4-Chloro-7-Nitrobenzofurazan induces ROS-mediated p53 dependent apoptosis in human fibrosarcoma cells.

BACKGROUND

Human fibrosarcoma is an aggressive soft tissue malignancy with limited targeted therapies, that prone to rapid metastasis and chemotherapy resistance. Targeting prooxidant pathways represents a promising clinical strategy for the control of fibrosarcoma.

OBJECTIVE

This study evaluated the anti-cancer efficacy and underlying molecular mechanism of 4-chloro-7-Nitrobenzofurazan (NBD) in human fibrosarcoma HT-1080 cells.

METHODS

Cell viability was determined by MTT assay in HT-1080 cells and normal lung fibroblast (IMR-90). Apoptotic populations and cell cycle progression were determined by flow cytometry using Annexin V-FITC/PI and propidium iodide (PI). Intracellular ROS levels were analyzed using DCFDA. Signaling pathways were assessed through p53/p21 luciferase reporter assay and Western blotting and validated using antioxidant control (Glutathione; GSH) and genetic control (p53 knockout HT-1080).

RESULTS

NBD selectively decreased HT-1080 cell viability (IC50 - 1.83 µM) while sparing the IMR-90 cells (IC50 - 34.02 µM). Flow cytometry analysis revealed concentration-dependent induction of late apoptosis and sub-G0/G1 phase DNA fragmentation. Mechanistically, NBD generated intracellular ROS and suppressed the catalase expression, disrupting the redox homeostasis. This oxidative stress induced a biphasic p21 response with potent p53 activation that shifted the cells from cytostatic arrest to apoptosis. Consequently, BCL-2 family proteins shifted toward the pro-apoptotic path, triggering executioner cleaved caspase-3 activation. ROS scavenging by GSH completely blocked p53 upregulation and caspase-3 cleavage, restoring cell viability. Furthermore, p53 KO completely diminished the NBD mediated caspase-3 activation and restored the cell survival.

CONCLUSION

NBD triggers selective apoptosis in HT-1080 cells via the ROS-induced p53 pathway, highlighting its potential as a redox-targeting therapeutic candidate.