Casticin induces leukemic cell death through apoptosis and mitotic catastrophe

Abstract

Casticin, a component from Vitex rotundifolia, widely used as an anti-inflammatory agent in Chinese traditional medicine, was reported to have anti-tumor activities. This study aims to examine the anti-leukemic activity of casticin on leukemia cells and its molecular mechanism. Cell viability was measured by MTT method; apoptosis and cell cycle arrest were determined by flow cytometry, AV-PI assay, and DNA fragmentation assay. Western blot were performed to measure the protein expression level. The cell morphology alteration was detected with immunofluorescent analysis and DAPI nuclear staining. Our results showed that the proliferation of leukemia cells, including K562, Kasumi-1, and HL-60, were inhibited by casticin in a time- and dose-dependent manner. The IC50, determined after 48 h incubation, was 5.95 microM, 4.82 microM, and 15.56 microM for K562, HL-60, and Kasumi-1, respectively. The cell cycle analysis demonstrated casticin treatment resulted in a significant G2/M accumulation, concomitant with upregulation of P21waf1 and P27kip1. The percentage of cells in G2/M increased with time of exposure and reached to its climax (75.3%) at 12 h after casticin treatment, and subsequently declined to 27% at 48 h. We found that casticin treatment induced remarkable apoptosis, evidenced by increased percentage of AV-positive PI-negative cells as well as the cleavage of PARP and caspase 3. In addition, DNA fragmentation assay showed the typical apoptotic DNA ladder in casticin-treated K562 cells. Mitotic catastrophe and decreased polymeric tubulin can also be observed in casticin-treated K562 cells. In addition, we found that PI3K/AKT pathway was activated; Ly294002, a PI3K/AKT specific inhibitor, can enhance the anti-leukemic effect of casticin. Taken together, these results demonstrated that casticin induced leukemic cell death via apoptosis and mitotic catastrophe, and could synergize with PI3K/AKT inhibitor, suggesting that casticin could be a promising therapeutic agent against leukemia.