Molecular mechanism of pancreatic tumor metastasis inhibition by Gd@C82(OH)22 and its implication for de novo design of nanomedicine

Abstract

Pancreatic adenocarcinoma is the most lethal of the solid tumors and the fourth-leading cause of cancer-related death in North America. Matrix metalloproteinases (MMPs) have long been targeted as a potential anticancer therapy because of their seminal role in angiogenesis and extracellular matrix (ECM) degradation of tumor survival and invasion. However, the inhibition specificity to MMPs and the molecular-level understanding of the inhibition mechanism remain largely unresolved. Here, we found that endohedral metallofullerenol Gd@C(82)(OH)(22) can successfully inhibit the neoplastic activity with experiments at animal, tissue, and cellular levels. Gd@C(82)(OH)(22) effectively blocks tumor growth in human pancreatic cancer xenografts in a nude mouse model. Enzyme activity assays also show Gd@C(82)(OH)(22) not only suppresses the expression of MMPs but also significantly reduces their activities. We then applied large-scale molecular-dynamics simulations to illustrate the molecular mechanism by studying the Gd@C(82)(OH)(22)-MMP-9 interactions in atomic detail. Our data demonstrated that Gd@C(82)(OH)(22) inhibits MMP-9 mainly via an exocite interaction, whereas the well-known zinc catalytic site only plays a minimal role. Steered by nonspecific electrostatic, hydrophobic, and specific hydrogen-bonding interactions, Gd@C(82)(OH)(22) exhibits specific binding modes near the ligand-specificity loop S1', thereby inhibiting MMP-9 activity. Both the suppression of MMP expression and specific binding mode make Gd@C(82)(OH)(22) a potentially more effective nanomedicine for pancreatic cancer than traditional medicines, which usually target the proteolytic sites directly but fail in selective inhibition. Our findings provide insights for de novo design of nanomedicines for fatal diseases such as pancreatic cancer.

Fig. 1.

Antitumor effect of Gd@C₈₂(OH)₂₂ nanoparticles on a JF305 pancreatic carcinoma model. (A) Tumor growth curves of the nude mice treated with saline, C₆₀(OH)₂₂ nanoparticles (1.0 μmol/kg per day), and Gd@C₈₂(OH)₂₂ nanoparticles (0.2 μmol/kg per day or 1.0 μmol/kg per day). (B) Photographs of representative tumors from mice 26 d after receiving JF305 cell implantation. (C) ESEM microphotograph of the surface of tumors. The saline group (a and e); C₆₀(OH)₂₂ (1.0 μmol/kg per day) group (b and f); Gd@C₈₂(OH)₂₂ (0.2 μmol/kg per day) group (c and g); Gd@C₈₂(OH)₂₂ (1.0 μmol/kg per day) group (d and h). The magnified image shows the detailed form of blood vessels of the surface of tumors in corresponding groups.

Fig. 2.

Influences of Gd@C₈₂(OH)₂₂ treatment on the protein expression and mRNA levels of MMP-2 and MMP-9 in tumor tissues. (A) Immunofluorescence results of MMP-2 and MMP-9 in JF305 pancreatic tumors (200×). (B) Semiquantitative RT-PCR analysis of the genes MMP-2 and MMP-9. Protein levels of MMP-2 (C) and MMP-9 (D) in the tumor of mice treated with saline and C₆₀(OH)₂₂ and Gd@C₈₂(OH)₂₂ nanoparticles quantified by ELISA. Results are expressed as means ± SD. *P < 0.05; **P < 0.01, significantly different from the saline group. (E) The combined activity of MMP-9/MMP-2 was determined by a MMP enzyme-activity assay. Gd@C₈₂(OH)₂₂ effectively inhibits MMP-9/-2 activity, whereas C₆₀(OH)₂₂ has little effect on them compared with control.

Fig. 3.

Molecular dynamics with MMP-9 and Gd@C₈₂(OH)₂₂. (A) X-ray crystal structure of the catalytic domain of MMP-9: two Zn²⁺ and three Ca²⁺ are depicted with orange and pink balls, respectively. (B) Endohedral metallofullerenol Gd@C₈₂(OH)₂₂, where the Gd atom is presented inside the fullerenol cage with a pink ball. (C) Molecular dynamics setup with a central MMP-9 surrounded by four Gd@C₈₂(OH)₂₂ at the tetrahedral corners solvated with about 22,000 water molecules in a 90 Å × 90 Å × 90 Å cubic box. (D) Characteristic temporal snapshots of Gd@C₈₂(OH)₂₂ binding onto MMP-9. The metallofullerenol Gd@C₈₂(OH)₂₂ can form an aggregate, followed by binding to MMP-9 on the hydrophobic patch near the ligand specificity loop S1′.

Fig. 4.

Binding free-energy landscapes and residue-specific contacts on MMP-9, as well as representative binding modes and pathway of Gd@C₈₂(OH)₂₂ on MMP-9. (A) Binding free-energy surface for fullerenol C₈₂(OH)₂₂ on MMP-9 shows a nonspecific binding mode (Left), and almost all surface residues of MMP-9 contribute to contact with C₈₂(OH)₂₂ (Right). (B) Metallofullerenol Gd@C₈₂(OH)₂₂ interacts with MMP-9 along a specified binding mode (Left) and contacts with only a specific set of residues near the ligand-specificity S1′ loop and SC loop (Right). A residue was assigned to be in a contact when any atom in the residue was within 5.0 Å of any atom of Gd@C₈₂(OH)₂₂ [or C₈₂(OH)₂₂]. The site participation is presented by the total number of frames of each residue in contact normalized by all frames and trajectories. (C, Left) Representative binding mode (a solid ball) showing that Gd@C₈₂(OH)₂₂ binds between the S1′ ligand-specificity loop (green ribbon) and the SC loop (purple ribbon), leading to the ligand binding groove. An alternative mode with a gray ball is shown that Gd@C₈₂(OH)₂₂ can bind at the back entrance of the S1′ cavity leading into the active site (ball and stick for active sites and orange ball for the catalytic Zn²⁺). (Right) Possible binding pathway: depending on major driving forces and duration time (only the first 100 ns is shown), the binding dynamics is characterized with three different phases. Phase I: a diffusion-controlled nonspecific electrostatic interaction; phase II: a transient nonspecific hydrophobic interaction; and phase III: a specific hydrophobic and hydrogen-bonded stable binding.