TAMR cells in comparison to TAMS cells constitutively express higher levels of prosurvival mediators and cholesterol-rich lipid microdomains. (a) TAMR cell lines MCF-7/TAMR and MCF-7/HER-2, as well as TAMS parental MCF-7/TAMS and MCF-7/Neo cells, were treated with TAM at 1 μM or VEH (ethanol) in steroid-depleted media containing 17β-estradiol (10^-9 M) for 2 days. Molecular profile of prosurvival mediators was determined with Western blot analyses. (pHER-1/tHER-1 and pHER-2/tHER-2 were not detected in the MCF-7/TAMS and MCF-7/Neo cells). (b) The expression of lipid microdomains was determined by staining the cells with fluorescein-tagged DiLC16 lipid-raft marker and viewed with a fluorescence microscope. (c) The expression of cholesterol-rich lipid microdomains was determined by staining cells with fluorescein-labeled filipin, a cholesterol marker, and viewed by using a fluorescence microscope. (a-c) Data are representative of a minimum of three independent experiments. DiIC-16, dialkylindocarbocyanine; HER, human epidermal growth factor; MCF-7/HER-2, Clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; MCF-7/TAMS, TAM-sensitive MCF-7/parental; pHER-1, phosphorylated-HER-1; pHER-2, phosphorylated-HER-2; TAM, tamoxifen; TAMR, tamoxifen resistant; TAMS, tamoxifen sensitive; VEH, vehicle control.

α-TEA alone and in combination with TAM induces apoptosis in TAMR cell lines.(a) TAM-sensitive MCF-7/parental cells and TAMR MCF-7/TAMR and MCF-7/HER-2 cells were treated with 10, 20, and 40 μM α-TEA for 1 day. Apoptosis was determined with Annexin V/PI. (b) MCF-7/TAMR and MCF-7/HER-2 cells were treated with 10 or 20 or 30 μM α-TEA, 0.5 or 1 or 1.5 μM TAM, and in combination for 1 day. Apoptosis was determined with annexin V/PI/FACS assay. (c) MCF-7/TAMR and MCF-7/HER-2 cells were treated separately and in combination with 20 μM α-TEA and 1 μM TAM for 1 day. Western blot analyses were performed to determine cleaved (c-) forms of caspases-8, 9, and PARP. Data in (a) and (b) are depicted as mean ± SD of three individual experiments. *Significantly different in comparison with TAM and α-TEA alone; P < 0.05. Data in (c) are representative of three individual experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; HER-2, epidermal growth-factor receptor-2 ErbB-2; TAM, tamoxifen; TAMR, tamoxifen resistant; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7.

Combination of α-TEA + TAM acts cooperatively to reduce prosurvival signaling in TAMR cells. (a, b) Western immunoblot analyses using aliquots of cell lysates from treated cells in Figure 3b were performed to assess prosurvival signaling mediators (a) and to assess antiapoptotic factors c-FLIP and Bcl-2 protein expression (b). (c) MCF-7/TAMR cells transfected with siRNAs to Akt-1 or c-FLIP, as well as control siRNA (labeled Control), were treated with α-TEA (20 μM) for 1 day. Western immunoblot analyses were performed to determine PARP, pJNK2/1, CHOP, DR5 (L/S), GRP 78, pAKT, and c-FLIP protein levels, with GAPDH serving as loading control. (a-c) Data are representative of three individual experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; Bcl-2, B-cell lymphoma 2; c-FLIP, cellular FLICE-inhibitory protein; CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein-78; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; pJNK, phosphorylated-c-Jun N-terminal kinase; siRNA, small interfering RNA; TAM, tamoxifen; TAMR, tamoxifen resistant.

Proposed signaling pathways modulated by α-TEA and TAM in TAMR cells. Based on published data and data presented here, a schematic diagram of the actions of α-TEA, MβCD, and TAM on proapoptotic and prosurvival signaling mediators in TAMR cells is depicted. (a) Proapoptotic pathway affected by α-TEA and α-TEA + TAM: α-TEA triggers DR5 extrinsic death receptor-mediated caspase-8/tBid/Bax/mitochondria/caspase-9-dependent apoptosis and caspase-8-mediated endoplasmic reticulum stress-dependent upregulation of JNK/CHOP/DR5 positive-feedback loop. Combination of α-TEA + TAM enhances these apoptotic pathways via suppression of Akt-mediated c-FLIP. (b) Prosurvival pathways affected by α-TEA, MβCD, and TAM. Both α-TEA and MβCD disrupt cholesterol-rich lipid microdomains, leading to suppression of crosstalk between mER-α and RTKs, reduction of total and phosphoprotein levels of RTKs (HER-1 and HER-2), decreased levels of pAkt and pERK1/2, as well as decreased levels of estrogen receptor-α activity via downregulation of nuclear estrogen receptor-α (nER-α) phosphorylation mediated by Akt and ERK (Ser-118 and Ser-167). Both Akt and ERK mediated downstream prosurvival/antiapoptotic mediators, and nER-α promoted proliferation/survival and inhibited apoptosis. In contrast, TAM stimulates survival signaling in TAMR cells. (c) Prosurvival pathways affected by combination of α-TEA or MβCD + TAM: α-TEA or MβCD + TAM cooperatively act to suppress prosurvival signaling, indicating that either α-TEA or MβCD restores TAM sensitivity by converting the TAM prosurvival (agonistic) actions to antisurvival (antagonistic) actions. c-FLIP, cellular FLICE-inhibitory protein; CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; HER-1, epidermal growth factor receptor ErbB-1; HER-2, epidermal growth factor receptor-2 ErbB-2; JNK, c-Jun N-terminal kinase; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; mER, membrane estrogen receptor; MβCD, methyl-b-cyclodextrin; nER, nuclear estrogen receptor; pAkt, phosphorylated-Akt; pERK1/2, phosphorylated-extracellular signal-regulated kinases 1 and 2; RTKs, receptor tyrosine kinases; TAM, tamoxifen; TAMR, tamoxifen resistant; tBid, truncated Bid; α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue.

Methyl-β-cyclodextrin (MβCD), a known cholesterol-rich membrane domain disruptor, reduces levels of prosurvival mediators and, in combination with TAM, induces apoptosis and reduces levels of prosurvival mediators. (a) MCF-7/TAMR cells were treated with MβCD at 2.5 and 5.0 μM for 1 day. Protein levels of prosurvival signaling mediators were determined with Western blot analyses. (b) MCF-7/TAMR and MCF-7/HER-2 cells were treated with TAM at 0.5 or 1 μM with and without MβCD at 1.25 or 2.5 μM for 1 day. Apoptosis was determined with Annexin V/PI. (c) Western blot analyses were performed to determine the prosurvival signaling mediators in both TAMR cell lines treated separately and in combination with 1 μM TAM and 2.5 μM MβCD for 1 day. Data in (a) and (c) are representative of two individual experiments. Data in (b) are depicted as mean ± SD of three individual experiments. *Significantly different in comparison with TAM or MβCD treatment alone; P < 0.05. TAM, tamoxifen; TAMR, tamoxifen resistant; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7.

α-TEA alone and in combination with TAM induces biomarkers of endoplasmic reticulum stress, and siRNA knockdowns show necessary roles for CHOP, DR5, and JNK. (a) Western immunoblot analyses, using aliquots of cell lysates from cells treated with 20 μM α-TEA and 1 μM TAM in Figure 3b, were performed to assess pJNK2/1, CHOP, and DR5 (L/S) protein levels, as well as endoplasmic reticulum stress marker GRP78 protein expression with GAPDH as loading control. (b) MCF-7/TAMR cells transfected with siRNAs to CHOP, DR5, and JNK as well as control siRNA (labeled Control) were treated with TAM (1 μM) + α-TEA (20 μM) for 1 day. Western immunoblot analyses were performed to determine degree of apoptosis, as measured by cleaved PARP (cPARP), pJNK2/1, CHOP, and DR5 (L/S) protein levels, with GAPDH serving as the loading control. Data from (a) and (b) are representative of three individual experiments. CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein-78; JNK, c-Jun N-terminal kinase; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; siRNA, small interfering RNA; TAM, tamoxifen; TAMR, tamoxifen resistant; α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue.

Cholesterol-rich lipid microdomains are involved in α-TEA + TAM circumvention of TAMR. (a) MCF-7/TAMR and MCF-7/HER-2 cells were treated with α-TEA at 20 μM for 15 hours. Identification of cholesterol-rich microdomains was determined by staining the cells with fluorescein-tagged cholesterol marker Filipin and images viewed by using a fluorescence microscope. (b) MCF-7/TAMR cells were pretreated with 10 μM exogenous cholesterol for 2 hours, followed by treatment with 20 μM α-TEA or 20 μM α-TEA plus 1 μM TAM for l day. Western blot analyses were performed to determine treatment effects on prosurvival signaling mediators and PARP cleavage. (a, b) Data are representative of three independent experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; TAM, tamoxifen; TAMR, tamoxifen resistant.