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. 2014 Oct:144 Pt B:500-12.
doi: 10.1016/j.jsbmb.2014.09.015. Epub 2014 Sep 19.

Differential regulation of estrogen receptors α and β by 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol}, a novel resveratrol analog

Amruta Ronghe  1 , Anwesha Chatterjee  1 , Bhupendra Singh  2 , Prasad Dandawate  3 , Leigh Murphy  4 , Nimee K Bhat  1 , Subhash Padhye  3 , Hari K Bhat  5
Affiliations

Differential regulation of estrogen receptors α and β by 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol}, a novel resveratrol analog

Amruta Ronghe et al. J Steroid Biochem Mol Biol. 2014 Oct.

Abstract

Breast cancer is the second leading cause of death among women in the United States. Estrogens have been implicated as major risk factors in the development of breast neoplasms. Recent epidemiologic studies have suggested a protective role of phytoestrogens in prevention of breast and other cancers. Resveratrol, a naturally occurring phytoestrogen found notably in red grapes, berries and peanuts, has been shown to possess potent anti-cancer properties. However, the poor efficacy of resveratrol has prevented its use in a clinical setting. In order to improve the efficacy of resveratrol, we have synthesized a small combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the growth of breast cancer cell lines. We have recently shown that one of the synthesized analogs, 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol} (HPIMBD), has better anti-cancer properties than resveratrol. The objective of this study was to investigate the differential regulation of estrogen receptors (ERs) α and β as a potential mechanism of inhibition of breast cancer by HPIMBD VSports手机版. Estrogen receptors α and β have been shown to have opposing roles in cellular proliferation. Estrogen receptor α mediates the proliferative responses of estrogens while ERβ plays an anti-proliferative and pro-apoptotic role. We demonstrate that HPIMBD significantly induces the expression of ERβ and inhibits the expression of ERα. HPIMBD also inhibits the protein expression levels of oncogene c-Myc and cell cycle protein cyclin D1, genes downstream to ERα and important regulators of cell cycle, and cellular proliferation. HPIMBD significantly induces protein expression levels of tumor suppressors p53 and p21 in MCF-7 cells. Additionally, HPIMBD inhibits c-Myc in an ERβ-dependent fashion in MCF-10A and ERβ1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this novel compound. Molecular docking studies confirm higher affinity for binding of HPIMBD in the ERβ cavity. Thus, HPIMBD, a novel azaresveratrol analog may inhibit the proliferation of breast cancer cells by differentially modulating the expressions of ERs α and β. .

Keywords: Breast cancer; Estrogen receptors; Resveratrol; Resveratrol analogs V体育安卓版. .

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Fig. 1
Fig. 1. HPIMBD significantly inhibits the proliferation of breast cancer cell lines and shows a dose- and time-dependent cytotoxicity
a) Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in the HPIMBD- or Res-treated cells by that in vehicle-treated cells X 100. Each experiment was performed in quadruplicate and data are expressed as percentage proliferation + SEM relative to respective vehicle-treated controls. b) LDH release assays were performed on non-neoplastic breast epithelial and breast cancer cell lines as described above. Percentage increase in LDH release was determined by dividing the difference of absorbances between HPIMBD- or Res-treated and vehicle-treated cells to the difference of absorbances between total intracellular LDH and vehicle-treated cells X 100. Each experiment was performed in triplicate and data are expressed as percentage LDH release + SEM relative to respective vehicle-treated controls (taken as 0%). c–h) Non-tumorigenic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of HPIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Non-neoplastic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 48 hours and BrdU incorporation assays were performed. Each experiment was performed in triplicate and the data are expressed as percentage BrdU incorporation + SEM. (*) indicates a P value <0.05 compared to controls.
Fig. 1
Fig. 1. HPIMBD significantly inhibits the proliferation of breast cancer cell lines and shows a dose- and time-dependent cytotoxicity
a) Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in the HPIMBD- or Res-treated cells by that in vehicle-treated cells X 100. Each experiment was performed in quadruplicate and data are expressed as percentage proliferation + SEM relative to respective vehicle-treated controls. b) LDH release assays were performed on non-neoplastic breast epithelial and breast cancer cell lines as described above. Percentage increase in LDH release was determined by dividing the difference of absorbances between HPIMBD- or Res-treated and vehicle-treated cells to the difference of absorbances between total intracellular LDH and vehicle-treated cells X 100. Each experiment was performed in triplicate and data are expressed as percentage LDH release + SEM relative to respective vehicle-treated controls (taken as 0%). c–h) Non-tumorigenic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of HPIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Non-neoplastic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 48 hours and BrdU incorporation assays were performed. Each experiment was performed in triplicate and the data are expressed as percentage BrdU incorporation + SEM. (*) indicates a P value <0.05 compared to controls.
Fig. 1
Fig. 1. HPIMBD significantly inhibits the proliferation of breast cancer cell lines and shows a dose- and time-dependent cytotoxicity
a) Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in the HPIMBD- or Res-treated cells by that in vehicle-treated cells X 100. Each experiment was performed in quadruplicate and data are expressed as percentage proliferation + SEM relative to respective vehicle-treated controls. b) LDH release assays were performed on non-neoplastic breast epithelial and breast cancer cell lines as described above. Percentage increase in LDH release was determined by dividing the difference of absorbances between HPIMBD- or Res-treated and vehicle-treated cells to the difference of absorbances between total intracellular LDH and vehicle-treated cells X 100. Each experiment was performed in triplicate and data are expressed as percentage LDH release + SEM relative to respective vehicle-treated controls (taken as 0%). c–h) Non-tumorigenic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of HPIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Non-neoplastic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 48 hours and BrdU incorporation assays were performed. Each experiment was performed in triplicate and the data are expressed as percentage BrdU incorporation + SEM. (*) indicates a P value <0.05 compared to controls.
Fig. 1
Fig. 1. HPIMBD significantly inhibits the proliferation of breast cancer cell lines and shows a dose- and time-dependent cytotoxicity
a) Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in the HPIMBD- or Res-treated cells by that in vehicle-treated cells X 100. Each experiment was performed in quadruplicate and data are expressed as percentage proliferation + SEM relative to respective vehicle-treated controls. b) LDH release assays were performed on non-neoplastic breast epithelial and breast cancer cell lines as described above. Percentage increase in LDH release was determined by dividing the difference of absorbances between HPIMBD- or Res-treated and vehicle-treated cells to the difference of absorbances between total intracellular LDH and vehicle-treated cells X 100. Each experiment was performed in triplicate and data are expressed as percentage LDH release + SEM relative to respective vehicle-treated controls (taken as 0%). c–h) Non-tumorigenic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of HPIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Non-neoplastic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 48 hours and BrdU incorporation assays were performed. Each experiment was performed in triplicate and the data are expressed as percentage BrdU incorporation + SEM. (*) indicates a P value <0.05 compared to controls.
Fig. 1
Fig. 1. HPIMBD significantly inhibits the proliferation of breast cancer cell lines and shows a dose- and time-dependent cytotoxicity
a) Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in the HPIMBD- or Res-treated cells by that in vehicle-treated cells X 100. Each experiment was performed in quadruplicate and data are expressed as percentage proliferation + SEM relative to respective vehicle-treated controls. b) LDH release assays were performed on non-neoplastic breast epithelial and breast cancer cell lines as described above. Percentage increase in LDH release was determined by dividing the difference of absorbances between HPIMBD- or Res-treated and vehicle-treated cells to the difference of absorbances between total intracellular LDH and vehicle-treated cells X 100. Each experiment was performed in triplicate and data are expressed as percentage LDH release + SEM relative to respective vehicle-treated controls (taken as 0%). c–h) Non-tumorigenic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of HPIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Non-neoplastic breast epithelial cell line and breast cancer cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 48 hours and BrdU incorporation assays were performed. Each experiment was performed in triplicate and the data are expressed as percentage BrdU incorporation + SEM. (*) indicates a P value <0.05 compared to controls.
Fig. 2
Fig. 2. HPIMBD induces mRNA and protein expression levels of ERβ
a) Breast cancer cell lines MCF-7, T47D and MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 24 hours. Total RNA was isolated and reverse transcribed to cDNA. Real-time quantitative PCR was performed to analyze the expression of cyclophilin and ERβ mRNA. Expression of ERβ mRNA was determined by dividing the number of cDNA molecules of ERβ by the number of cDNA molecules of cyclophilin, a housekeeping gene. Fold change was determined by dividing the expression of ERβ in the HPIMBD- or Res-treated cells by the expression of ERβ in vehicle-treated cells. Each experiment was performed in quadruplicate and the data are expressed as fold change + SEM relative to control. b) Non-neoplastic breast epithelial MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM doses of Res or HPIMBD for up to 72 hours. Proteins were isolated and western blot analyses were performed as described in the Materials and Methods section. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. Representative western blots are shown for each cell line mentioned above at the time point of maximal induction of ERβ in that cell line (MCF10A = 24 hours; MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 = 12 hours; MCF-7 and T47D = 48 hours). c) MCF-7, T47D and MDA-MB-231 cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD up to 72 hours and a time-course study was performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes compared to vehicle-treated controls were calculated from four individual experiments. The bar graph represents fold change in ERβ protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls. d) MDA-MB-231 cells were treated with vehicle or 25 to 100 μM doses of HPIMBD and a dose-response study was performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ protein expression (Mean + SEM) compared to vehicle-treated control were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 2
Fig. 2. HPIMBD induces mRNA and protein expression levels of ERβ
a) Breast cancer cell lines MCF-7, T47D and MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 24 hours. Total RNA was isolated and reverse transcribed to cDNA. Real-time quantitative PCR was performed to analyze the expression of cyclophilin and ERβ mRNA. Expression of ERβ mRNA was determined by dividing the number of cDNA molecules of ERβ by the number of cDNA molecules of cyclophilin, a housekeeping gene. Fold change was determined by dividing the expression of ERβ in the HPIMBD- or Res-treated cells by the expression of ERβ in vehicle-treated cells. Each experiment was performed in quadruplicate and the data are expressed as fold change + SEM relative to control. b) Non-neoplastic breast epithelial MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM doses of Res or HPIMBD for up to 72 hours. Proteins were isolated and western blot analyses were performed as described in the Materials and Methods section. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. Representative western blots are shown for each cell line mentioned above at the time point of maximal induction of ERβ in that cell line (MCF10A = 24 hours; MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 = 12 hours; MCF-7 and T47D = 48 hours). c) MCF-7, T47D and MDA-MB-231 cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD up to 72 hours and a time-course study was performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes compared to vehicle-treated controls were calculated from four individual experiments. The bar graph represents fold change in ERβ protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls. d) MDA-MB-231 cells were treated with vehicle or 25 to 100 μM doses of HPIMBD and a dose-response study was performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ protein expression (Mean + SEM) compared to vehicle-treated control were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 2
Fig. 2. HPIMBD induces mRNA and protein expression levels of ERβ
a) Breast cancer cell lines MCF-7, T47D and MDA-MB-231 were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 24 hours. Total RNA was isolated and reverse transcribed to cDNA. Real-time quantitative PCR was performed to analyze the expression of cyclophilin and ERβ mRNA. Expression of ERβ mRNA was determined by dividing the number of cDNA molecules of ERβ by the number of cDNA molecules of cyclophilin, a housekeeping gene. Fold change was determined by dividing the expression of ERβ in the HPIMBD- or Res-treated cells by the expression of ERβ in vehicle-treated cells. Each experiment was performed in quadruplicate and the data are expressed as fold change + SEM relative to control. b) Non-neoplastic breast epithelial MCF-10A and breast cancer cell lines MCF-7, T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 were treated with vehicle (DMSO), 50 μM doses of Res or HPIMBD for up to 72 hours. Proteins were isolated and western blot analyses were performed as described in the Materials and Methods section. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. Representative western blots are shown for each cell line mentioned above at the time point of maximal induction of ERβ in that cell line (MCF10A = 24 hours; MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 = 12 hours; MCF-7 and T47D = 48 hours). c) MCF-7, T47D and MDA-MB-231 cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD up to 72 hours and a time-course study was performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes compared to vehicle-treated controls were calculated from four individual experiments. The bar graph represents fold change in ERβ protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls. d) MDA-MB-231 cells were treated with vehicle or 25 to 100 μM doses of HPIMBD and a dose-response study was performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ protein expression (Mean + SEM) compared to vehicle-treated control were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 3
Fig. 3. HPIMBD inhibits mRNA and protein expression levels of ERα
a) Breast cancer cell lines MCF-7 and T47D were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 24 hours. Total RNA was isolated and reverse transcribed to cDNA. Real-time quantitative PCR was performed to analyze the expression of cyclophilin and ERα mRNA. Expression of ERα mRNA was determined as described in the Materials and Methods section. Each experiment was performed in quadruplicate and the data are expressed as fold change + SEM relative to control. b) Breast cancer cell lines MCF-7 and T47D were treated with vehicle (DMSO), 50 μM Res or HPIMBD for up to 72 hours. Proteins were isolated and western blot analyses were performed. Fold changes in ERα (Mean + SEM) compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. Representative western blots are shown for MCF-7 and T47D cell lines after 48 hours of treatments, which is the time point of maximal inhibition of ERα in both the cell lines post-HPIMBD treatment. c) MCF-7 and T47D cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD for up to 72 hours and a time-course study was performed. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes compared to vehicle-treated controls were calculated from four individual experiments. The bar graph represents fold change in ERα protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls. d) T47D cells were treated with vehicle or 25 to 100 μM doses of HPIMBD for 48 hours and a dose-response study was performed. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERα protein expression (Mean + SEM) compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 3
Fig. 3. HPIMBD inhibits mRNA and protein expression levels of ERα
a) Breast cancer cell lines MCF-7 and T47D were treated with vehicle (DMSO), 50 μM Res or HPIMBD for 24 hours. Total RNA was isolated and reverse transcribed to cDNA. Real-time quantitative PCR was performed to analyze the expression of cyclophilin and ERα mRNA. Expression of ERα mRNA was determined as described in the Materials and Methods section. Each experiment was performed in quadruplicate and the data are expressed as fold change + SEM relative to control. b) Breast cancer cell lines MCF-7 and T47D were treated with vehicle (DMSO), 50 μM Res or HPIMBD for up to 72 hours. Proteins were isolated and western blot analyses were performed. Fold changes in ERα (Mean + SEM) compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. Representative western blots are shown for MCF-7 and T47D cell lines after 48 hours of treatments, which is the time point of maximal inhibition of ERα in both the cell lines post-HPIMBD treatment. c) MCF-7 and T47D cell lines were treated with vehicle (DMSO), 50 μM Res or HPIMBD for up to 72 hours and a time-course study was performed. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes compared to vehicle-treated controls were calculated from four individual experiments. The bar graph represents fold change in ERα protein expression (Mean + SEM) in respective cell lines treated with Res or HPIMBD compared to vehicle-treated controls. d) T47D cells were treated with vehicle or 25 to 100 μM doses of HPIMBD for 48 hours and a dose-response study was performed. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERα protein expression (Mean + SEM) compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 4
Fig. 4. HPIMBD inhibits protein expression levels of c-Myc and cyclin D1, induces p53 and p21 in MCF-7 cells
a) Breast cancer cell line MCF-7 was treated with vehicle, 50 μM Res or HPIMBD for 48 hours. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in c-Myc or cyclin D1 protein expression (Mean + SEM) treated with Res or HPIMBD compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. b) Breast cancer cell line MCF-7 was treated with vehicle, 50 μM Res or HPIMBD for 12 hours. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in p53 or p21 protein expression (Mean + SEM) treated with Res or HPIMBD compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 5
Fig. 5. HPIMBD inhibits the expression of oncogene c-Myc in breast cancer cell lines
a) Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 cells were treated with vehicle (DMSO), 50 μM Res or HPIMBD. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in c-Myc protein expression (Mean + SEM) treated with Res or HPIMBD compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. b) MCF10A, ERβ1-transfected MDA-MB-231, MCF-7 and T47D cells were transfected with either 1 nmol/l of scrambled small interfering RNA or siERβ for 48 hours, and subsequently treated with 50 μM HPIMBD for 12 hours (ERβ1-transfected MDA-MB-231), 24 hours (MCF-10A) or 48 hours (MCF-7 and T47D). The treatment time points are based on maximal induction time of ERβ for respective cell lines following HPIMBD treatment. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ or c-Myc protein expression (Mean + SEM) treated with scrambled, siERβ or HPIMBD compared to vehicle-controls were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 5
Fig. 5. HPIMBD inhibits the expression of oncogene c-Myc in breast cancer cell lines
a) Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines T47D, MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 cells were treated with vehicle (DMSO), 50 μM Res or HPIMBD. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in c-Myc protein expression (Mean + SEM) treated with Res or HPIMBD compared to vehicle-treated controls were calculated from four individual experiments and are given at the top of each blot. b) MCF10A, ERβ1-transfected MDA-MB-231, MCF-7 and T47D cells were transfected with either 1 nmol/l of scrambled small interfering RNA or siERβ for 48 hours, and subsequently treated with 50 μM HPIMBD for 12 hours (ERβ1-transfected MDA-MB-231), 24 hours (MCF-10A) or 48 hours (MCF-7 and T47D). The treatment time points are based on maximal induction time of ERβ for respective cell lines following HPIMBD treatment. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in ERβ or c-Myc protein expression (Mean + SEM) treated with scrambled, siERβ or HPIMBD compared to vehicle-controls were calculated from four individual experiments and are given at the top of each blot. (*) indicates a P value <0.05 compared to controls.
Fig. 6
Fig. 6. Binding of Res and its analog HPIMBD into the active site of ERβ, as assessed by computer modeling studies
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