2-aryl benzimidazole conjugate induced apoptosis in human breast cancer MCF-7 cells through caspase independent
V Lakshma Nayak1, Narayana Nagesh2, A Ravikumar1,3
1Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India.
Abstract:
Apoptosis is a representative form of programmed cell death, which has been assumed to be critical for cancer prevention. Thus, any agent that can induce apoptosis may be useful for cancer treatment and apoptosis induction is arguably the most potent defense against cancer promotion. In our previous studies, 2-aryl benzimidazole conjugates were synthesized and evaluated for their antiproliferative activity and one of the new molecule (2f) was considered as a potential lead. This lead molecule showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The results of the present study revealed that this compound arrested the cell cycle at G2/M phase. Topoisomerase II inhibition assay and Western blot analysis suggested that this compound effectively inhibits topoisomerase II activity which leads to apoptotic cell death. Apoptosis induction in MCF-7 cells was further confirmed by loss of mitochondrial membrane potential (∆Ψm), release of cytochrome c from mitochondria, an increase in the level of apoptosis inducing factor (AIF), generation of reactive oxygen species (ROS), up regulation of proapoptotic protein Bax and down regulation of anti apoptotic protein Bcl-2. Apoptosis assay using Annexin V-FITC assay also suggested that this compound induced cell death by apoptosis. However, compound 2f induced apoptosis could not be reversed by Z-VAD-FMK (a pan-caspase inhibitor) demonstrated that the 2f induced apoptosis was caspase independent. Further, 2f treatment did not activate caspase-7 and caspase-9 activity, suggesting that this compound induced apoptosis in breast cancer cells via a caspase independent pathway. Most importantly, this compound was less toxic towards non-tumorigenic breast epithelial cells, MCF-10A. Furthermore, docking studies also support the potentiality of this molecule to bind to the DNA topoisomerase II.
Insights
A novel 2-aryl benzimidazole derivative (2f) effectively induces caspase-independent apoptosis in human breast cancer cells by inhibiting topoisomerase II. This compound shows potential as a less toxic breast cancer therapeutic agent.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Apoptosis, or programmed cell death, is crucial for cancer prevention and a potential target for cancer treatment.
- 2-Aryl benzimidazole conjugates have shown antiproliferative activity in previous studies.
- Identifying novel agents that induce apoptosis is essential for developing effective cancer therapies.
Purpose of the Study:
- To investigate the antiproliferative and apoptosis-inducing mechanisms of a novel 2-aryl benzimidazole derivative (compound 2f) against human breast cancer cells (MCF-7).
- To determine if the induced apoptosis is caspase-dependent or independent.
- To assess the compound's toxicity towards non-tumorigenic breast epithelial cells.
Main Methods:
- Cell cycle arrest analysis at G2/M phase.
- Topoisomerase II inhibition assay and Western blot analysis.
- Mitochondrial membrane potential (∆Ψm) assessment, cytochrome c release, AIF and ROS levels, and Bax/Bcl-2 protein expression analysis.
- Annexin V-FITC assay for apoptosis detection.
- Caspase activity assays and treatment with a pan-caspase inhibitor (Z-VAD-FMK).
- Cytotoxicity assessment on MCF-10A cells and molecular docking studies.
Main Results:
- Compound 2f demonstrated significant antiproliferative activity against MCF-7 cells.
- The compound induced cell cycle arrest at the G2/M phase and inhibited topoisomerase II activity.
- Apoptosis induction was confirmed through various assays, including loss of mitochondrial membrane potential, cytochrome c release, increased AIF and ROS, and altered Bax/Bcl-2 expression.
- Importantly, the apoptosis induced by compound 2f was found to be caspase-independent, as it was not reversed by Z-VAD-FMK and did not activate caspase-7 or caspase-9.
- Compound 2f exhibited lower toxicity towards non-tumorigenic MCF-10A cells compared to cancer cells.
- Docking studies supported the compound's ability to bind to DNA topoisomerase II.
Conclusions:
- Compound 2f is a potent inducer of caspase-independent apoptosis in human breast cancer cells, primarily through topoisomerase II inhibition.
- The selective toxicity towards cancer cells and lower toxicity towards normal cells suggest its potential as a targeted breast cancer therapeutic.
- Further investigation into compound 2f as a lead for novel breast cancer drug development is warranted.
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