell cycle arrest in the G2/M phase of HeLa cells, and down-regulating the expression of cyclin-dependent kinase 1 (CDK1) and cyclin B1. Moreover, FA-HSA-metallodrug complex promotes HeLa cells apoptosis through intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.Selectivity and efficiency of anticancer copper agents are improved through use of a folic acid-functionalized human serum albumin (FA-HSA) carrier.