[Show abstract][Hide abstract] ABSTRACT: Cancer gene therapy represents a new and promising therapeutic modality for various types of cancer. Two or more anti-oncogenes carried by a single vector could theoretically improve treatment efficacy, reduce side-effects from vectors, and have a satisfactory clinical application prospect; however, this has seldom been studied in breast cancer. The inhibitor of growth 4 (ING4), as a member of the inhibitor of growth tumor suppressor family has potent inhibitory effects on a variety of tumors. Interleukin‑24 (IL-24) has also shown broad spectrum and tumor-specific antitumor activities. In this study, we aimed to prove the enhanced antitumor activity of adenovirus-mediated ING4/IL-24 double tumor suppressor gene co-transfer in human breast cancer cells. We assessed the combined effect of the ING4/IL-24 bicistronic adenovirus (Ad-ING4-IL-24) in vitro and in vivo on MDA-MB-231 human breast cancer cells by detecting and comparing the apoptotic status in the bicistronic anti-oncogene group (Ad-ING4-IL-24) and in the ING4 or IL-24 single anti-oncogene groups, and also investigated the possible underlying mechanism. Our results showed that the bicistronic adenovirus-mediated ING4 and IL-24 co-expression induced additive growth suppression and apoptosis as well as an overlapping effect on the upregulation of p21, p27 and Bax, and the downregulation of Bcl-2 and survivin in MDA-MB‑231 human breast cancer cells in vitro or in vivo. Moreover, Ad-ING4-IL-24 treatment additively reduced CD34 expression and the microvessel density in MDA-MB-231 xenografted tumors in athymic nude mice, which correlated with the decreased expression of the vascular endothelial growth factor. The enhanced antitumor activity on breast cancer elicited by Ad-ING4-IL-24 was closely associated with the activation of the apoptotic pathways and the additive inhibition of tumor angiogenesis.