Cyproheptadine activates the mitochondrial pathway of caspase activation. (A) OCI-MY5 myeloma cells (2 × 10⁴) were treated with increasing concentrations of cyproheptadine (CYP) with and without the pan-caspase inhibitor z-VAD-fmk (100 μM). Forty-eight hours after incubation, cell viability was measured by MTS assay. Data represent the mean percentage plus or minus SD of viable cells compared with control treated controls. (B) OCI-MY5 and OPM1 myeloma cells and OCI-AML2 and CEM leukemia cells were treated with increasing concentrations of cyproheptadine. Twenty-four hours after treatment, total cellular protein was isolated and analyzed by SDS-PAGE/immunoblotting using anti–caspase-3 (Pro-C3), anti–caspase-8 (Pro-C8), anti–caspase-9 (Pro-C9), and anti–β-actin. Cle-C8 indicates cleaved caspase-8. (C) U266 and NB4 cells (2 × 10⁶) were treated with cyproheptadine (CYP, 20 μM) for increasing times. After incubation, cells were harvested and stained with DiIC₁(5) and PI to measure mitochondrial membrane potential (ΔΨ_M) and plasma membrane integrity, respectively. Results were analyzed by flow cytometry. (D) MDAY-D2 and LP-1 cells (2 × 10⁶ cells) were treated with increasing concentrations of cyproheptadine. Forty-eight hours after incubation, cells were harvested and stained with DiIC₁(5) and PI to measure mitochondrial membrane potential (ΔΨ_M). Results were analyzed by flow cytometry.