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Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3
Meng-Chieh Lin1, Guan-Yu Chen1, Hsin-Hsien Yu2,3
1School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
Aims:
Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro.
Methods:
MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry.
Results:
Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells.
Conclusion:
Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.
Insights
Benzamil, a sodium-calcium exchange blocker, effectively inhibits osteosarcoma cell growth by inducing apoptosis. This drug also potentiates chemotherapy by disrupting integrin signaling and mitochondrial function.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Osteosarcoma is a prevalent bone cancer in children and adolescents.
- Investigating novel therapeutic agents for osteosarcoma is crucial.
Purpose of the Study:
- To evaluate the in vitro therapeutic potential of benzamil, an amiloride analogue and sodium-calcium exchange blocker, against osteosarcoma.
- To elucidate the molecular mechanisms underlying benzamil's anti-osteosarcoma activity.
Main Methods:
- Osteosarcoma cell lines (MG63 and U2OS) were treated with benzamil.
- Cell viability, apoptosis markers (TUNEL, PARP cleavage, caspase-7, annexin V/PI), intracellular calcium, integrin expression, FAK/STAT3 activation, mitochondrial function (TMRE, ATP), and anti-apoptotic protein levels were assessed.
- Western blot and flow cytometry were primary analytical techniques.
Main Results:
- Benzamil significantly suppressed osteosarcoma cell viability by inducing apoptosis.
- Benzamil reduced cell surface integrin expression (α5, αV, β1), inhibited FAK and STAT3 phosphorylation, and impaired mitochondrial function and ATP production.
- Benzamil decreased levels of anti-apoptotic proteins (XIAP, Bcl-2, Bcl-xL) and enhanced cisplatin- and methotrexate-induced apoptosis.
Conclusions:
- Benzamil demonstrates significant anti-osteosarcoma activity through apoptosis induction.
- The mechanism involves the inhibition of integrin/FAK/STAT3 signaling, leading to mitochondrial dysfunction and ATP depletion.
- Benzamil shows promise as a therapeutic agent and chemosensitizer for osteosarcoma.
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