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Antiestrogen-binding site ligands induce autophagy in myeloma cells that proceeds through alteration of cholesterol metabolism.

Sola B, Poirot M, de Medina P, Bustany S, Marsaud V, Silvente-Poirot S, Renoir JM - Oncotarget (2013)

Bottom Line: Autophagy was recognized by monodansylcadaverin staining, detection of autophagosomes by electronic microscopy, and detection of the cleaved form of the microtubule-associated protein light chain 3.Moreover, AEBS ligands restored sensitivity to dexamethasone in resistant MM cells.Since we showed previously that AEs arrest MM tumor growth in xenografted mice, we propose that AEBS ligands may have a potent antimyeloma activity alone or in combination with drugs used in clinic.

View Article: PubMed Central - PubMed

Affiliation: Normandie University, UNICAEN EA4652, Caen, France.

ABSTRACT
Multiple myeloma (MM) is a malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Despite extensive efforts to design drugs targeting tumoral cells and their microenvironment, MM remains an incurable disease for which new therapeutic strategies are needed. We demonstrated here that antiestrogens (AEs) belonging to selective estrogen receptor modulators family induce a caspase-dependent apoptosis and trigger a protective autophagy. Autophagy was recognized by monodansylcadaverin staining, detection of autophagosomes by electronic microscopy, and detection of the cleaved form of the microtubule-associated protein light chain 3. Moreover, autophagy was inhibited by drugs such as bafilomycin A1 and 3-methyladenosine. Autophagy was mediated by the binding of AEs to a class of receptors called the antiestrogen binding site (AEBS) different from the classical estrogen nuclear receptors. The binding of specific ligands to the AEBS was accompanied by alteration of cholesterol metabolism and in particular accumulation of sterols: zymostenol or desmosterol depending on the ligand. This was due to the inhibition of the cholesterol-5,6-epoxide hydrolase activity borne by the AEBS. We further showed that the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathway mediated autophagy signaling. Moreover, AEBS ligands restored sensitivity to dexamethasone in resistant MM cells. Since we showed previously that AEs arrest MM tumor growth in xenografted mice, we propose that AEBS ligands may have a potent antimyeloma activity alone or in combination with drugs used in clinic.

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OHT and AEBS ligands induce the accumulation of cholesterol precursors in HMCLsA) RPMI 8226 and LP-1 cells were treated with EtOH or OHT (10 μM) for 48 h and cytospinned. Slides were stained with filipin and analyzed. Cells containing free sterols are colored in blue. Cells were visualized in the ultraviolet range using a x40 objective on a Zeiss LSM 510 microscope (Göettingen, Germany). B) The sterols accumulated in RPMI 8226 and LP-1 cells after 48 h incubation with SERMs, SERDs and selective AEBS/ChEH ligands were determined and quantified. Analyses were performed by HPLC and GC/MS as described in the Methods section. The cholesterol intermediates were quantified as a percent by the weight of total sterol. The amount of zymostenol and desmosterol was quantified by references to an external standard. C) HMCLs were treated with EtOH, OHT (10 μM), Tam (10 μM), RU 58668 (10 μM) or PBPE (40 μM) for 24 h and incubated with 0.6 μM [14C]-EC for 48 h and ChEH activity was assayed by measuring the conversion of CE into CT by TLC. A representative autoradiogram from three independent experiments is shown. D) RPMI 8226 and LP-1 cells were pre-incubated with 500 μM Vit E, 1 mM NAC, 50 nM BAF A1 or 10 mM 3-MA and then challenged with OHT (10 μM) or PBPE (40 μM) for 72 h. Cell death was determined by Trypan blue exclusion. Data were expressed as the percentage of cell death relative to control cells that received OHT (10 μM) or PBPE (40 μM). Experiments were repeated three times in duplicate with comparable results. The data present ed here are the means ± SEM of all experiments. *, p<0.001.
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Figure 3: OHT and AEBS ligands induce the accumulation of cholesterol precursors in HMCLsA) RPMI 8226 and LP-1 cells were treated with EtOH or OHT (10 μM) for 48 h and cytospinned. Slides were stained with filipin and analyzed. Cells containing free sterols are colored in blue. Cells were visualized in the ultraviolet range using a x40 objective on a Zeiss LSM 510 microscope (Göettingen, Germany). B) The sterols accumulated in RPMI 8226 and LP-1 cells after 48 h incubation with SERMs, SERDs and selective AEBS/ChEH ligands were determined and quantified. Analyses were performed by HPLC and GC/MS as described in the Methods section. The cholesterol intermediates were quantified as a percent by the weight of total sterol. The amount of zymostenol and desmosterol was quantified by references to an external standard. C) HMCLs were treated with EtOH, OHT (10 μM), Tam (10 μM), RU 58668 (10 μM) or PBPE (40 μM) for 24 h and incubated with 0.6 μM [14C]-EC for 48 h and ChEH activity was assayed by measuring the conversion of CE into CT by TLC. A representative autoradiogram from three independent experiments is shown. D) RPMI 8226 and LP-1 cells were pre-incubated with 500 μM Vit E, 1 mM NAC, 50 nM BAF A1 or 10 mM 3-MA and then challenged with OHT (10 μM) or PBPE (40 μM) for 72 h. Cell death was determined by Trypan blue exclusion. Data were expressed as the percentage of cell death relative to control cells that received OHT (10 μM) or PBPE (40 μM). Experiments were repeated three times in duplicate with comparable results. The data present ed here are the means ± SEM of all experiments. *, p<0.001.

Mentions: The presence of MLBs and UVs in OHT-treated cells could reflect an increasing mass of free sterols and lipids in OHT-treated cells. Detection of free sterols in HMCLs was achieved using filipin labeling. As shown Figure 3A, perinuclear vesicles were stained by filipin in OHT- but not in EtOH- or RU 58668-treated cells. As previously described for BC cells [12], we confirmed that Tam, OHT, benzylphenoxy-ethyl-pyrrolidin (PBPE) and RU 39411 bind to the AEBS with high affinity in HMCLs while RU 58668 had no measurable affinity (Figure 3B). Free sterols accumulated also in HMCLs treated with OHT and AEBS ligands (Supplementary Figure 2). Tam and PBPE induced the accumulation zymostenol, OHT and RU 39411 induced the accumulation of desmosterol and, as expected, RU 58668 did not induce free sterol accumulation (Figure 3B). Moreover, OHT and PBPE induced the accumulation of sphingomyelin, which was correlative with the increase of cellular free sterols (Supplementary Figure 2). Altogether, these data established that MLBs content found in HMCLs is similar to that found in BC cells [14]. Neutral lipids analyses showed that OHT and PBPE treatments induced the accumulation of triacylglycerol (TG) in cells explaining the presence of UVs (Supplementary Figure 2). We recently reported that TG biosynthesis and accumulation was mainly due to the inhibition of ChEH carried out by the AEBS, which led also to the accumulation of 5,6-ECs [18]. We next evaluated the activity of the ChEH enzyme and firstly verified that ChEH activity was measurable in each cell line. As shown Figure 3C, 5,6-EC accumulated in OHT-, PBPE- and Tam-treated cells indicating that ChEH activity was inhibited by these drugs. By contrast, its activation product, CT accumulated in EtOH- and RU 58668-treated cells (Figure 3C). We found that, in HMCLs, AEBS ligands inhibited ChEH in a dose-dependent fashion with submicromolar concentrations of IC50 except for RU 58668 which did not inhibit ChEH up to 10 μM (Table 1).


Antiestrogen-binding site ligands induce autophagy in myeloma cells that proceeds through alteration of cholesterol metabolism.

Sola B, Poirot M, de Medina P, Bustany S, Marsaud V, Silvente-Poirot S, Renoir JM - Oncotarget (2013)

OHT and AEBS ligands induce the accumulation of cholesterol precursors in HMCLsA) RPMI 8226 and LP-1 cells were treated with EtOH or OHT (10 μM) for 48 h and cytospinned. Slides were stained with filipin and analyzed. Cells containing free sterols are colored in blue. Cells were visualized in the ultraviolet range using a x40 objective on a Zeiss LSM 510 microscope (Göettingen, Germany). B) The sterols accumulated in RPMI 8226 and LP-1 cells after 48 h incubation with SERMs, SERDs and selective AEBS/ChEH ligands were determined and quantified. Analyses were performed by HPLC and GC/MS as described in the Methods section. The cholesterol intermediates were quantified as a percent by the weight of total sterol. The amount of zymostenol and desmosterol was quantified by references to an external standard. C) HMCLs were treated with EtOH, OHT (10 μM), Tam (10 μM), RU 58668 (10 μM) or PBPE (40 μM) for 24 h and incubated with 0.6 μM [14C]-EC for 48 h and ChEH activity was assayed by measuring the conversion of CE into CT by TLC. A representative autoradiogram from three independent experiments is shown. D) RPMI 8226 and LP-1 cells were pre-incubated with 500 μM Vit E, 1 mM NAC, 50 nM BAF A1 or 10 mM 3-MA and then challenged with OHT (10 μM) or PBPE (40 μM) for 72 h. Cell death was determined by Trypan blue exclusion. Data were expressed as the percentage of cell death relative to control cells that received OHT (10 μM) or PBPE (40 μM). Experiments were repeated three times in duplicate with comparable results. The data present ed here are the means ± SEM of all experiments. *, p<0.001.
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Figure 3: OHT and AEBS ligands induce the accumulation of cholesterol precursors in HMCLsA) RPMI 8226 and LP-1 cells were treated with EtOH or OHT (10 μM) for 48 h and cytospinned. Slides were stained with filipin and analyzed. Cells containing free sterols are colored in blue. Cells were visualized in the ultraviolet range using a x40 objective on a Zeiss LSM 510 microscope (Göettingen, Germany). B) The sterols accumulated in RPMI 8226 and LP-1 cells after 48 h incubation with SERMs, SERDs and selective AEBS/ChEH ligands were determined and quantified. Analyses were performed by HPLC and GC/MS as described in the Methods section. The cholesterol intermediates were quantified as a percent by the weight of total sterol. The amount of zymostenol and desmosterol was quantified by references to an external standard. C) HMCLs were treated with EtOH, OHT (10 μM), Tam (10 μM), RU 58668 (10 μM) or PBPE (40 μM) for 24 h and incubated with 0.6 μM [14C]-EC for 48 h and ChEH activity was assayed by measuring the conversion of CE into CT by TLC. A representative autoradiogram from three independent experiments is shown. D) RPMI 8226 and LP-1 cells were pre-incubated with 500 μM Vit E, 1 mM NAC, 50 nM BAF A1 or 10 mM 3-MA and then challenged with OHT (10 μM) or PBPE (40 μM) for 72 h. Cell death was determined by Trypan blue exclusion. Data were expressed as the percentage of cell death relative to control cells that received OHT (10 μM) or PBPE (40 μM). Experiments were repeated three times in duplicate with comparable results. The data present ed here are the means ± SEM of all experiments. *, p<0.001.
Mentions: The presence of MLBs and UVs in OHT-treated cells could reflect an increasing mass of free sterols and lipids in OHT-treated cells. Detection of free sterols in HMCLs was achieved using filipin labeling. As shown Figure 3A, perinuclear vesicles were stained by filipin in OHT- but not in EtOH- or RU 58668-treated cells. As previously described for BC cells [12], we confirmed that Tam, OHT, benzylphenoxy-ethyl-pyrrolidin (PBPE) and RU 39411 bind to the AEBS with high affinity in HMCLs while RU 58668 had no measurable affinity (Figure 3B). Free sterols accumulated also in HMCLs treated with OHT and AEBS ligands (Supplementary Figure 2). Tam and PBPE induced the accumulation zymostenol, OHT and RU 39411 induced the accumulation of desmosterol and, as expected, RU 58668 did not induce free sterol accumulation (Figure 3B). Moreover, OHT and PBPE induced the accumulation of sphingomyelin, which was correlative with the increase of cellular free sterols (Supplementary Figure 2). Altogether, these data established that MLBs content found in HMCLs is similar to that found in BC cells [14]. Neutral lipids analyses showed that OHT and PBPE treatments induced the accumulation of triacylglycerol (TG) in cells explaining the presence of UVs (Supplementary Figure 2). We recently reported that TG biosynthesis and accumulation was mainly due to the inhibition of ChEH carried out by the AEBS, which led also to the accumulation of 5,6-ECs [18]. We next evaluated the activity of the ChEH enzyme and firstly verified that ChEH activity was measurable in each cell line. As shown Figure 3C, 5,6-EC accumulated in OHT-, PBPE- and Tam-treated cells indicating that ChEH activity was inhibited by these drugs. By contrast, its activation product, CT accumulated in EtOH- and RU 58668-treated cells (Figure 3C). We found that, in HMCLs, AEBS ligands inhibited ChEH in a dose-dependent fashion with submicromolar concentrations of IC50 except for RU 58668 which did not inhibit ChEH up to 10 μM (Table 1).

Bottom Line: Autophagy was recognized by monodansylcadaverin staining, detection of autophagosomes by electronic microscopy, and detection of the cleaved form of the microtubule-associated protein light chain 3.Moreover, AEBS ligands restored sensitivity to dexamethasone in resistant MM cells.Since we showed previously that AEs arrest MM tumor growth in xenografted mice, we propose that AEBS ligands may have a potent antimyeloma activity alone or in combination with drugs used in clinic.

View Article: PubMed Central - PubMed

Affiliation: Normandie University, UNICAEN EA4652, Caen, France.

ABSTRACT
Multiple myeloma (MM) is a malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Despite extensive efforts to design drugs targeting tumoral cells and their microenvironment, MM remains an incurable disease for which new therapeutic strategies are needed. We demonstrated here that antiestrogens (AEs) belonging to selective estrogen receptor modulators family induce a caspase-dependent apoptosis and trigger a protective autophagy. Autophagy was recognized by monodansylcadaverin staining, detection of autophagosomes by electronic microscopy, and detection of the cleaved form of the microtubule-associated protein light chain 3. Moreover, autophagy was inhibited by drugs such as bafilomycin A1 and 3-methyladenosine. Autophagy was mediated by the binding of AEs to a class of receptors called the antiestrogen binding site (AEBS) different from the classical estrogen nuclear receptors. The binding of specific ligands to the AEBS was accompanied by alteration of cholesterol metabolism and in particular accumulation of sterols: zymostenol or desmosterol depending on the ligand. This was due to the inhibition of the cholesterol-5,6-epoxide hydrolase activity borne by the AEBS. We further showed that the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathway mediated autophagy signaling. Moreover, AEBS ligands restored sensitivity to dexamethasone in resistant MM cells. Since we showed previously that AEs arrest MM tumor growth in xenografted mice, we propose that AEBS ligands may have a potent antimyeloma activity alone or in combination with drugs used in clinic.

Show MeSH
Related in: MedlinePlus