Enhancing effects of indirubin on the arsenic disulfide-induced apoptosis of human diffuse large B-cell lymphoma cells

The aim of the present study was to investigate the indirubin-enhanced effects of arsenic disulfide (As(2)S(2)) on the proliferation and apoptosis of diffuse large B-cell lymphoma (DLBCL) cells in order to identify an optimum combination therapy. The human DLBCL cells, LY1 and LY8, were treated with different concentrations of indirubin for 24, 48 and 72 h. Next, the cells were treated with 10 μM As(2)S(2) or a combination of 10 μM As(2)S(2) and 20 μM indirubin for 48 h. Cell proliferation inhibition was detected using cell counting kit-8 and cell apoptosis was determined using flow cytometry. The expression levels of Bcl-2, Bcl-2-associated X protein (Bax) and caspase-3 were analyzed by quantitative polymerase chain reaction (qPCR) and western blotting. The DLBCL cell viability exhibited no significant changes at 24, 48 or 72 h with increasing indirubin concentration. In addition, the apoptotic rates of the LY1 and LY8 cells demonstrated no noticeable effects at 48 h with increasing indirubin concentration. Following treatment with the combination of indirubin and As(2)S(2), the inhibitory and apoptotic rates of the cells were notably increased compared with those of the As(2)S(2)-treated group. The qPCR results revealed that indirubin alone had no enhancing effect upon the Bax/Bcl-2 mRNA expression ratio and caspase-3 mRNA expression. Western blot analysis revealed that indirubin alone had an enhancing effect upon the Bax/Bcl-2 protein ratio and procaspase-3 protein expression. In addition, the results demonstrated that the 21-KDa Bax protein was proteolytically cleaved into an 18-KDa Bax in the DLBCL cells treated with the combination of indirubin and As(2)S(2). Indirubin alone did not inhibit proliferation or induce the apoptosis of the LY1 and LY8 cells. However, the combination of indirubin and As(2)S(2) yielded enhancing effects. Therefore, the results of the present study demonstrated that with regard to antitumor activities, As(2)S(2) served as the principal drug, whereas indirubin served as the adjuvant drug. The enhancing effect was due, in part, to the induction of the mitochondrial apoptotic pathway, which involves the cleavage of Bax.