Biochemical correlates of the antitumor and antimitochondrial properties of gossypol enantiomers

Racemic gossypol has been shown to have antitumor properties that may be due to its a bility to uncouple tumor mitochondria or to its inhibitory effects on a variety of nonmitochondrial enzymes. We have studied the antimitochondrial and enzyme-inhibiting properties of gossypol in human carcinoma cell lines of breast (MCF-7, T47-D), ovarian (OVCAR-3) colon (HCT-8), and pancreatic (MiaPaCa) origin by comparing the effects of its purified (+)- and (-)-enantiomers. (-)-Gossypol shows up to 10-fold greater antiproliferative activity than (+)-gossypol in the cancer cell lines and in normal hematopoietic stem cells grown in vitro, with IC₅₀ values ranging from 1.5 to 4.0 μM for the cancer cells and from 10 to 20 μM for the human marrow stem cells. As well, multidrug-resistant MCF/Adr cells appear more resistant to (-)-gossypol than their parental cell line. Electron microscopy indicates that the earliest ultrastructural change in tumor cells exposed to a cytotoxic (10 μM) concentration of (-)-gossypol is the selective destruction of their mitochondria. Consistent with this observation, ³¹P magnetic resonance spectroscopy detects pronounced changes in tumor cell high energy phosphate metabolism within 24 hr of (-)-gossypol treatment, manifest by 1.6- to >50-fold differential reductions in the intracellular ratios of ATP/ P_i, relative to (+)-gossypol-treated cell lines; the magnitude of these antimitochondrial effects correlates with the antiproliferative activity of (-)-gossypol. Northern blot RNA analyses suggest that treatment with a 5-10 μM dose of (-)-gossypol induces a transient increase in the expression of heat shock gene products, particularly hsp-70 transcripts. The mean 5-fold increase in (-)-gossypol-induced hsp-70 mRNA appears coincident with a comparable heat-stimulated increase in transcript levels, as compared with control or (+)-gossypol-treated cells. The enzyme-inhibiting properties of gossypol enantiomers were compared in cell-free assays measuring glutathione-S-transferase-α, -μ, and n activities, calmodulin stimulation of cyclic nucleotide phosphodiesterase, and protein kinase C activity. Both enantiomers are near equivalent antagonists of calmodulin stimulation and protein kinase C activity, exceeding the potency of known inhibitors such as phenothiazines by as much as 50-fold. In contrast, (-)-gossypol is a 3-fold more potent inhibitor of glutathione-S-transferase-α and -π isozyme activity, resulting in IC₅₀ values of 1.6 and 7.0 μM, respectively, for these two isozymes. Because of the enhanced resistance of MCF/Adr cells to (-)-gossypol, which may be related to their increased glutathione-S-transferase and protein kinase C content, (-)-gossypol should be evaluated for its potential to modify the cytotoxic resistance of human carcinoma cells to other chemotherapeutic agents. Furthermore, the above newly described (+)- and (-)-gossypol effects may be useful in directing structure-function studies using chiral-specific gossypol derivatives, in order to develop more selective and potent antimitochondrial chemotherapeutic agents.