Gossypol increases expression of the pro-apoptotic BH3-only protein NOXA through a novel mechanism involving phospholipase A2, cytoplasmic calcium, and endoplasmic reticulum stress

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.

Keywords: Apoptosis; B Cell Lymphoma 2 (BCL2); Endoplasmic Reticulum Stress (ER Stress); Leukemia; NOXA; Phospholipase A.

FIGURE 1.

Gossypol induces ER stress, NOXA, and sensitizes NB4 cells to ABT-737. A, NB4 cells were incubated with 0–40 μm gossypol for 6 h; NOXA expression was analyzed by Western blotting. B, NB4 cells were incubated with 0–100 nm ABT-737 with or without 20 μm gossypol for 6 h. PARP cleavage was used as a marker of apoptosis. C, NB4 cells were transfected with 3 μg of siRNA (control or NOXA), incubated for 48 h, and then incubated with gossypol, ABT-737, or both in combination for 6 h. PARP cleavage and NOXA were assessed by Western blotting. D, NB4 cells were incubated with 20 μm gossypol for 0–6 h. Protein levels were analyzed by Western blotting. The mobility shift in PERK (arrow) is indicative of phosphorylation and activation. E, NB4 cells were transfected with control siRNA or siATF4 plus siATF3 and incubated for 48 h. Transfected cells were then incubated with gossypol or bortezomib for 6 h and probed for the indicated proteins.

FIGURE 2.

Gossypol increases cytoplasmic calcium and calcium-dependent NOXA. A, NB4 cells were incubated with INDO-1 AM for 1 h and analyzed for cytoplasmic calcium concentrations. Gossypol or thapsigargin was added after 100 s. Error bars represent 1 S.E. (n = 3). B, NB4 cells were incubated with the indicated concentrations of BAPTA AM for 1 h and then treated with gossypol. The indicated proteins were assessed by Western blotting.

FIGURE 3.

Gossypol increases PLA2 activity, which is required for increased calcium and NOXA. A, NB4 cells were incubated with PLA2 substrate for 5 min and then analyzed for PLA2 activity. After a 30-s baseline read, the run was halted, gossypol was added, and the run was restarted after a 10-s delay (dotted line). PLA2 activity was plotted after subtracting the background signal. B, PLA2 activity was quantified following a 30-s incubation with gossypol. Error bars represent S.E. (n = 2). C, NB4 cells were incubated with the indicated concentrations of aristolochic acid for 1 h, treated with gossypol for 30 s, and then analyzed for PLA2 activity. Error bars represent S.E. (n = 2). D, NB4 cells were incubated with 2 mm EGTA, 10 μm BAPTA AM, or both for 1 h, treated with gossypol for 30 s, and then analyzed for PLA2 activity. E, NB4 cells were incubated with INDO-1 AM and the indicated concentrations of aristolochic acid for 1 h and then analyzed for cytoplasmic calcium concentration. Gossypol was added after 30 s. F, NB4 cells were incubated with the indicated concentrations of aristolochic acid for 1 h, treated with gossypol for 6 h, and assessed for NOXA protein by Western blotting. G, THP1 (left) and U937 (right) cells were analyzed for PLA2 activity (top) or cytoplasmic calcium (bottom) following the addition of gossypol (5, 10, or 20 μm).

FIGURE 4.

Treatment with LPA and AA mimics effects of gossypol. A, NB4 cells were incubated with INDO-1 AM for 1 h and analyzed for cytoplasmic calcium. The indicated concentrations of LPA and AA were added after 30 s. B, NB4 cells were incubated with the indicated concentrations of LPA, AA, or LPA plus AA for 6 h, and analyzed for NOXA protein by Western blotting. C, NB4 cells were incubated with 50 μm LPA and AA for the indicated times and assessed for the indicate proteins by Western blotting. The mobility shift in PERK (arrow) is indicative of phosphorylation and activation. D, NB4 cells were incubated with or without 100 μm aristolochic acid for 1 h and analyzed for cytoplasmic calcium. Error bars represent S.E. (n = 3). LPA and AA were added after 30 s. E, NB4 cells were incubated with or without 100 μm aristolochic acid for 1 h, treated with the indicated concentrations of LPA and AA, and NOXA expression was analyzed by Western blotting.

FIGURE 5.

Gossypol derivatives increase PLA2 activity, cytoplasmic calcium, NOXA protein, and overcome stroma-mediated resistance to the PLA2 inhibitor ABT-199 in CLL. A, NB4 cells were incubated with the indicated concentrations of apogossypol or AT-101 for 6 h. The indicated proteins were analyzed by Western blotting. B, NB4 cells were incubated with the indicated concentrations of apogossypol or AT-101 for 45 s and analyzed for PLA2 activity. Error bars represent S.E. (n = 2). C, NB4 cells were incubated with INDO-1 AM for 1 h and analyzed for cytoplasmic calcium. Error bars represent 1 S.E. (n = 3). The indicated concentrations of apogossypol or AT-101 were added after 30 s. D, CLL cells (n = 4) were incubated with drugs immediately (left) or co-cultured on CD154⁺ stroma overnight (right) and incubated with the indicated concentrations of ABT-199 and AT-101. Percentage survival is plotted as the percentage of cells that do not exhibit condensed chromatin as assessed by Hoechst 33342 dye. Error bars represent 1 S.E.