afimoxifene and Multiple-Myeloma

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Humans; Liposomes; Mice; Multiple Myeloma; Receptors, Estrogen; Tamoxifen; Xenograft Model Antitumor Assays

To determine the better liposomal formulation incorporating the active metabolite of tamoxifen, 4-hydroxy-tamoxifen (4HT) and the biological impact of 4HT-pH-gradient liposomes on response to in vivo treatment.. Several pegylated liposomes were formulated by varying the composition of lipids, increasing external pH from 7.4 to 9.0 and doubling the lipid concentration. Dipalmitoylphosphatidylcholine / cholesterol / distearoylphosphoethanolamine poly(ethylene)glycol liposomes (DL-9 liposomes) were chosen for their physico-chemical properties. Toxicity and release kinetics were assessed in breast cancer MCF-7 as well as in multiple myeloma (MM) cells. In vivo antitumor activity and bio-distribution were measured in the RPMI8226 MM model.. Compared to conventional non-pH-gradient liposomes, 4HT-DL-9 liposomes resulted in concentration of up to 1 mM 4HT, greater stability, relative toxicity and slow 4HT release. Intravenous injections of 4HT-DL-9 liposomes at 4 mg/kg/week blocked MM tumor growth. Ki67 and CD34 labeling decreased in treated tumors, concomitantly with increase of activated caspase-3 supporting a cell proliferation arrest, a decrease of tumor vasculature and the induction of tumor cell death.. This antitumor effect was assumed to be the result of a modified biodistribution of 4HT once trapped in DL-9 liposomes. Such 4HT-containing pH-gradient Stealth nanocarriers could be helpful for MM treatment.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Liposomes; Mice; Mice, Nude; Multiple Myeloma; Proton-Motive Force; Tamoxifen; Xenograft Model Antitumor Assays

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.. 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.. 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.. 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.

Topics: Apoptosis; Caspase 9; Caspases; Cell Cycle Proteins; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Estrogen Antagonists; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Membrane Potentials; Mitochondria; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured; Tumor Suppressor Proteins

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.

Topics: Apoptosis; Breast Neoplasms; Capsules; Cell Cycle; Cell Division; Cell Line, Tumor; Drug Carriers; Estrogen Receptor Modulators; Female; Humans; Liposomes; Multiple Myeloma; Tamoxifen; Transfection

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.

Topics: Apoptosis; Cell Line, Tumor; Dose-Response Relationship, Drug; G1 Phase; Humans; Kinetics; Multiple Myeloma; Receptors, Estrogen; Tamoxifen

Previous studies have suggested that multiple myeloma (MM) cells express estrogen receptors (ER). In the present study, we characterized the effects of estrogen agonists and antagonists (anti-estrogens [AE]) on growth of MM cell lines and MM patient cells. In addition to antagonizing estrogen binding to ER, AE can trigger apoptosis. Hence, we also determined whether estrogens or AE altered MM cell survival. Immunoblotting showed that ER-alpha is expressed in 4 of 5 MM cell lines (ARH-77, RPMI 8226, S6B45, and U266, but not OCI-My-5 cells), as well as in freshly isolated MM cells from 3 of 3 patients. 17beta-estradiol (E2) did not significantly alter proliferation of MM cell lines or MM patient cells. In contrast, two structurally distinct AE, tamoxifen (TAM) and ICI 182,780 (ICI), significantly inhibited the proliferation of all 5 MM cell lines and MM cells from 2 of 2 patients (IC50, 2 to 4 micromol/L). Proliferation of these cell lines was also inhibited by the hydroxylated TAM derivative, 4-hydroxytamoxifen (4HTAM), although this derivative was less potent than TAM (IC50, 3 to 25 micromol/L). In contrast, the dehalogenated TAM derivative toremifene (TOR) did not inhibit MM cell proliferation. We next examined the effects of these agents on MM cell survival. TAM, ICI, and, to a lesser extent, 4HTAM and TOR triggered apoptosis in both ER-alpha-positive as well as ER-alpha-negative MM cell lines and patient MM cells, evidenced both by fluorescence-activated cell sorting (FACS) analysis using propidium iodide staining and the TUNEL assay. TAM-induced growth inhibition and apoptosis of ER-alpha-positive S6B45 MM cells was not blocked by coculture with excess E2. TAM-induced apoptosis of S6B45 MM cells was also unaffected by addition of exogenous interleukin-6. Importantly, both the inhibition of MM cell proliferation and the induction of MM cell apoptosis were achieved at concentrations of TAM (0.5 and 5.0 micromol/L) that did not significantly alter in vitro growth of normal hematopoietic progenitor cells. Similar plasma levels of TAM have been achieved using high-dose oral TAM therapy, with an acceptable toxicity profile. These studies therefore provide the rationale for trials to define the utility of AE therapy in MM.

Topics: Apoptosis; Breast Neoplasms; Cell Division; Cell Survival; Coloring Agents; Estradiol; Estrogen Antagonists; Flow Cytometry; Fulvestrant; Hematopoietic Stem Cells; Humans; Immunoblotting; Multiple Myeloma; Propidium; Receptors, Estrogen; Tamoxifen; Toremifene; Tumor Cells, Cultured