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Objective: Leaves of Bichofia javanica (BJ) have been traditionally used for many ailments including cancer. In the present study, antileukemic activity of the leaf extract was evaluated on human leukemic cell lines. Materials and Methods: Human leukemic cell lines U937, K562, and HL60 were purchased from National Facility for Animal Tissue and Cell Culture, Pune, India. The cells were routinely maintained in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 in air. The methanol extract of BJ (MEBJ) was dissolved in PBS and used at the concentrations of 5, 10, and 15 μg/ml for cell viability and cytotoxicity studies (MTT assay). Cell counts were made in quadruplicate samples at the interval of 24, 48, and 72 h and cytarabine (20 μg/ml) served as standard drug. The apoptotic pathway of cytotoxicity was assessed by DNA agarose gel electrophoresis technique and confirmed by fluorescence and confocal microscopic methods at the concentration of 10 μg/ml. Results: MEBJ showed significant cytotoxicity (P<0.001) in leukemic cell lines in the invitro cell proliferation assay. IC50 of MEBJ was very low (3.5 μg/ml) at 72 h in the HL60 cell line. The apoptotic pathway of cytotoxicity was observed at 10 μg/ml of MEBJ by the fragmented DNA pattern in the apoptosis assay, chromatin condensation, and apoptotic body formation as revealed in the fluorescence and confocal microscopic studies. Conclusion: The present findings support the ethno-medicinal use of BJ for cancer by mediating through the apoptosis pathway. [ABSTRACT FROM AUTHOR]