RESUMO
BACKGROUND: Breast cancer is a commonly diagnosed cancer worldwide. Human MutT homolog 1 (MTH1) is found to be elevated in breast tumors and cancer cells need MTH1 for survival. Pharmacological inhibition of MTH1 may be potentially beneficial in the treatment of breast cancer. METHODS: MA-24 was screened by malachite green colorimetric assay for MTH1 inhibitors and the kinetic characteristics of MA-24 were assessed. The features of MA-24's binding with MTH1 were ascertained through molecular docking, and the cytotoxic activity of MA-24 was validated in vitro and in vivo. Target engagement assays, comet assay, and Western blot confirmed the intracellular target and mechanism of MA-24. RESULTS: MA-24 shows potent antitumor bioactivity both in vitro and in vivo. MA-24 competitively inhibited the MTH1 and further induced DNA strand breaks, leading to increased apoptosis of cancer cells depending on the upregulation of the cleaved-caspase 3-cleaved-PARP axis. In particular, MA-24 exhibited a powerful efficacy and safety in vivo (tumor growth inhibition rate: 61.8%). CONCLUSIONS: MA-24 possesses a broad spectrum of breast cancer cytotoxicity and offered valuable insights for overcoming the challenges of chemotherapy-related toxicity, which holds great potential for the further development MA-24 as an anti-cancer drug.
RESUMO
Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumourtargeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an NiNTA column and a G50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by highperformance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec1, respectively. The antihMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.
Assuntos
Anticorpos Monoclonais/imunologia , Enzimas Reparadoras do DNA/imunologia , Enzimas Reparadoras do DNA/metabolismo , Desenvolvimento de Medicamentos/métodos , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular Tumoral , Clonagem Molecular , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios Enzimáticos/métodos , Humanos , Oxirredução , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.