Jasmonates are a group of plant stress hormones, which play a role in activation of apoptosis and defense mechanisms. In vitro, it has been found that methyl jasmonate MJ induces death of lymphoma and leukemia cells by independent mechanisms involving mitochondrial damage at the level of the oxidative phosphorilation.
Since CLL is a suitable model to study the effect of MJ on circulating leukemic cells, we analyzed the in vitro apoptosis and necrosis of B-CLL cells treated with MJ by PI/ Annexin V analysis and cytotoxicity by tetrazolium-based assay (XTT test). In comparison normal lymphocytes from healthy donors were also analyzed.
Interestingly, normal lymphocytes were not affected al all by methyl jasmonate. Blood samples from 28 CLL patients in advanced stages not treated or at least 4 weeks after cessation of treatment were selected and incubated with MJ. The proportion of cells undergoing apoptosis or necrosis following 4 hour of incubation was measured by flow cytometry and considered as positive if >35% cells death occurred. Following 24 hours incubation cells vialability was measured using XTT Cell Proliferation kit assay by Elisa. Cytotoxicity (%) was calculated by the ratio of optical density of control cells - drug-treated cells/ control cells ×100 (IC50). The titration of drug showed that 1 mM of MJ was the most effective to induce apoptosis of CLL cells and has no effect on normal lymphocytes. In 90% of samples the specific cells death tested by Annexin V was higher than 35%. A significant cytotoxicity was observed in 60% of the cases with an optimal titration of the drug for XTT test of 0.5 mM. Correlation between the two methods was found in 80% of the cases analyzed.
Previous experiments showed that MJ induces cytochrome C release selectively in mitochondria isolated from CLL cells and high glucose levels inhibit MJ-induced ATP depletion. Furthermore MJ induces mitochondrial membrane depolarization in CLL cells. Altogether these experiments showed that MJ kills B-CLL cells through an original pathway inducing bioenergitic cell collapse. The cytotoxic effect and specific cell death tested in the present analysis support the concept that MJ is a potential drug to treat B-CLL. A clinical trial is currently carried out in our Institution.
Disclosures: R.B. is working in Sepal Pharma.; Sepal Pharma for experiments.