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Adriamycin (ADM) is a first‑line agent administered during the therapeutic regimes against osteosarcoma. Clinical administration of ADM produces systemic toxicity and resistance in patients, which restricts its applicability. In the present study the effects of phenethyl isothiocyanate (PEITC) on ADM‑induced apoptosis in osteosarcoma cells was evaluated. Using U2‑OS osteosarcoma cell line cells, treatment with PEITC or ADM for 24 h was observed to dose‑dependently inhibit proliferation of U2‑OS cells with half maximal inhibitory concentration (IC50) values of 5.33 µM and 10.32 µg/ml, respectively. When U2‑OS cells were treated with a combination of the two agents, the inhibition was apparently enhanced, as the IC50 values decreased to 2 µM for PEITC and 1 µg/ml for ADM. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling revealed that treatment with PEITC or ADM alone reduced the viability of the U2‑OS cells. Furthermore, the viability of the U2‑OS cells was additionally reduced when treatment was with PEITC and ADM together. Supporting this finding, the activity and expression of caspase‑3 were observed to be enhanced in the U2‑OS cells following treatment with either PEITC or ADM, or a combination of the two. These results clearly indicate that PEITC enhances ADM‑induced apoptosis in osteosarcoma cells.