Mechanistic Insights into the Antileukemic Activity of ... - Ingenta Connect

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Mechanistic Insights into the Antileukemic Activity of Hyperforin C. Billard, F. Merhi and B. Bauvois* Centre de Recherche des Cordeliers, Equipe 18, INSERM U872, Paris, France; Université Pierre et Marie Curie and Université Paris-Descartes, UMRS 872, Paris, France Abstract: Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) – conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells – two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Keywords: Apoptosis, drug resistance, hyperforin, leukemia, proliferation, signaling. INTRODUCTION Hypericum perforatum L. is a perennial flowering plant commonly known as St John's wort (SJW). It is extensively cultivated in Europe and extracts of its dried flowers, leaves and aerial parts have been used for centuries as medicinal treatments for skin wounds, depression and inflammatory, respiratory and infectious diseases [1-4]. The major known constituents of SJW extracts include naphtodianthrones (hypericin, pseudohypericin, protohypericin and protopseudohypericin), phloroglucinols (hyperforin/HYP, adhyperforin, hyperfirin and adhyperfirin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, and I3,II8-biapigenin) and phenolic acids (chlorogenic acid and 3-O-coumaroylquinic acid) [5-8]. The biological and pharmacological properties of purified hypericin, pseudohypericin, quercitin and hyperforin have attracted much attention and have been discussed in a number of excellent reviews [2, 3, 8-16]. For example, quercetin exerts anti-infectious, antithrombotic, antiinflammatory and antimetastatic effects [11, 13, 15, 16], while hypericins exhibit antidepressant, antitumor and photosensitizer activities [12]. The following section summarizes hyperforin's biological activities, effects on normal leukocytes and data of prospective trials in humans and in animal models. We then review recent data on the in vitro effects of pure HYP and the compound's mechanisms of action in chronic lymphoid leukemia (CLL) and acute

*Address correspondence to this author at the Centre de Recherche des Cordeliers UMRS 872, Equipe 18, 15 rue de l'Ecole de Médecine, F-75270 Paris cedex 06, France; Tel: +33 144 278 188; Fax: +33 144 278 161; E-mail: [email protected] 1873-5576/13 $58.00+.00

myeloid leukemia (AML) – diseases that are both characterized by their resistance to cell death and chemotherapy. After a brief description of novel HYP derivatives, we lastly consider the remaining challenges and controversies in this field. H3C H3C H3C

CH3

CH3 O

H3C H3C

OH O O CH3

CH3

CH3 H3C

Fig. (1). The chemical structure of hyperforin. 5-hydroxy-6Rmethyl-1R,3,7S-tris(3-methyl-2-butenyl)-5S-(2-methyl-1-oxopropyl)6R-(4-methyl-3-pentenyl)-bicyclo[3.3.1]non-3-ene-2,9 dione. C35 H52O4: 536 g/mol.

GENERAL CHARACTERISTICS OF HYPERFORIN Hyperforin is a polyprenylated acylphloroglucinol derivative (Fig. 1). It consists of a phloroglucinol skeleton with five lipophilic isoprene moieties, which are derived entirely or predominantly (>98%) via the deoxyxylulose phosphate pathway [17]. The first prenylation step is catalyzed by a soluble, ion-dependent dimethylallyltransferase [18]. The HYP skeleton is formed from isobutyryl© 2013 Bentham Science Publishers

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Current Cancer Drug Targets, 2013, Vol. 13, No. 1

CoA and three molecules of malonyl-CoA by isobutyrophenone synthase [19]. Hyperforin displays antidepressant activity [9, 10] and other neurological effects; as such, it has potential value in the treatment of Alzheimer's disease [20]. The compound also exerts antibacterial, anti-inflammatory and antitumor properties [2, 3, 8]. At the cellular level, HYP reduces the growth of various human tumor cell lines [2, 21], cancer invasion, metastasis [22] and angiogenesis [23, 24] (with IC50 values ranging from 3 and 15 µM). It has broadspectrum anti-angiogenic activity (in the 1-10 µM range) in endothelial cells and the innate immune cells that interact with the latter [23-25]. An unsuspected pro-inflammatory effect of HYP (2-10 µM) is suggested in modulating interleukin-8 and ICAM-1 in human intestinal epithelia cells and primary hepatocytes [26]. Hyperforin is a key factor in SJW's induction of cytochrome P450 (CYP) metabolic enzymes (CYP3A4 and CYP2C9 in particular) and Pglycoprotein (P-gp) [14, 27, 28]. CYP3A and CYP2C9 can metabolize around half of all drugs and thus alter the latter's clinical efficacy. P-gp is an ABC transporter involved in multidrug resistance (MDR). Hyperforin (in the 0.2 nM-1 µM range) acts on the pregnane X receptor (PXR) to upregulate the transcription of P-gp in intestinal LS180 cells [29, 30] and CYP enzymes (CYP3A, CYP2B and CYP2C) [31-33]. In the same concentration range, HYP acts as a potent inhibitor of MDR1 expression and CYP3A4 and CYP1A1 activities [34-36]. EFFECTS OF HYPERFORIN ON NORMAL LEUKOCYTES Regarding its in vitro anti-inflammatory properties, HYP inhibits the proliferative response of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin A (PHA) in a dose-dependent manner (with an IC50 of around 2 µM at 24 h) and is free of toxic effects at concentrations up to 180 µM [37]. Moreover, HYP downregulates the effector functions (i.e. IFN-γ production and chemokine receptor CXCR3 expression) of lymphocytes activated by PHA and IL-2 in a dose-dependent manner (0.6-2.5 µM) [38]. In neutrophils and monocytes, HYP blocks induced Ca2+ mobilization (IC50 0.4-4 µM), which is coupled to reactive oxygen species formation and elastase release [39]. It is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase activities (IC50 1-3 µM) involved in the formation of proinflammatory eicosanoids (such as PGE2) from arachidonic acid [40, 41]. In accordance, HYP suppresses PGE2 biosynthesis in vivo [42]. Lastly, HYP blocks chemotaxis and chemo-invasion (IC50 1 µM for both) of neutrophils and monocytes [24, 43]. The effect of HYP on blood hematopoietic progenitors and bone marrow cells colony forming units remains to be investigated. EFFECTS OF HYPERFORIN IN HUMANS AND ANIMAL MODELS Initial studies of the pharmacokinetic profile of HYP in humans after oral administration of an alcoholic SJW extract have demonstrated oral bioavailability of the phloroglucinol [10, 44, 45]. No signs of major dose-limiting toxicity of

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HYP, such as body weight loss, myelosuppression, gastrointestinal and hematological toxicities, were observed [21, 24, 46]. With regard to safety concerns, as evoked above, HYP is a key constituent of SJW responsible for the induction of CYP drug-metabolizing enzymes involved in the metabolism of pharmaceutical drugs [27, 28]. Results from 26 clinical trials employing low-HYP extract content (< 4 mg/day equivalent to 7.5 µmol/day) showed no significant effect on the CYP3A drug-metabolizing enzyme in contrast to extracts containing higher amounts of HYP extract content (21-55 mg/day equivalent to 40-103 µmol/day) [27]. For example, administration of SJW preparations with high (21 mg/day) HYP content to renal transplant patients for 14 days in addition to their regular regimen of cyclosporine affected the extent of the pharmacokinetic interaction between cyclosporine and SJW [47]. In contrast, coadministration of low (0.3 mg/day) HYP content did not significantly affect cyclosporine pharmacokinetics and did not require cyclosporine dose adjustments compared with baseline [47]. Therefore, it can be expected that further clinical trials with low doses of pure HYP should reduce the frequency of drug interactions involving HYP. A limited number of studies have documented the in vivo properties of pure HYP following administration in animals. Although caution should be exercised in extrapolating results of animal models to humans, these studies validate the effects observed in vitro and suggest the therapeutic efficacy of HYP in neurologic, neoplastic and inflammatory sites (Table 1) [21, 24, 25, 42, 48-54]. As regards humans, a recent study has evidenced the anti-inflammatory potency of HYP (in a cream formulation 1.5% w/v) in reducing UVBinduced skin erythema [46]. BACKGROUND INFORMATION ON LEUKEMIAS Chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) are the two most common types of adult leukemia in the United States and Europe. Both diseases show a peculiar resistance to chemotherapies. CLL is characterized by clonal expansions of CD5+ B lymphocytes accumulating in the blood. The accumulation of leukemic cells, which are mostly quiescent, results mainly from their inability to develop the apoptotic program and an excess of survival signals delivered by the tumor microenvironment although proliferating pools exist in the bone marrow and lymph nodes [55, 56]. Despite recent therapeutic advances with the combination of purine analogs, alkylating agents and monoclonal antibodies (fludarabine, cyclophosphamide and rituximab, or allied agents), nearly all patients relapse and CLL remains an incurable disease [57, 58]. Acute myeloid leukemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. The latter are used to define distinct AML subfamilies [59-61]. Leukemia cells are unable to undergo (i) growth arrest, (ii) terminal differentiation, (iii) apoptosis in response to appropriate environmental stimuli, and disseminate from the bone marrow into peripheral tissues [59-61]. The conventional chemotherapeutic approach for AML patients is based on treatment combining an anthracycline with cytarabine [62]. However, AML therapy remains a challenge

In Vitro Antileukemic Mechanisms of Hyperforin

Table 1.


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In vivo effects of pure, native hyperforin in animal models.

In Vivo Effects

Animal

Trial

HYP Dosage

References

Mouse

Circular activity cages

1-10 mg/kg i.p.

[48]

Mouse

Brain membrane fluidity

15 mg/kg oral

[49]

Rat

Hippocampal acetylcholine release

1-10 mg/kg i.p.

[122]

Neurological activity - locomotor - modulation of neurotransmission

2+

2+

Mouse

Brain Ca and Zn storage

4 mg/kg i.p. (daily, 4 weeks)

[123]

Rat

MT-450 cell tumor growth and vascularization

0.2 µmol s.c. (daily, 2 weeks)

[21, 25, 51, 53]

- anti-angiogenic - anti-inflammatory

Mouse

OKS-Imm cell tumor growth and inflammatory cellular infiltrate

0.15 µmol i.p. (3 times a week) 1.9 nmol p.t.

[24]

- anti-angiogenic

Mouse

PMN-triggered neoangiogenesis

0.15 µmol i.p.

[50]

Mouse

Croton-oil-induced ear edema

0.25 µmol/cm2

[52]

Mouse

Intratracheal instillation of bleomycin

0.15 µmol i.p.

[50]

Rat

Carrageenan-induced pleurisy

4 mg/kg, i.p.

[42, 54]

Mouse

Carrageenan-induced pleurisy

1 mg/kg i.p.

[42]

Antitumor activity - antiproliferative - anti-angiogenic - anti-lymphoangiogenic

Anti-inflammatory

PMN: polymorphonuclear cells; i.p.: intraperitoneal; i.v.: intravenous; p.t.: peritumor; s.c.: subcutaneous; 1 mg of HYP is equivalent to 1.87 µmol.

for clinicians because a large number of patients are refractory to primary therapies or relapse later. New drugs are currently in clinical evaluation including inhibitors of tyrosine kinases, farnesyltransferase inhibitors, histone deacetylase inhibitors or deoxyadenosine analogs [59, 61, 62]. Given that both CLL and AML are characterized by a deficiency in apoptotic cell death, strategies for overcoming this deficiency have attracted much attention. The characterization of new agents capable of re-activating apoptosis is therefore relevant and natural products appear as good candidates. MECHANISMS OF HYPERFORIN'S ANTILEUKEMIC ACTIVITIES Apoptosis Induction in CLL Apoptosis is regulated by a complex network of interactions between pro- and anti-apoptotic proteins. Apoptotic signals can be initiated by external stimuli (such as cytotoxic drugs) and induce changes in mitochondrial membrane permeability (via the mitochondrial or intrinsic pathway). As a consequence, cytochrome c is released into the cytoplasm and then binds to and induces a conformational change in APAF-1 -resulting in the formation of the "apoptosome" complex [63]. The apoptosome recruits and activates caspase-9, which in turns activates effector caspases-3, -6 and -7. Mitochondrial membrane permeability is strictly regulated by proteins of the Bcl-2 family. This family comprises three classes: the prosurvival members, the pro-apoptotic members (all of which have Bcl-2 homology domains BH1 to 4) and the BH3-only members (having only the BH3 homology domain). The functional activity of the

prosurvival proteins, such as Bcl-2, Bcl-xL and Mcl-1, is to sequester the pro-apoptotic members Bak and Bax. When stimulated, the BH3-only proteins (Puma, Bad, Noxa, so on) bind to prosurvival proteins, which results in the liberation and activation of Bak and Bax responsible for mitochondrial membrane permeabilization. In CLL, leukemic cells over-express many anti-apoptotic proteins (including some members of the Bcl-2 family) [6466]. Our laboratory was the first to show that HYP is capable of inducing the mitochondrial apoptosis pathway in CLL cells (IC50: 3-4 µM), as evidenced by phosphatidylserine externalization, DNA fragmentation, disruption of the mitochondrial transmembrane potential, caspase-3 activation and cleavage of the caspase substrate PARP-1 [67]. Hyperforin downregulates Bcl-2, Mcl-1 and two other proteins that are over-expressed by CLL cells: the cell cycle inhibitor p27kip1 (through caspase-dependent cleavage into a p23 form) and type 2 nitric oxide (NO) synthase [67]. Inhibition of the latter reduces the production of NO [67], which displays anti-apoptotic properties in CLL cells [68] through S-nitrosylation of the active site of caspases [69, 70]. The observed, protective effect of the general caspase inhibitor z-VAD-fmk indicates that HYP-promoted apoptosis of CLL cells is mostly a caspase-dependent process. Furthermore, normal B lymphocytes from healthy donors appear to be much less sensitive to HYP-induced apoptosis (with an IC50 of around 15 µM) than CLL cells are [67]. Recently, it was reported that expression levels of Noxa (a pro-apoptotic, BH3-only member of the Bcl-2 family) are inversely related to CLL survival capacity [71]. Our group has shown that (i) HYP enhances Noxa protein expression during CLL cell apoptosis [72] and (ii) upregulated Noxa

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interacts with Mcl-1, leading to the displacement of Bak from its complex with Mcl-1, and to Bak activation [73]. Interestingly, the fact that Noxa silencing partly reduces HYP-mediated apoptosis highlights the Noxa’s pivotal role in the pro-apoptotic effect of HYP on CLL cells [73]. Inhibition of Proteasome Activity in CLL The proteasome is a multi-catalytic enzyme found in all eukaryotic cells. It degrades many intracellular proteins (including pro-apoptotic regulators) and is now acknowledged to be a promising therapeutic target [74]. The inhibition of proteasome activity by bortezomib and other proteasome inhibitors is known to result in the accumulation of Noxa [71]. Our group has reported that HYP inhibits the chymotrypsin activity of the proteasome in CLL cells, with inhibition peaking at concentrations of 30 µM [73]. Given that the phloroglucinol does not modulate Noxa mRNA, this inhibition of proteasomal activity might be responsible for the HYP-mediated enhancement of Noxa expression. A number of plant-derived compounds are natural proteasome inhibitors (such as terpenoids and polyphenols including flavonoids) and some can induce apoptosis and even upregulate Noxa. In contrast to bortezomib, HYP does not have a detectable enhancing effect on the expression of the prosurvival protein Mcl-1 in CLL cells - at least at the concentrations that produce maximum Noxa upregulation and apoptosis induction (≤18 µM). These data appear to be of considerable interest because (i) Mcl-1 is the crucial antiapoptotic protein in CLL and (ii) increased Mcl-1 expression is a major obstacle to the clinical use of bortezomib in this leukemia. It remains to examine whether HYP upregulates Noxa through the same mechanism as bortezomib and other proteasome inhibitors. Proliferation Arrest and Apoptosis Induction in AML In 2003, Hostanska et al. were the first to show that HYP inhibited the proliferation of human myeloid leukemia cell lines K562 and U937 cells, with growth inhibition (GI50) values of around 15 µM at 48h [75]. Our study confirmed that highly purified HYP induced a time- and dosedependent growth arrest in U937 cells. Moreover, we demonstrated that HYP produced cell cycle arrest in the G2/M and sub-G1 phases in AML cell lines characterized by distinct French-American-British (FAB) subtypes, namely U937 cells (FAB M5/monoblast; p53 null), HL-60 (M2/myeloblast; p53 null), NB4 (M3/promyelocyte; p53+) and OCI-AML3 (M4/myelomonocyte; p53+) [76]. The GI50 concentrations ranged from 2.5 to 4 µM at 48 h. The lack of a relationship between p53 status and the inhibition of cell proliferation strongly suggested that HYP operates through a p53-independent mechanism in these AML cell lines. Hyperforin-induced growth arrest was accompanied by apoptotic cell death and directly interfered with cell survival via the modulation of apoptotic/survival pathways. In U937 cells, HYP stimulated caspase-9, -8 and -3 activities and induced PARP-1 cleavage. Apoptosis was blocked by both z-VAD-fmk and selective inhibitors of caspase-9 and -3 [75, 76]. In the same concentration range, HYP induced apoptosis in primary AML blasts (regardless of their FAB status) but normal peripheral blood mononuclear cells were not affected

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[76]. We further showed that HYP did not activate the caspases directly [76]. Apoptosis in U937 cells was accompanied by upregulation of pro-apoptotic Noxa and downregulation of anti-apoptotic Bcl-2 (thus directly affecting the mitochondrial death pathway) [76]. In K562 cells, HYP treatment arrested cells at G1 and again induced caspase-dependent apoptosis via the mitochondrial pathway [77]. In contrast, the specific caspase-8 inhibitor z-IETDfmk could not inhibit HYP-induced apoptosis in U937 cells, which strongly suggests that caspase-8 was not involved in the cell death process [76]. Moreover, HYP affected neither Fas levels nor the amounts of secreted tumor necrosis factorα (TNF-α) in U937 cells - indicating that the compound did not trigger the death receptor Fas/CD95/APO-1 pathway (extrinsic pathway of apoptosis). The serine/threonine protein kinase B (also referred to as AKT1) has well-documented oncogenic potential and prosurvival activities that can counteract the apoptosis induced by cancer drugs [78]. Activated AKT1 signaling protects AML cells from apoptosis [79]. Furthermore, AKT1 negatively regulates apoptosis of AML cells through phosphorylation of the Bcl-2 family member Bad on Ser136 [80]. This results in Bad inactivation, since only nonphosphorylated Bad can trigger apoptosis [81]. We have shown for the first time that HYP downregulates the level of the active form of AKT1 through dephosphorylation (at Ser473) in U937 cells [76]. By using an ELISA assay and recombinant AKT1 protein, we further demonstrated that HYP directly inhibits AKT1 kinase activity in a cell-free system. As a consequence of AKT inhibition, HYP blocks the phosphorylation of Bad (at Ser136) and thus promotes its activation [76]. Anti-angiogenic Effects in CLL and AML The gelatinase matrix metalloproteinases (MMP)-2 and 9 are secreted as inactive pro-enzymes (proMMP-2: 72 kDa; proMMP-9: 92 kDa) and are processed into their active forms (MMP-2: 65 kDa; MMP-9: 82 kDa) by various activation machineries [82]. Thanks to their proteolytic activities, MMP-2 and MMP-9 play major but indirect roles in cell signaling by controlling the bioavailability and bioactivity of molecules that target specific receptors involved in cell growth, migration, inflammation and angiogenesis [82, 83]. In vitro data also support the hypothesis whereby gelatinase pro-enzymes contribute to tumor cell motility [84, 85]. Accordingly, recent evidence shows that binding between cell-surface integral proteins and progelatinases may directly influence tumor cell behavior and activate the classical signaling pathways involved in cell growth, migration, survival and angiogenesis [86]. Serum levels of proMMP-9 and vascular endothelial growth factor (VEGF) are significantly higher in patients with CLL than in healthy individuals [87, 88]. Primary leukemic cells produce and release VEGF and proMMP-9 [88]. The latter is involved in the migration of CLL cells in vitro [89, 90]. Moreover, when bound to its docking receptors α4β1 integrin and CD44 at the surface of CLL cells, proMMP-9 can induce an intracellular signaling pathway that favors cell survival [91]. We found that HYP inhibited proMMP-9 production by reducing the number of

In Vitro Antileukemic Mechanisms of Hyperforin

CLL cells secreting proMMP-9 but not by reducing proMMP-9 transcription [92]. MMP-9 inhibition was also associated with a decrease in VEGF production [92]. Furthermore, VEGF participates in the transcriptional upregulation of proMMP-9 in CLL cells [88]. Although a causal relationship remains to be evidenced, it is possible that HYP-elicited VEGF inhibition results in the late-stage downregulation of MMP-9 transcript observed in CLL cells. In contrast to normal myeloid precursors, AML cells were initially found to secrete significant levels of proMMP2 and/or proMMP-9 [85, 93]. Both these proMMPs are thought to be involved in AML cell migration [85, 94, 95]. Acute myeloid leukemia cells produce and release VEGF, which in turn stimulates AML cell growth [96]. Our own study detected the release of VEGF (5-288 pg/ml) and proMMP-2 (0.1-13 ng/ml) by primary AML cells, whereas proMMP-9 production was only detectable in 62% of cases (0.1-51 ng/ml) (Merhi et al., personal communication). Although HYP (2 µM) did not alter the levels of proMMP and VEGF transcripts, it did inhibit the production of proMMP-2 in most samples and marginally decreasing the levels of VEGF and proMMP-9. Unexpectedly, HYP induced the appearance of a truncated form of MMP-9 (85 kDa), the importance of which in AML remains to be defined. Accordingly, bone marrow AML blasts have been shown to express an 85 kDa proMMP-9 resulting from deglycosylation [97]. Reversal of Multidrug Resistance in CLL and AML The ABC transporters are involved in the phenomenon of MDR, which represents a crucial problem for cancer chemotherapy. These transmembrane proteins export drugs from the cell and their expression during therapy is responsible for the emergence of MDR in a variety of tumors [98]. The prototype ABC transporter is P-gp, a product of the MDR1 (or ABCB1) gene that functions as an ATP-dependent efflux pump. Multidrug resistance is a major cause of treatment failure in AML [99]. While studying HYP's effects on leukemia cells, our laboratory showed that the compound inhibited P-gp's functional activity in CLL cells and in a daunorubicin-resistant, P-gp-over-expressing variant of the leukemic myeloid HL-60 cell line (HL-60/DBR) [100]. Primary CLL cells and HL-60/DBR cells also expressed breast cancer resistance protein (BCRP) [100] - another ABC transporter (encoded by the ABCG2 gene) [101]. Hyperforin was also able to lower BCRP activity in these cells [100]. These data suggest that HYP could be of value in modulating the MDR phenotype in leukemic cells. STABLE DERIVATIVES OF HYPERFORIN The potential clinical applications of purified HYP are limited by its poor solubility and stability in aqueous solution and its sensitivity to light and oxygen [53, 102]. This instability is related to the presence of reactive functional groups, i.e. the enolized and oxidation-prone βdiketone moiety and the prenyl side chains [103]. Various synthetic derivatives of HYP have greater stability, solubility and pharmacological activity and have thus been studied for potential antidepressant, anti-inflammatory, antitumor and anti-angiogenic uses (both in vitro and in animal models).


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Hyperforin-DCHA The dicyclohexylammonium salt of HYP (HYP-DCHA) has been tested successfully for its antidepressant activity in rats [104] and anti-inflammatory activity in mice [50]. In fact, HYP-DCHA blocks the IL-6 release normally triggered by inflammatory mediators [105]. In vitro, the compound (i) triggers apoptosis in murine and human tumor cell lines [22, 106], (ii) inhibits migration of the latter in Matrigel®-coated Transwell® chambers and (iii) decreases proMMP-2 and -9 secretion. In vivo¸ HYP-DCHA reduces the number of metastasis in mice grafted with the C-26 and B16-LU8 tumors [22]. The compound's inhibition of neutrophil chemotaxis and migration is accompanied by the blockade of leukocyte elastase-triggered activation of proMMP-9 [50, 103]. Oxidized Forms of Hyperforin The major degradation products of HYP in acidic aqueous solutions include furo-HYP and furo-HYP hydroperoxide [102, 107, 108]. They are formed by a mutual, oxidative interaction between the enol moiety and the prenyl chains [107, 108]. Furo-HYP is reportedly a potent inhibitor of CYP3A4 [35] but appears to be a much less potent anti-angiogenic compound than HYP is [103]. Recently, tetrahydro-HYP and octahydro-HYP-DCHA (stable derivatives of HYP, obtained by oxidative modification) were reported to be potent inhibitors of angiogenesis in vitro, as demonstrated by inhibition of endothelial cell growth/migration and microtubule formation on Matrigel [103]. Aristoforin O-(carboxymethyl)-HYP (also referred to as aristoforin) is highly stable and more soluble in aqueous solution than HYP. It retains the biological activities of the parent compound (and notably the antitumor properties) but is less toxic in animals [51]. Aristoforin has a strong inhibitory effect on the in vitro proliferation of MT-450 mammary carcinoma cells [51]. In thoracic duct ring outgrowth assays, both HYP and aristoforin suppressed lymphatic capillary outgrowth. Both compounds were able to inhibit tumorinduced lymphangiogenesis in a rat model [53]. Acetylate Hyperforin Another stable derivative, acetylate HYP (ace-HYP), can modulate α-secretase-mediated amyloid precursor protein (APP) processing via a protein kinase C signaling pathway in epithelial cells transfected with APP cDNA [109]. AceHYP exhibits antiproliferative effects in various malignant cells lines, including the K562 cell line that is representative of chronic myeloid leukemia [106]. Ace-HYP's efficacy has been demonstrated in animal models that are sensitive to antidepressant and anxiolytic drugs [110]. CONCLUSIONS AND PERSPECTIVES The main problem in chemotherapy of CLL or AML is intrinsic or acquired MDR by leukemic cells, which resist apoptosis as a result of survival, proliferation, migration and angiogenesis signals provided by the tumor

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microenvironment. Hence, reducing side-effects, optimizing efficacy and identifying new molecular targets become a major goal in the development of novel apoptosis inducers. In view of HYP's low toxicity for normal blood cells and its anti-inflammatory properties, the compound appeared to be an attractive candidate for the development of novel, antileukemic drugs. The data accumulated over the last six years proves that HYP is able to (i) inhibit the expression of mediators involved in the chemoresistance of leukemic cells (such as P-gp) and in angiogenesis, survival and migration (VEGF and proMMPs) and (ii) effectively target signaling pathways related to tumor cell proliferation and survival/ apoptosis. Table 2 summarizes the effects of HYP in CLL and AML and specifies the identified molecular mechanisms and targets. Importantly, HYP stimulates the expression of Noxa, a pro-apoptotic BH3-only protein from the Bcl-2 family that is crucial in fine-tuning cell death decisions by specifically inhibiting the prosurvival activity of Mcl-1 and by targeting the latter protein for proteasomal degradation [111]. The development of BH3 mimetics capable of mimicking Noxa and effectively antagonizing Mcl-1 offers a novel approach to the treatment of CLL [112, 113]. The clinical use of HYP in CLL patients can also be considered. The demonstration that HYP is a direct inhibitor of AKT1's kinase activity is of particular interest. As a result, the proapoptotic function of Bad (a downstream target of AKT1) is promoted. Thus, the potential therapeutic value of HYP in AML is related to its ability to inhibit a protein kinase that is crucially involved in the modulation of cell proliferation, survival and death pathways [79, 114, 115]. Despite these in vitro observations, HYP's therapeutic potential has yet to be evaluated in vivo in animal models of CLL or AML. However, several aspects require further investigation. One major concern with pharmacological use of HYP relates Table 2.

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to its interference with other therapeutic agents via activation of CYP450 enzymes (notably CYP3A4) and/or P-gp. Clinical studies of low HYP contents (