Mechanism of RIP2 enhancing stemness of glioma cells induces temozolomide resistance

by Selleckchem | Jan 06 2024

Aims: We aimed to investigate the role of receptor-interacting protein 2 (RIP2) in regulation of stemness of glioma cells and chemotherapy resistance.

Methods: Plasmid transfection was used to overexpress RIP2. Chemical inhibitors were used to inhibit RIP2 or NF-κB activity. Cancer stemness of glioma cells was investigated by sphere formation assays, clone formation assays, and xenograft tumor formation assays. The expression of RIP2, p-NF-κB, IκBα, CD133, or SOX-2 was detected by Western blotting and immunofluorescence. Apoptosis was detected by flow cytometry. Immunohistochemical staining was used to detect the expression of RIP2, CD133, and SOX-2 in xenograft tumor tissue. The effect of the RIP2/NF-κB pathway on temozolomide (TMZ) resistance was evaluated by xenograft tumor assay.

Results: Transfection with RIP2 plasmid enhanced the sphere formation capability of U251 cells, clone formation capability, and xenograft tumor formation capability. RIP2 could mediate TMZ resistance by upregulating the expression of CD133 and SOX-2 by activating the NF-κB pathway. Both RIP2 inhibitor GSK583 and the NF-κB inhibitor SC75741 could reverse the resistance of U251 cells to TMZ.

Conclusion: RIP2 mediates TMZ resistance by regulating the maintenance of stemness in glioma cells through NF-κB. Interventions targeting the RIP2/NF-κB pathway may be a new strategy for TMZ-resistant gliomas.

Comments:

This study explores the impact of receptor-interacting protein 2 (RIP2) on glioma cell stemness and their resistance to chemotherapy, particularly temozolomide (TMZ). Here's a breakdown of the key findings and methods:

### **Key Methods Used:**
- **Plasmid Transfection:** To overexpress RIP2 in glioma cells.
- **Chemical Inhibitors:** Used to inhibit RIP2 or NF-κB activity.
- **Assays:**
- **Sphere Formation Assays:** Investigating cancer stemness.
- **Clone Formation Assays:** Assessing cell proliferation and survival.
- **Xenograft Tumor Formation Assays:** Analyzing tumor growth in vivo.
- **Analysis Techniques:**
- **Western Blotting:** Determining protein expression levels.
- **Immunofluorescence:** Visualizing protein expression.
- **Flow Cytometry:** Detecting apoptosis in cells.
- **Immunohistochemical Staining:** Assessing protein expression in tumor tissue.

### **Key Results:**
- **RIP2 Overexpression Effects:**
- Enhanced sphere formation, clone formation, and xenograft tumor formation capabilities in U251 cells.
- **Role in TMZ Resistance:**
- RIP2 mediated TMZ resistance by increasing CD133 and SOX-2 expression via NF-κB activation.
- Inhibition of RIP2 or NF-κB reversed TMZ resistance in U251 cells.

### **Conclusion:**
- **RIP2/NF-κB Pathway:** Mediates TMZ resistance by regulating glioma cell stemness.
- **Therapeutic Implications:** Targeting the RIP2/NF-κB pathway might offer a novel strategy for overcoming TMZ-resistant gliomas.

This study's findings suggest that manipulating the RIP2/NF-κB pathway could be a promising avenue for developing therapies to combat TMZ resistance in gliomas.