4-Chloro-7-Nitrobenzofurazan induces ROS-mediated p53 dependent apoptosis in human fibrosarcoma cells
E H T Thulshan Jayathilaka1, Moon-Moo Kim2
1Department of Cosmetics Engineering, Graduate School of Applied Chemistry·Food Engineering, Dong-Eui University, 176 Eomgwang-ro, Jin- gu, Busan, 47340, Republic of Korea.
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.
Insights
4-chloro-7-Nitrobenzofurazan (NBD) selectively targets human fibrosarcoma cells by inducing oxidative stress and apoptosis. This redox-targeting strategy shows promise for treating aggressive fibrosarcoma, a cancer with limited therapeutic options.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Human fibrosarcoma is an aggressive malignancy with poor therapeutic options.
- Targeting prooxidant pathways offers a potential strategy for fibrosarcoma treatment.
Purpose of the Study:
- To evaluate the anti-cancer effects of 4-chloro-7-Nitrobenzofurazan (NBD) on human fibrosarcoma HT-1080 cells.
- To elucidate the molecular mechanisms underlying NBD's anti-cancer activity.
Main Methods:
- Cell viability assessed via MTT assay in HT-1080 and IMR-90 cells.
- Apoptosis and cell cycle analyzed by flow cytometry.
- Intracellular reactive oxygen species (ROS) and signaling pathways (p53/p21) were evaluated using DCFDA, Western blotting, and reporter assays.
Main Results:
- NBD selectively reduced HT-1080 cell viability (IC50=1.83 µM) while sparing normal cells (IC50=34.02 µM).
- NBD induced apoptosis and G0/G1 cell cycle arrest by increasing intracellular ROS and suppressing catalase.
- NBD activated the p53 pathway, leading to caspase-3 activation and apoptosis, which was reversed by antioxidants or p53 knockout.
Conclusions:
- NBD induces selective apoptosis in fibrosarcoma cells through a ROS-dependent p53 pathway.
- NBD demonstrates potential as a novel redox-targeting therapeutic agent for fibrosarcoma.
