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Updated: Feb 13, 2026
In vitro Cell Migration and Invasion Assays
Published on: June 1, 2014
Caspase-3 regulates the migration, invasion and metastasis of colon cancer cells
Min Zhou1,2, Xinjian Liu2, Zonghai Li1
1State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Abstract:
Caspase-3 (CASP3) is a major mediator of apoptosis activated during cellular exposure to cytotoxic drugs, radiotherapy or immunotherapy. It is often used as a marker for efficacy of cancer therapy. However, recent reports indicate that caspase-3 has also non-apoptotic roles such as promotion of tumor relapse and tumor angiogenesis. Therefore, the roles of caspase-3 in tumor progression remain to be defined clearly. In our study, we established caspase-3 knockout (KO) colon cancer cell lines by use of the CRISPR technology. In vitro, caspase-3 knockout HCT116 cells were significantly less clonogenic in soft agar assays. They were also significantly less invasive and more sensitive to radiation and mitomycin C than control cells. In vivo, CASP3KO cells formed tumors at rates similar to control cells but were significantly more sensitive to radiotherapy. They were also less prone to pulmonary metastasis when inoculated either subcutaneously or intravenously. At the mechanistic level, caspase-3 gene knockout appeared to cause reduced EMT phenotypes when compared to parental HCT116 cells. Indeed, they showed significantly increased E-cadherin expression, reduced N-cadherin, Snail, Slug and ZEB1 expression than control cells. Therefore, therapeutic targeting of caspase-3 may not only increase the sensitivity of cancer cell to chemotherapy and radiotherapy, but also inhibit cancer cell invasion and metastasis.
Insights
Caspase-3 (CASP3) promotes cancer progression and metastasis. Removing CASP3 in colon cancer cells reduced their ability to grow, invade, and spread, suggesting CASP3 as a potential therapeutic target.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Caspase-3 (CASP3) is a key apoptosis mediator and cancer therapy efficacy marker.
- Emerging evidence suggests CASP3 also drives tumor relapse and angiogenesis, necessitating clarification of its role in tumor progression.
Purpose of the Study:
- To investigate the multifaceted roles of caspase-3 in colon cancer progression.
- To determine the impact of caspase-3 knockout on cancer cell behavior in vitro and in vivo.
Main Methods:
- CRISPR-Cas9 technology was used to generate caspase-3 knockout (CASP3KO) HCT116 colon cancer cell lines.
- In vitro assays assessed clonogenicity, invasion, and sensitivity to radiation and mitomycin C.
- In vivo studies evaluated tumor formation, radiotherapy sensitivity, and pulmonary metastasis.
- Mechanistic analysis focused on epithelial-mesenchymal transition (EMT) markers.
Main Results:
- CASP3KO cells exhibited significantly reduced clonogenicity, invasion, and increased sensitivity to radiation and mitomycin C in vitro.
- In vivo, CASP3KO tumors were more sensitive to radiotherapy and showed reduced pulmonary metastasis.
- Caspase-3 gene knockout led to decreased EMT phenotypes, evidenced by increased E-cadherin and reduced N-cadherin, Snail, Slug, and ZEB1 expression.
Conclusions:
- Caspase-3 plays a significant role in promoting colon cancer cell growth, invasion, and metastasis.
- Targeting caspase-3 may enhance sensitivity to cancer therapies and inhibit tumor progression and metastasis.
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