L-Gossypol Has Significant In Vitro Activity Against Primary Acute Myeloid Leukaemia Cells, Inducing Apoptosis through the Intrinsic Pathway.

Abstract

The potential anti-tumor effect of the polyphenolic cotton oilseed extract gossypol has been known for a number of years. Studies in cell lines and primary tumor material have suggested both antiproliferative effects and the induction of apoptosis (Huang et al. Anticancer research 26:3a) with activity being seen against even multidrug resistant tumors. Although the mechanism of action of gossypol has not yet been well-characterised, there is some evidence that it may block the binding of the apoptotic regulator BCL-2 with the pro-apoptotic proteins BAX and BAD (Prada et al. blood 106:11). Acute myeloid leukaemia (AML) is characterised by accumulation of primitive blasts in bone marrow and peripheral blood. The disease is highly heterogeneous and inherently resistant to therapy, dysregulation of apoptosis frequently leading to a survival advantage in blast cells. We studied the in vitro activity of gossypol against cryopreserved primary blasts obtained from 44 AML patients. Cells were incubated for 48 hours with a range of concentrations of gossypol, this being followed by MTS cytotoxicity assay. IC50 concentrations ranged from 792nM to in excess of 100 mM (median 22.791 mM). Statistical analysis showed no significant variation in drug sensitivity according to sex, age, presenting white cell count, FLT3 mutation status, karyotype or clinical outcome measures. The in vitro combination of gossypol with the cytotoxic agent cytosine arabinoside (AraC) was assessed in 40 primary samples using the median effects principle of Chou and Talalay. The median combination index of 0.946 suggested an overall additive effect when the agents were combined, although numbers were insufficient to establish this conclusively (95% CI for median 0.84–1.31). Flow cytometric annexin V binding assay confirmed apoptosis to be the principal mode of cell death in response to gossypol, cell viability obtained using this method correlating well with that obtained using the MTS assay. Gossypol caused downstream activation of Caspase 3/7 via Caspase 9 as determined by luminometric analysis, indicating that the intrinsic pathway is the main inducer of this cycle. Gossypol is effective in inducing apoptosis in primary AML blasts and there is potential for greater efficacy when used in combination with cytotoxic chemotherapy. Further in vitro development of this agent is now required to further define its future clinical role.