16-Hydroxycleroda-3,13-dien-15,16-olide deregulates PI3K and Aurora B activities that involve in cancer cell apoptosis

Abstract

The PI3K-AKT pathway and Aurora kinase play essential roles in such cellular processes as cell survival, angiogenesis, and differentiation, and are usually expressed at maximum levels during cancer cell proliferation. The present study investigated the effect of the natural compound, 16-hydroxycleroda-3,13-dien-15,16-olide (PL3), on regulating the PI3K-AKT pathway and Aurora B, which led to cancer cell apoptosis. PL3 acts as a PI3K inhibitor by influencing cell survival, signaling transduction, and cell cycle progression. It was observed that PL3 targeted and induced dephosphorylation of the PI3K pathway, degradation of Aurora B and mitotic-related gene expressions, and sequentially shut down the cell cycle. This eventually resulted in cell death. As Aurora B was downregulated, spindle dysfunction and destruction of the G₂/M phase checkpoint resulted in DNA-damaged cells undergoing apoptosis. Moreover, PL3 also resensitized T315I-mutated Bcr-ABL⁺ BA/F3 cells to improve the cytotoxicity of Imatinib in Imatinib-resistant cell line. Taken together, PL3 can perturb the PI3K-AKT pathway and Aurora B resulting in gene silencing and cell cycle disturbance. It was demonstrated that PL3 acted like a novel small-molecule PI3K modulator, thereby potentially contributing to cancer chemotherapy and combination medication.

Introduction

The Aurora family can be classified into three members, Aurora A, B, and C, which are essential for cell-cycle regulation. The family also has serine/threonine kinase activity which modifies microtubes during chromosome movement and segregation. Aurora A is localized in centrosomes and is important for maturation, spindle assembly, and metaphase I spindle orientation. Aurora B is localized to microtubules near kinetochores, which are highly expressed and play key roles in ensuring the genetic stability of cell division (Gully et al., 2010). Chromosome segregation is a critical step in maintaining gene stability, and the overexpression of Aurora B is required to coordinate cellular process (Schmitt et al., 2002). Anticancer reagents can kill cells by destroying the spindle checkpoint integrity.

It is known that PI3K-AKT and Aurora kinases are important targets for interventions by cancer therapies, and scientists recently found cross-talk between the PI3K-AKT pathway and Aurora kinase activation (Liu et al., 2008, Yao et al., 2009). Phosphatidylinositol (PI) 3-kinases (PI3Ks) are now well-known as important regulators of cell survival, differentiation, angiogenesis, cell invasion, and metastasis (Kim and Chung, 2002). They were found to be activated during colony stimulating factor (CSF)-1-mediated cell proliferation and survival, and required for prevention of apoptosis in various types of cells by regulating growth factors (Scheid et al., 1995, Takashima et al., 1996, Varticovski et al., 1989). There are three classes of PI3Ks, classes I, II, and III with regulatory subunits for each according to their respective structural characteristics and substrate specificity (Fruman et al., 1998, Liu et al., 2009). Class I PI3K is the most commonly studied, and is directly activated by cell surface receptors (Fruman et al., 1998). The serine/threonine kinase AKT was identified as having a crucial role amongst PI3Ks and preventing apoptosis by targeting Bad, caspase-9 and Forkhead transcription factors. Both phosphorylation events on T308 and S473 are required for full activation of AKT (Alessi et al., 1997). Activated AKT is initially translocated to plasma membranes, and has the capacity to phosphorylate a variety of substrate proteins in order to perform its functions in cells, which are involved in apoptosis signaling transduction (Alessi et al., 1996, Engelman et al., 2006).

A wide variety of natural compounds are toxic to cancer cells, but their mechanisms of toxicity differ. 16-Hydroxycleroda-3,13-dien-15,16-olide (PL3), one of the major clerodane diterpenoid compounds isolated from the bark of Polyalthia longifolia, was reported to exhibit anti-inflammatory activity against fMLP/CB-induced superoxide generation by neutrophils. In addition to the anti-inflammatory effect, it also showed cytotoxicity towards breast cancer cells and hepatoma carcinoma cells (Chang et al., 2006, Shih et al., 2010). In a previous study, 22 compounds were isolated from P. longifolia (Chang et al., 2006). Increasingly, cytotoxic diterpenes were isolated from this plant. However, the mechanism of action of these treasured chemicals against cancer is still unclear. In the present study, the effects of PL3 on various cancer cell lines were investigated, and its possible molecular mechanism of apoptosis was determined.