Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

Abstract

Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first smallmolecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.

Polo-like kinase 1 inhibitors, mitotic stress and the tumor suppressor p53

“…29,30 However, we and others observed that Plk1 inhibitors affect both tumor cells with functional or deficient p53 as well as primary/normal non-transformed proliferating cells. 16,19,32,38 In the previous work, based on different cancer cell lines, we have demonstrated that cancer cells with wild type p53 actually responded more sensitively to Plk1 inhibitors, as evidenced by a high degree of apoptosis induction. 15 In the present work, we have focused on the question whether mitotic stress influences the effect of Plk1 inhibitors in cancer cells in context of the p53 status.…”

“…As indicated in the figure legend, all mitotic stress inducers were used in a low dose after performing dose-kinetics, so that they induce mitotic stress but not yet apoptosis during pretreatment. BI 2536 and BI 6727, 12,32,33 two of the most intensively studied kinase domain inhibitors, and Poloxin, the selective PBD inhibitor, 16,17 were taken as representatives of Plk1 inhibitors.…”

“…In fact, multiple small-molecule inhibitors targeting the enzymatic kinase domain and the regulatory Polobox binding domain (PBD) have been recently developed and characterized. 1,[7][8][9][10][11][12][13][14][15][16][17][18][19] In particular, BI 2536 and BI 6727 are the most intensively investigated Plk1 inhibitors. [20][21][22][23][24][25] Poloxin, the first reported non-peptidic inhibitor of the PBD of Plk1, shows its specificity and anti-proliferative activity in vitro as well as in vivo.…”

“…[20][21][22][23][24][25] Poloxin, the first reported non-peptidic inhibitor of the PBD of Plk1, shows its specificity and anti-proliferative activity in vitro as well as in vivo. [15][16][17] While the preclinical data of Plk1 inhibitors are encouraging, the clinical results are rather less inspiring, showing limited anticancer activity. 20,23,26,27 It is of importance to identify the molecules and mechanisms responsible for the sensitivity of Plk1 inhibitors.…”

B-cell lymphoma 6 promotes proliferation and survival of trophoblastic cells

“…Cell cycle profiles were analyzed using a FACSCalibur (BD Biosciences, Heidelberg) as described. 51,52 Briefly, cells were harvested, washed with PBS, fixed in chilled 70% ethanol at 4 C for at least 30 min, treated with 1 mg/ml of RNase A (Sigma-Aldrich) and stained with 100 mg/ml of propidium iodide for 30 min. DNA content was determined by FACS.…”