During the recent years, HF was shown, in addition, to exhibit antitumoral activity, as attested by its antiproliferative, proapoptotic, anti-invasive and antimetastasic effects.

Antiproliferative and proapoptotic effects

Alcoholic extracts of SJW induce dose-dependent growth arrest of several human malignant cells. Most experiments, however, have been performed with transformed cell lines, and it should be recalled that the sensitivity of normal and transformed cells can vary to a large extent. The leukemia cell lines K562 and U937 were the more sensitive and HF was suggested to be, at least partly, responsible for these effects.60 Indeed, purified HF inhibited the growth of these cells at GI(50) values about 12.5 μ M.61 Of note, these concentrations are rather high and may be cytotoxic in medium and long-term cultures. HF was reported to inhibit the growth of various other human and rat tumor cell lines in vivo and to elicit characteristic features of apoptosis in vitro.62 HF stimulated caspase-3 and -9 activity in MT-450 carcinoma cells and apoptosis was blocked by the caspase inhibitor zVAD.fmk. The intrinsic or mitochondrial pathway was involved, inasmuch as HF induced a dissipation of the mitochondrial transmembrane potential Δψ m and the release of cytochrome c from mitochondria. HF treatment inhibited the growth of these cells in rats without any signs of acute toxicity.63 It is worth noting that other lipophilic compounds, in addition to HF, may be involved in apoptosis induction by SJW extracts in various cancer cells.64

Our group observed recently that HF displayed ex vivo proapoptotic activities in B-CLL patients' cells.1 HF elicited the externalization of phosphatidylserine, DNA fragmentation and dissipation of the transmembrane mitochondrial potential Δψ m . HF stimulated caspase-3 activity and induced the caspase-dependent cleavage of poly-ADP ribose polymerase-1 and of two antiapoptotic proteins, Mcl-1 and Bcl-2. These two proteins are involved in the control of mitochondrial permeability and the release of apoptogenic molecules, and thus play important roles in the resistance of B-CLL cells to various chemotherapeutic agents. HF-induced apoptosis was associated with a downregulation of iNOS expression and of NO production by the leukemic cells. We showed previously that the endogenous production of NO by B-CLL cells is a component of their resistance to spontaneous apoptosis.65 This inhibition of the NO pathway could be a cause of apoptosis, inasmuch as HF-elicited apoptosis could be partially reverted by low concentrations of a NO donor. This would suggest that HF-induced suppression of NO release is a molecular switch contributing to subsequent apoptosis, perhaps through a release of the blocking effect of NO on the active site of caspases.66 We also observed that p27kip1, an inhibitor of cyclin-dependent kinase overexpressed in B-CLL, was downregulated by HF through a caspase-dependent pathway.1 The susceptibility of B-CLL to fludarabine in vitro is inversely correlated with p27kip1 levels, suggesting that this protein may be involved in the cell cycle arrest and in the resistance of patients to chemotherapy.67 By promoting the cleavage of p27kip1, HF could allow the cells to enter the cell cycle and therefore to become susceptible to a treatment with classical cell cycle-dependent chemotherapeutic agents.

Anti-invasive and antiangiogenic effects

Neo-angiogenesis, the formation of new blood vessels from the existing ones, plays a crucial role in the development of tumors by providing to cancer cells their necessary oxygen supply. Recent work has shown that HF displays both anti-invasive and antiangiogenic properties in vitro and in vivo. It was first reported that low concentrations of a dicyclohexylammonium salt of HF (DCHA-HF) inhibited the chemoinvasion of murine (C-26) and human (HT-1080) tumor cell lines through reconstituted basement membrane. The latter occured via the impairment of proteinases involved in extracellular matrix (ECM) degradation, elastase being inhibited to a much higher degree than cathepsin G and urokinase, and via a reduction in the secretion of matrix metalloproteinases 2 and 9 (MMP-2 and-9), two ECM remodeling enzymes. Daily intraperitoneal administration of 300 nmol of HF was shown to reduce the size and number of metastasis in two murine models of metastatic dissemination (C-26 and B16-LU8), with concomitant reduction of inflammatory infiltration and neovascularization.62 The anti-angiogenic properties of HF were confirmed by the observation that it inhibited the growth of endothelial cells in vitro, prevented the formation of capillary tubes by bovine aortic endothelial cells cultured on Matrigel and by the chorioallantoic membrane assay in vivo. HF also dramatically inhibited urokinase and MMP-2, two enzymes involved in the degradation of the basement membrane and of components of the ECM, an early step in the process of angiogenesis.68 HF also blocked microvessel formation of human dermal microvascular endothelial cells on ECM and reduced their proliferation in the absence of apoptosis induction.69 To evaluate the antiangiogenic activity of HF in vivo, peritumoral injections of HF in rats subcutaneously grafted with MT-450 mammary carcinoma cells were found to result in an inhibition of tumor growth, induction of apoptosis in tumor cells and reduced tumor vascularization. In addition to the induction of tumor cell apoptosis, HF can also suppress angiogenesis by a direct, non-toxic effect on endothelial cells.

Treatment of B-CLL cells with HF resulted in a marked inhibition of their capacity to secrete MMP-9, an essential component in the neo-angiogenesis through degradation of the ECM. The phloroglucinol impaired the production of the latent 92 kDa proenzyme and this was associated with a decrease in vascular endothelial growth factor release by the leukemic cells. Moreover, HF prevented the formation of micro-tubules by human bone marrow endothelial cells (HBMEC) cultured on Matrigel, evidencing its capacity to inhibit vessel formation.2 These results confirm the growing interest of HF as a potential drug for the treatment of cancer and the prevention of metastasis dissemination.

Reversal of P-gp activity

HF inhibits the functional activity of P-gp in B-CLL cells in vitro, as estimated by the enhanced uptake of rhodamine123. Similar effects were observed in a P-gp-overexpressing variant of the HL-60 cell line resistant to daunorubicin (Quiney et al., submitted). These results confirm that HF inhibits P-gp functional activity in vitro in different cell types. They suggest that HF could help revert the MDR phenotype and justify testing the pharmacological activity of this molecule in combination with classical chemotherapeutic agents.

Anticarcinogenic effect

As related previously, SJW extracts and HF modulate several enzymes of the CYP450 family, causing a risk of interference with the administration of other drugs. However, this modulation could be beneficial in some instances, inasmuch as SJW extracts and HF are potent inhibitors of carcinogen formation from benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1, a major human procarcinogen-activating enzyme, HF acting as a competitive inhibitor.70 SJW and HF may therefore be worthy of further evaluation for cancer chemopreventive potential.