ROS mediated ER stress induces Bax-Bak dependent and independent apoptosis in response to Thioridazine
Mahendra Seervi1, Ankita Rani1, Abhay K Sharma1
1Department of Botany/Biotechnology, Patna University, Patna, Bihar, India.
Abstract:
A dopamine receptor antagonist, Thioridazine (TDZ) is known for its cytotoxic activity against various cancers and its role in combinational chemotherapy is being actively investigated. Several molecular targets of TDZ have been studied to delineate its anticancer activities, with contrasting findings in different cancer types. Moreover, the underlying mechanism of cell death from TDZ treatment is not well defined. In the current study, we studied TDZ mediated cell death mechanism employing cervical cancer cells. TDZ treatment induced nuclear condensation, mitochondrial membrane potential loss, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3 substantiating mitochondrial pathways of apoptosis in cells. TDZ induced ROS generation and up-regulation of ER stress linked proteins, such as CHOP, BiP etc. ER stress and apoptosis caused by TDZ were prevented by ROS inhibitor N-acetyl-L-cysteine (NAC) and protein synthesis inhibitor cycloheximide. In TDZ mediated cytocidal cellular process, autophagy acted as a cell survival factor as the inhibition of autophagy by 3-Methyladenine resulted in increased cell death. TDZ induced apoptosis was associated with decreased Bcl-2 expression and the overexpression of Bcl-2 resulted in inhibition of apoptosis. Studies in Bax-Bak knock-out cell model indicated that TDZ trigger both the Bax-Bak dependent and independent apoptosis through ROS. In the presence of Bax and Bak, cells are more sensitised to death than in the absence of these proteins. Both Bax-Bak dependent and independent apoptosis were significantly inhibited by ROS inhibitor NAC. Conclusively, TDZ induced Bax-Bak dependent and independent apoptosis by enhancing ROS production followed by ER stress.
Insights
Thioridazine (TDZ) triggers cervical cancer cell death via apoptosis, involving mitochondrial pathways, reactive oxygen species (ROS), and endoplasmic reticulum (ER) stress. Autophagy inhibition enhances TDZ
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Thioridazine (TDZ), a dopamine receptor antagonist, exhibits cytotoxic effects against various cancers.
- Its precise anticancer mechanisms and cell death pathways remain incompletely understood.
- Investigating TDZ's role in cervical cancer is crucial for potential therapeutic applications.
Purpose of the Study:
- To elucidate the mechanism of Thioridazine (TDZ)-induced cell death in cervical cancer cells.
- To identify key molecular pathways, including apoptosis, ROS generation, and ER stress, involved in TDZ's cytotoxic effects.
- To assess the role of autophagy and Bcl-2 family proteins in TDZ-mediated cell death.
Main Methods:
- Cervical cancer cell lines were treated with Thioridazine (TDZ).
- Apoptosis was assessed via nuclear condensation, mitochondrial membrane potential, cytochrome c release, and caspase activation.
- Reactive oxygen species (ROS) generation, ER stress markers (CHOP, BiP), autophagy, and Bcl-2 family proteins were analyzed.
- ROS inhibitor N-acetyl-L-cysteine (NAC), protein synthesis inhibitor cycloheximide, autophagy inhibitor 3-Methyladenine, and Bax-Bak knock-out models were employed.
Main Results:
- TDZ induced apoptosis through mitochondrial pathways, evidenced by caspase-9 and caspase-3 activation.
- TDZ treatment led to increased ROS generation and ER stress.
- Autophagy inhibition enhanced TDZ-induced cell death, indicating a pro-survival role for autophagy.
- TDZ decreased Bcl-2 expression, and its overexpression inhibited apoptosis.
- TDZ triggered both Bax-Bak dependent and independent apoptosis, significantly mediated by ROS.
Conclusions:
- Thioridazine (TDZ) induces apoptosis in cervical cancer cells via ROS production, leading to ER stress and mitochondrial pathway activation.
- Autophagy acts as a survival mechanism, while Bcl-2 downregulation promotes TDZ-induced cell death.
- TDZ elicits both Bax-Bak dependent and independent apoptotic pathways, highlighting its complex cytotoxic mechanism.
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