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Published on: July 12, 2018
The Inhibitory Effects of Anti-GPC3 Antibody on Wnt/β-Catenin Signaling Pathway as a Biological Therapy in Liver
Qin Gan1, Jia Shao2, Tingli Sun3
1Department of Hepatobiliary and Pancreatic Surgery, Jiujiang First People's Hospital, Jiujiang City Key Laboratory of Cell Therapy, Jiujiang, 332000, Jiangxi, China.
Abstract:
To investigate the effects of anti-GPC3 antibody on the Wnt/catenin pathway in liver cancer biology, thus providing a new target for the biological treatment of the disease. A total of 12 BALB/C experimental nude mice were selected as experimental objects. The mice were all male, weighed 15-20 g and aged 4-5 weeks. First, mouse liver cancer models were constructed. Then, the HepG2 liver cancer cells in logarithmic growth period were inoculated into the caudal veins of mouse models. On the 3rd day after inoculation, the tumor formations of nude mice were observed. Second, the anti-GPC3 antibody was designed and constructed, and the activity of anti-GPC3 antibody was detected. The mouse models were divided into the experimental group (EG) and the control group (CG), with 6 mice in each group. In the EG, mice were injected with 3 mg/kg of anti-GPC3 antibody in the caudal veins once a day for three consecutive days, while in the CG mice were injected with the same amount of saline as the control. Mice in both groups received 3 injections in total. After the last administration, all mice were euthanized using the decapitation method following anesthesia with ethyl ether. The liver cancer cells of nude mice were extracted and cultured in DMEM medium. The effects of anti-GPC3 antibody on Wnt/β-catenin in liver cancer nude mice and the effects of anti-GPC3 antibody on epithelial-mesenchymal transition (EMT) in mouse liver cancer cells were observed. The bodyweight, liver weight and index of mice in the EG increased significantly (P < 0.05). The serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) levels of mice in the EG were reduced than those in the CG (P < 0.05). Compared with the CG, the superoxide dismutase (SOD) concentration increased, while the malonaldehyde (MDA) concentration decreased in the liver tissues of mice in the EG (P < 0.05). There was binding activity between GPC3 recombinant protein and the antibody; the affinity constant was KD = 1.4 × 10-6 M, compared with the commercial anti-GPC3 antibody, the affinity constant was lower. The interference effects of siRNA on anti-GPC3 antibody were detected by Western Blot. After the injection of anti-GPC3 antibody, the expression of β-catenin siRNA in liver cancer cells decreased significantly. The optical microscope images of mouse liver cancer cells in groups were compared. Through down-regulating the Wnt/β-catenin by anti-GPC3 antibody, the morphology of epithelial cells was maintained, the cells were arranged orderly, the cubic structure was kept stable, and the occurrence of EMT was reduced. However, in the CG, the structure of mouse liver cancer cells was disordered, the obvious EMT occurred (P < 0.05). Through down-regulating the expression of Wnt/β-catenin by anti-GPC3 antibody, the invasiveness, and metastasis of liver cancer cells could be effectively inhibited. Compared with the CG, the number of cells passing through the chamber was significantly reduced (P < 0.05). The anti-GPC3 antibody had inhibitory effect on Wnt/β-catenin signaling pathway. The occurrence of EMT and the invasiveness of liver cancer cells were inhibited effectively. Therefore, the anti-GPC3 antibody could be used as a reference for clinical molecular targeted therapy of liver cancer.
Insights
This study shows that anti-GPC3 antibody treatment inhibits liver cancer progression by down-regulating the Wnt/β-catenin pathway, reducing epithelial-mesenchymal transition (EMT), and decreasing cancer cell invasiveness.
Area of Science:
- Oncology
- Molecular Biology
- Immunotherapy
Background:
- Hepatocellular carcinoma (HCC) remains a significant global health challenge.
- The Wnt/β-catenin signaling pathway is frequently dysregulated in liver cancer, promoting tumor growth and metastasis.
- Glypican-3 (GPC3) is an oncofetal protein overexpressed in HCC, making it a promising therapeutic target.
Purpose of the Study:
- To investigate the therapeutic potential of an anti-GPC3 antibody in liver cancer.
- To elucidate the effects of anti-GPC3 antibody on the Wnt/β-catenin pathway and epithelial-mesenchymal transition (EMT).
- To evaluate the impact of anti-GPC3 antibody on liver cancer cell invasiveness and metastasis.
Main Methods:
- Construction of mouse liver cancer models using HepG2 cells inoculated into nude mice.
- Administration of anti-GPC3 antibody or saline (control) via caudal vein injection.
- Assessment of tumor formation, liver function markers (ALT, AST, ALP), oxidative stress markers (SOD, MDA), GPC3-antibody binding affinity, Wnt/β-catenin pathway activity, EMT markers, and cell invasiveness.
Main Results:
- Anti-GPC3 antibody treatment led to significant increases in body and liver weight, alongside reduced liver enzymes (ALT, AST, ALP) and improved oxidative stress markers (increased SOD, decreased MDA) in mice.
- The anti-GPC3 antibody demonstrated binding activity to GPC3 recombinant protein with a KD of 1.4 × 10-6 M.
- Treatment significantly downregulated Wnt/β-catenin signaling, suppressed EMT, maintained epithelial cell morphology, and markedly reduced the invasiveness and metastasis of liver cancer cells.
Conclusions:
- Anti-GPC3 antibody effectively inhibits liver cancer progression by targeting the Wnt/β-catenin pathway.
- The antibody suppresses EMT and reduces cancer cell invasiveness and metastasis.
- Anti-GPC3 antibody shows promise as a novel molecular targeted therapy for liver cancer.
