Trans-resveratrol, its dimer epsilon-viniferin and two preparations of vineatrol (a grape-derived polyphenol fraction isolated from vine-shots extracts) were compared for their effects on the proliferation and survival of normal and leukemic human lymphocytes. The two different batches of vineatrol (vineatrol 10 and 25%) was obtained by HPLC fractionation and contained 10 and 25% trans-resveratrol, respectively. The different polyphenols were added to cultures of leukemic cells from chronic B cell malignancies (B-cell chronic lymphocytic leukemia, B-CLL or hairy cell leukemia, HCL) or normal peripheral blood-derived mononuclear cells (PBMC) as a control. The different polyphenols displayed anti-proliferative effect on the leukemic cells, as estimated by the observed inhibition of tritiated thymidine uptake and the reduction of cell recovery. Vineatrol 10% was the most potent whereas vineatrol 25% and resveratrol displayed comparable activity, epsilon-viniferin only exhibiting slight effets. The same order of potency was observed for their capacity to induce apoptosis in leukemic B cells. In contrast, the survival of normal peripheral blood mononuclear cells (PBMC) was little affected in the presence of these polyphenolic compounds and higher concentrations were required in order to elicit cell death. Polyphenol-driven apoptosis in chronic leukemic B cells was shown to correlate with an activation of caspase 3, a drop in the mitochondrial transmembrane potential, a reduction in the expression of the anti-apoptotic protein bcl-2, as well as a reduction in the expression of the inducible nitric oxide synthase (iNOS). Our data therefore indicate that vine-shoots may be a convenient and natural source of material for the purification of resveratrol and other polyphenolic compounds of putative therapeutic interest.

Summary. Trans-resveratrol was analysed for its apoptotic and growth inhibitory activity in human B-cell lines derived from chronic B-cell malignancies (WSU-CLL and ESKOL), and in leukaemic lymphocytes from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Resveratrol displayed antiproliferative activity on both B-cell lines, as estimated by the decrease in cell recovery and inhibition of thymidine uptake. Furthermore, resveratrol induced apoptosis in the two cell lines as well as in B-CLL patients' cells, as evidenced by the increase in annexin V binding, caspase activation, DNA fragmentation and decrease of the mitochondrial transmembrane potential DY m . We previously reported that nitric oxide (NO), endogenously released by an iNO synthase (iNOS) spontaneously expressed in these leukaemic cells, contributed to their resistance towards apoptosis. We show here that resveratrol inhibited both iNOS protein expression and in situ NO release in WSU-CLL, ESKOL and B-CLL patients'cells. In addition, Bcl-2 expression was also inhibited by resveratrol. Thus, downregulation of the two anti-apoptotic proteins iNOS and Bcl-2 can contribute to the apoptotic effects of resveratrol in leukaemic B cells from chronic leukaemia. Our data suggest that this drug is of potential interest for the therapy of B-CLL.

Even though the capacity of B-CLL leukemic cells to proliferate has been underestimated until recently, the accumulation of tumor cells in patients mostly results from a defect in the apoptotic program. Several mechanisms can account for this deficient cell death pathway. These include overexpression of anti-apoptotic molecules such as members of the Bcl-2 family, which control the opening of the mitochondrial transition permeability pore, and of the IAP (inhibitors of apoptosis) family, which inhibit the activity of caspases. The latter is also suppressed by nitric oxide (NO) released through an inducible NO synthase present in the leukemic cells. The activity of the receptors with a death domain (Fas, TRAIL) is impaired, thus contributing to the resistance to spontaneous and/or drug-induced apoptosis. Interferons as well as several cytokines and angiogenic factors are also involved in the failure of programmed cell death, either by providing efficient signals for survival (BAFF) or by counteracting the apoptotic process. A better knowledge of the mechanisms of survival and escape from apoptosis of B-CLL cells has led to the proposal of new drugs that selectively interfere at the different steps of these cascades. Their study is complicated by the lack of suitable cell lines and pre-clinical models. Nevertheless, some of these chemotherapeutic agents appear to be promising, provided they are correctly targeted to the leukemic cells.