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Objective To evaluate Carissa spinarum stem isolate for its anti-cancer therapeutic potential. Methods The n-butanol fraction of aqueous extract from Carissa spinarum stem was assessed for its cytotoxic and pro-apoptotic activity. Key findings We report for the first time the anti-cancer potential of C. spinarum stem aqueous extract (CSE) and its n-butanol fraction (CSF). Both inhibited cell proliferation of various human cancer cell lines in which leukaemia HL-60 cells treated with CSF showed maximum growth inhibition having an inhibitory concentration (IC50) value of 34.58 ± 0.91 µg/ml. In addition, CSF induced concentration-dependent apoptosis in HL-60 cells as measured by various end-points (e.g. Annexin V binding, DNA laddering, apoptotic body formation and an increase in hypodiploid subG0 DNA content). Moreover, persistent levels of reactive oxygen species caused translocation of Bax to mitochondria and Bcl-2 degradation, which led to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. These events were associated with significant activation of caspase-3, caspase-6 and caspase-9 leading to poly (ADP-ribose) polymerase cleavage. Conclusion All the above parameters revealed that CSF induced apoptosis through the mitochondrial dependent pathway in HL-60 cells. [ABSTRACT FROM AUTHOR]