Abstract
Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154(+) stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.
Highlights
The mechanism through which gossypol mediates anti-cancer activity is poorly understood
We demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA
NOXA Is Required for Gossypol-mediated Sensitization to ABT-737—We have reported previously that gossypol induces NOXA and sensitizes the acute pro-myelocytic leukemia line
Summary
The mechanism through which gossypol mediates anti-cancer activity is poorly understood. Results: Gossypol rapidly activates phospholipase A2, increases cytoplasmic calcium, endoplasmic reticulum stress, and NOXA, and sensitizes cells to apoptosis. We demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. A related, orally bioavailable compound, navitoclax (ABT-263), has completed phase I clinical trials in CLL [2] and small cell lung cancer [3] This compound has demonstrated efficacy, resistance can occur when cancer cells rely on alternative BCL2 family members, such as MCL1 and BFL1 [4]. We reported previously that several putative BH3 mimetics, including gossypol, activate the integrated stress response (ISR) rather than directly inhibiting the anti-apoptotic BCL2 proteins [25]. We identify phospholipase A2 (PLA2) as a critical and rapidly activated target of gossypol, which leads to an increase in cytoplasmic calcium, activation of ER stress, and the induction of NOXA
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