Indirubin-3′-monoxime, a CDK Inhibitor Induces Growth Inhibition and Apoptosis-independent Up-regulation of Survivin in Transitional Cell Cancer

Abstract

In traditional Chinese Medicine, the preparation Danggui Longhui Wan has been used for years in the treatment of chronic myelocytic leukemia. The compound indirubin has been shown to be the active constituent. A cell permeable derivative, indirubin-3′-monoxime, is a selective and potent inhibitor of cyclin-dependent kinases (cdk). The ability of indirubin-3′-monoxime to induce apoptosis and tumor cell death in transitional cell cancer cell lines was investigated here. The growth-inhibitory properties were evaluated by EZ4U, a cytotoxic assay; apoptosis induction was determined by immunoblotting of cleaved PARP and flow cytometry of Annexin-V/PI staining during treatment. To evaluate further the underlying molecular action of indirubin-3′-monoxime on the cell cycle, the levels of cdk-1 and survivin, a mitotic spindle checkpoint and apoptosis-regulating protein, respectively, were additionally determined by flow cytometry and immunoblotting. The results indicated that indirubin-3′-monoxime induced reversible growth arrest in all four cell lines and an increase of apoptosis in two of them. The treatment with indirubin-3′-monoxime increased the expression of survivin almost four times in the RT4 cells and more than doubled it in the RT112 and T24 cells. In the SUP cells, the expression of survivin increased more than seven-fold after 72-h incubation. No clear correlation between the low apoptosis induction rate and extent of survivin expression was found. Cdk expression was not significantly altered by indirubin-3′-monoxime. In summary, indirubin-3′-monoxime might be a promising candidate for targeted cancer therapy, however, its molecular action remains to be further evaluated.