Improved antitumoral properties of pure antiestrogen RU 58668-loaded liposomes in multiple myeloma.

In most of multiple myeloma (MM) cells, the "pure" antiestrogen (AE) RU 58668 (RU) induced either a G1-arrest (LP-1, OPM-2, NCI-H929, U266 cells) or apoptosis (RPMI 8226 cells). In RPMI 8226 cells, RU activates a caspase-dependent cell death pathway leading to the release of cytochrome c, the decrease of the essential MM survival factor Mcl-1, the cleavage of Bid and the activation of caspases-3 and -8. Incorporation of RU in pegylated cholesterol-containing liposomes allowed a controlled RU release, improving its anti-proliferative and apoptotic effects in cells. In RPMI 8226 xenografts, i.v. injected RU-liposomes but not free RU, exhibited antitumor activity. In vivo, RU-liposomes triggered the mitochondrial death pathway, concomitantly with a down-regulation of Mcl-1 and Bid cleavage. The decrease of CD34 immunoreactivity indicated a reduction of angiogenesis. The decrease of VEGF secretion in vitro supported a direct effect of RU on angiogenesis. These pro-apoptotic and antiangiogenic effects were explained by a prolonged exposure to the drug and to the endocytosis capacity of liposomes which might increase RU uptake and bypass a membrane export of free RU. Thus, these combined enhanced activities of RU-liposomes support that such a delivery of an AE may constitute a strategy of benefit for MM treatment.

4-Hydroxytamoxifen inhibits proliferation of multiple myeloma cells in vitro through down-regulation of c-Myc, up-regulation of p27Kip1, and modulation of Bcl-2 family members.

PURPOSE: Multiple myeloma is an incurable B-cell malignancy requiring new therapeutic strategies. Our approach was to analyze the in vitro effects of a selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT), on six multiple myeloma cell lines. EXPERIMENTAL DESIGN: Cultured multiple myeloma cells were treated with various 4-OHT concentrations and the cellular response was studied: cell proliferation, cell viability, induction of apoptosis, caspase activities, and expression of signaling proteins. RESULTS: We found that pharmacologic concentrations of 4-OHT inhibit cell proliferation (4 of 6 cell lines). This inhibition is achieved by two independent events: a block at the G(1) phase of the cell cycle and the induction of apoptotic death. The cellular response to 4-OHT depends on the presence of functional estrogen receptors. 4-OHT treatment activates an intrinsic mitochondrial caspase-9-dependent pathway but not the Fas/FasL death pathway. Signaling pathways known to be involved in the survival and/or proliferation of multiple myeloma cells are not affected by 4-OHT treatment. 4-OHT-induced G(1) arrest is accompanied by the up-regulation of the cell cycle inhibitor p27(Kip1) and the down-regulation of c-Myc. Among the Bcl-2 family members tested, the proapoptotic BimS protein is induced whereas the antiapoptotic protein Mcl-1 is decreased. CONCLUSIONS: Although the effects of 4-OHT are observed at micromolar concentrations, cellular mechanisms responsible for G(1) arrest, as well as apoptosis induction, are similar to those observed in breast cancer cells. Our data support the concept that 4-OHT may represent an alternative approach to inhibit proliferation and induce apoptosis of multiple myeloma cells.

Estrogen receptor alpha and beta subtype expression and transactivation capacity are differentially affected by receptor-, hsp90- and immunophilin-ligands in human breast cancer cells.

In MCF-7 (estrogen receptor (ER)+) and in MDA-MB-231 (ER-) cells stably transfected with either estrogen receptor alpha (ERalpha) or beta (ERbeta) subtype (MDA-MB-231 stably transfected with the mouse ERalpha cDNA (MERA) and MDA-MB-231 stably transfected with the human ERbeta cDNA (HERB), respectively) N-term heat shock protein of 90kDa (hsp90) ligands (geldanamycin and radicicol) and C-term hsp90 ligands (novobiocin) decrease the basal and estradiol (E(2))-induced transcription activity of ER on an estrogen responsive element (ERE)-LUC reporter construct concomitantly with or 1h after E(2) treatment. All hsp90 ligands induced an E(2)- and MG132-inhibited decrease of both ER cell content. However, the kinetics of these degradations are slower than those induced by the selective estrogen receptor down-regulator RU 58668 (RU). This suggests that inhibition of the hsp90 ATPase activity targets both ERs to the 26S proteasome and that hsp90 interacts with both ER subtypes. Rapamycin (Rapa) and cyclosporin A (CsA), ligands of immunophilins FK506 binding protein (FKBP52) and cyclophilin of 40kDa (CYP40) interacting in separate ER-hsp90 complexes, both induced a proteasomal-mediated degradation of ERs but not of their cognate immunophilin. Moreover, they also decrease the E(2)-induced luciferase transcription but weaker than RU and hsp90 ligands. Fluorescence activated cell sorter (FACS) analysis revealed a blockade of cell progression by RU and 4-hydroxy-tamoxifen at the G(1) phase of the cell cycle and an induction of apoptosis in MCF-7 cells. Rapa and mainly CsA (but not FK506) and hsp90 ligands promote by their own apoptosis in MCF-7, in MERA, and in HERB cells and in MDA-MB-231 ER-null cells. These data suggest that (1) hsp90, as for all steroid receptors, acts as a molecular chaperone for ERbeta; (2) ER-ligands (except tamoxifen), hsp90- and immunophilin-ligands (except FK506) target the two ER subtypes to a proteasome-mediated proteolysis via different signalling pathways; (3) hsp90- and immunophilin-ligands Rapa and CsA, alone or in association with anti-estrogens such as RU, may constitute a potential therapeutic strategy for breast cancer treatment.

Innovative drug delivery nanosystems improve the anti-tumor activity in vitro and in vivo of anti-estrogens in human breast cancer and multiple myeloma.

Anticancer drug efficiency is governed by its bioavailability. In order to increase this parameter, we synthesized several injectable and biodegradable systems based on incorporation of anti-estrogens (AEs) in nanoparticles (NPs) and liposomes were synthesized. Both nanospheres (NS) and nanocapsules (NCs, polymers with an oily core in which AEs were solubilized) incorporated high amounts of 4-hydroxy-tamoxifen (4-HT) or RU 58668 (RU). Physico-chemical and biological parameters of these delivery systems, and coupling of polyethylene-glycol chains on the NP surface revealed to enhance the anti-tumoral activity of trapped AEs in a breast cancer MCF-7 cell xenograft model and to induce apoptosis. These features correlated with an augmentation of p21(Waf-1/Cip1) and of p27(Kip1) and a concomitant decrease of cyclin D1 and E in tumor extracts. Liposomes containing various ratios of lipids enhanced the apoptotic activity of RU in several multiple myeloma (MM) cell lines tested by flow cytometry. MM cell lines expressed both estrogen receptor alpha and beta subtypes except Karpas 620. Karpas 620 cells which did not respond to AEs became responsive following ER cDNA transfection. A new MM xenograft model was generated after s.c. injection of RPMI 8226 cells in nude mice. RU-loaded liposomes, administered i.v. in this model, at a dose of 12mgRU/kg/week, induced the arrest of tumor growth contrary to free RU or to empty liposomes. Thus, the drug delivery of anti-estrogens enhances their ability to arrest the growth of tumors which express estrogen receptors and are of particular interest for estrogen-dependent breast cancer treatment. In addition it represents a new potent therapeutic approach for multiple myeloma.

Various phosphorylation pathways, depending on agonist and antagonist binding to endogenous estrogen receptor alpha (ERalpha), differentially affect ERalpha extractability, proteasome-mediated stability, and transcriptional activity in human breast cancer cells.

Estrogen receptor-alpha (ER) is down-regulated in the presence of its cognate ligand, estradiol (E2), as well as in the presence of antiestrogens, through the ubiquitin proteasome pathway. Here, we show that, at pharmacological concentrations, the degradation rate of pure antagonist/endogenous ER complexes from human breast cancer MCF-7 cells is 10 times faster than that of ER-E2 complexes, while 4-hydroxy-tamoxifen (4-OH-T)-ER complexes are stable. Whereas pure antagonist-ER complexes are firmly bound to a nuclear compartment from which they are not extractable, the 4-OH-T-ER accumulates in a soluble cell compartment. No difference was observed in the fate of ER whether bound to pure antiestrogens ICI 182,780 or RU 58668. Cycloheximide experiments showed that, while the proteasome-mediated destruction of E2-ER (unlike that of RU 58668- and ICI 182,780-ER) complexes could implicate (or not) a protein synthesis-dependent process, both MAPKs (p38 and ERKs p44 and p42) are activated. By using a panel of kinase inhibitors/activators to study the impact of phosphorylation pathways on ER degradation, we found that protein kinase C is an enhancer of proteasome-mediated degradation of both ligand-free and ER bound to either E2, 4-OH-T, and pure antagonists. On the contrary, protein kinase A, MAPKs, and phosphatidyl-inositol-3 kinase all impede proteasome-mediated destruction of ligand free and E2-bound ER while only MAPKs inhibit the degradation of pure antiestrogens/ER species. In addition, no correlation was found between the capacity of kinase inhibitors to affect ER stability and the basal or E2-induced transcription. These results suggest that, in MCF-7 breast cancer cells, ER turnover, localization, and activity are maintained by an equilibrium between various phosphorylation pathways, which are differently modulated by ER ligands and protein kinases.

The selective estrogen receptor modulator 4-hydroxy tamoxifen induces G1 arrest and apoptosis of multiple myeloma cell lines.

Multiple myeloma (MM) is an incurable hematological malignancy for which new therapeutic strategies should be envisaged. The selective estrogen receptor modulator (SERM), 4-hydroxy tamoxifen (4-OHTam), in the range of 1 to 10 micro M, was able to impair the cell proliferation of MM cell lines. This was achieved by blocking cells at the G1 phase of the cell cycle and by inducing apoptosis. This cellular response was observed in five out of six tested cell lines, all five expressing both alpha and beta estrogen receptor forms. No modifications of Bcl-2, Bcl-X, and Bax levels were observed, as well as no changes in Pi3K/Akt and JAK/STAT pathways that are often constitutively active in these cells. The signalization of 4-OHTam-induced cell death needs further investigation.