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. 2016 Jun 15:301:1-13.
doi: 10.1016/j.taap.2016.03.003. Epub 2016 Mar 9.

4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, 1 a novel resveratrol analog, differentially regulates estrogen receptors α and β in breast cancer cells

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

"VSports注册入口" 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, 1 a novel resveratrol analog, differentially regulates estrogen receptors α and β in breast cancer cells

Amruta Ronghe (V体育安卓版) et al. Toxicol Appl Pharmacol. .

Abstract

Breast cancer is a public health concern worldwide. Prolonged exposure to estrogens has been implicated in the development of breast neoplasms. Epidemiologic and experimental evidence suggest a chemopreventive role of phytoestrogens in breast cancers. Resveratrol, a naturally occurring phytoestrogen, has been shown to have potent anti-cancer properties. However, poor efficacy and bioavailability have prevented the use of resveratrol in clinics. In order to address these problems, we have synthesized a combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the proliferation of breast cancer cells. We have recently shown that 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), has better anti-cancer properties than resveratrol and any other resveratrol analog we have synthesized so far. The objective of this study was to investigate the regulation of estrogen receptors (ERs) α and β by TIMBD in breast cancer cell lines. We demonstrate that TIMBD significantly induces the mRNA and protein expression levels of ERβ and inhibits that of ERα. TIMBD inhibits mRNA and protein expression levels of oncogene c-Myc, and cell cycle protein cyclin D1, which are important regulators of cellular proliferation VSports手机版. TIMBD significantly induces protein expression levels of tumor suppressor genes p53 and p21 in MCF-7 cells. TIMBD 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 analog. ERβ plays a partial role in inhibition of proliferation by TIMBD while ERα overexpression does not significantly affect TIMBD's inhibition. .

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

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Conflict of interest statement

Conflict of Interest Statement:

All authors declare that they have no conflicts of interest with the contents in this article with respect to third party financial support, financial relationships, sources of revenue, sponsor interactions, patents or other affiliations.

Figures

Fig. 1
Fig. 1. TIMBD significantly inhibits 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) or 50 μM Res or TIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in Res- or TIMBD-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 breast epithelial and breast cancer cell lines as described in the Materials and Methods section. Percentage increase in LDH release was determined by dividing the difference of absorbances between Res-, TIMBD- 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) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of TIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 respective controls.
Fig. 1
Fig. 1. TIMBD significantly inhibits 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) or 50 μM Res or TIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in Res- or TIMBD-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 breast epithelial and breast cancer cell lines as described in the Materials and Methods section. Percentage increase in LDH release was determined by dividing the difference of absorbances between Res-, TIMBD- 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) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of TIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 respective controls.
Fig. 1
Fig. 1. TIMBD significantly inhibits 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) or 50 μM Res or TIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in Res- or TIMBD-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 breast epithelial and breast cancer cell lines as described in the Materials and Methods section. Percentage increase in LDH release was determined by dividing the difference of absorbances between Res-, TIMBD- 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) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of TIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 respective controls.
Fig. 1
Fig. 1. TIMBD significantly inhibits 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) or 50 μM Res or TIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in Res- or TIMBD-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 breast epithelial and breast cancer cell lines as described in the Materials and Methods section. Percentage increase in LDH release was determined by dividing the difference of absorbances between Res-, TIMBD- 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) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of TIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 respective controls.
Fig. 1
Fig. 1. TIMBD significantly inhibits 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) or 50 μM Res or TIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in Res- or TIMBD-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 breast epithelial and breast cancer cell lines as described in the Materials and Methods section. Percentage increase in LDH release was determined by dividing the difference of absorbances between Res-, TIMBD- 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) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of TIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 respective controls.
Fig. 1
Fig. 1. TIMBD significantly inhibits 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) or 50 μM Res or TIMBD for 72 hours and MTT assays were performed. Percentage proliferation was determined by dividing the absorbance in Res- or TIMBD-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 breast epithelial and breast cancer cell lines as described in the Materials and Methods section. Percentage increase in LDH release was determined by dividing the difference of absorbances between Res-, TIMBD- 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) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or graded doses of TIMBD for up to 72 hours and LDH release assays were performed. Percentage increase in LDH release was determined as described above. i) Breast epithelial and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 respective controls.
Fig. 2
Fig. 2. TIMBD induces mRNA and protein expression levels of ERβ
a) MCF-7, T47D and MDA-MB-231 cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 Res- or TIMBD-treated cells by the expression of ERβ in vehicle-treated cells (taken as 1). Each experiment was performed in quadruplicate and the data are expressed as fold change ± SEM relative to control. b) MCF-10A and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM doses of Res or TIMBD 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 or transgene tagged ERβ1 protein expression (Mean ± SEM) in ERβ1-transfected MDA-MB-231 cells treated with Res or TIMBD compared to vehicle-treated controls of the same cell type were calculated from four individual experiments and the mean values 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 (MCF-10A = 24 hours; MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 = 12 hours; MCF-7 and T47D = 48 hours). The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. c) MCF-7, T47D and MDA-MB-231 cell lines were treated with vehicle (DMSO) or 50 μM TIMBD 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 TIMBD compared to vehicle-treated controls. d) MDA-MB-231 cells were treated with vehicle or 25 to 100 μM doses of TIMBD for 12 hours 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 the mean values are given at the top of each blot. (*) indicates a P value <0.05 compared to respective controls.
Fig. 2
Fig. 2. TIMBD induces mRNA and protein expression levels of ERβ
a) MCF-7, T47D and MDA-MB-231 cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 Res- or TIMBD-treated cells by the expression of ERβ in vehicle-treated cells (taken as 1). Each experiment was performed in quadruplicate and the data are expressed as fold change ± SEM relative to control. b) MCF-10A and breast cancer cell lines were treated with vehicle (DMSO) or 50 μM doses of Res or TIMBD 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 or transgene tagged ERβ1 protein expression (Mean ± SEM) in ERβ1-transfected MDA-MB-231 cells treated with Res or TIMBD compared to vehicle-treated controls of the same cell type were calculated from four individual experiments and the mean values 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 (MCF-10A = 24 hours; MDA-MB-231, vector-transfected and ERβ1-transfected MDA-MB-231 = 12 hours; MCF-7 and T47D = 48 hours). The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. c) MCF-7, T47D and MDA-MB-231 cell lines were treated with vehicle (DMSO) or 50 μM TIMBD 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 TIMBD compared to vehicle-treated controls. d) MDA-MB-231 cells were treated with vehicle or 25 to 100 μM doses of TIMBD for 12 hours 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 the mean values are given at the top of each blot. (*) indicates a P value <0.05 compared to respective controls.
Fig. 3
Fig. 3. TIMBD inhibits mRNA and protein expression levels of ERα
a) MCF-7 and T47D cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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) MCF-7 and T47D cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 the mean values 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 the breast cancer cell lines post-TIMBD treatment. The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. c) MCF-7 and T47D cell lines were treated with vehicle (DMSO) or 50 μM TIMBD for up to 72 hours and a time-course study was performed. Proteins were isolated and western blot analyses were performed. Intensities of 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 TIMBD compared to vehicle-treated controls. d) T47D cells were treated with vehicle or 25 to 100 μM doses of TIMBD 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 the mean values are given at the top of each blot. (*) indicates a P value <0.05 compared to respective controls.
Fig. 3
Fig. 3. TIMBD inhibits mRNA and protein expression levels of ERα
a) MCF-7 and T47D cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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) MCF-7 and T47D cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 the mean values 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 the breast cancer cell lines post-TIMBD treatment. The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. c) MCF-7 and T47D cell lines were treated with vehicle (DMSO) or 50 μM TIMBD for up to 72 hours and a time-course study was performed. Proteins were isolated and western blot analyses were performed. Intensities of 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 TIMBD compared to vehicle-treated controls. d) T47D cells were treated with vehicle or 25 to 100 μM doses of TIMBD 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 the mean values are given at the top of each blot. (*) indicates a P value <0.05 compared to respective controls.
Fig. 4
Fig. 4. TIMBD inhibits mRNA and protein expression levels of c-Myc and cyclin D1 in breast cancer cells
a) MCF-7, T47D and MDA-MB-231 cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 c-Myc mRNA. mRNA expression levels of c-Myc and cyclophilin were 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) MCF-10A and breast cancer cell lines were treated with either vehicle (DMSO) or 50 μM Res or TIMBD for up to 72 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 protein expression (Mean ± SEM) treated with TIMBD compared to vehicle-treated controls were calculated from four individual experiments and the mean values are given at the top of each blot. The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. c) MCF-7, T47D and MDA-MB-231 cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 cyclin D1 mRNA. Expression of cyclin D1 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. d) MCF-10A and breast cancer cell lines were treated with either vehicle (DMSO) or 50 μM Res or TIMBD for up to 72 hours. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in cyclin D1 protein expression (Mean ± SEM) treated with TIMBD compared to vehicle-treated controls were calculated from four individual experiments and the mean values are given at the top of each blot. The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. (*) indicates a P value <0.05 compared to respective controls.
Fig. 4
Fig. 4. TIMBD inhibits mRNA and protein expression levels of c-Myc and cyclin D1 in breast cancer cells
a) MCF-7, T47D and MDA-MB-231 cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 c-Myc mRNA. mRNA expression levels of c-Myc and cyclophilin were 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) MCF-10A and breast cancer cell lines were treated with either vehicle (DMSO) or 50 μM Res or TIMBD for up to 72 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 protein expression (Mean ± SEM) treated with TIMBD compared to vehicle-treated controls were calculated from four individual experiments and the mean values are given at the top of each blot. The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. c) MCF-7, T47D and MDA-MB-231 cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD 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 cyclin D1 mRNA. Expression of cyclin D1 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. d) MCF-10A and breast cancer cell lines were treated with either vehicle (DMSO) or 50 μM Res or TIMBD for up to 72 hours. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in cyclin D1 protein expression (Mean ± SEM) treated with TIMBD compared to vehicle-treated controls were calculated from four individual experiments and the mean values are given at the top of each blot. The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. (*) indicates a P value <0.05 compared to respective controls.
Fig. 5
Fig. 5. TIMBD induces protein expression levels of tumor suppressor genes p53 and p21 in MCF-7 breast cancer cells
a) MCF-7 breast cancer cells were treated with vehicle (DMSO) or 50 μM Res or TIMBD. Proteins were isolated and western blot analyses were performed. Intensities of the bands were quantified and normalized to α-tubulin. Fold changes in p53 and p21 protein expression (Mean ± SEM) treated with TIMBD compared to vehicle-treated controls were calculated from four individual experiments and the mean values are given at the top of each blot. The vertical lines in the boxes indicate that intervening lanes have been removed. Each individual western blot panel compares signals from protein samples loaded on the same gel. b) MCF-7 cells were treated with either vehicle (DMSO) or 50 μM Res or TIMBD up to 24 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. The bar graph represents fold change in p53 or p21 protein expression levels (Mean ± SEM) in respective cell lines treated with Res or TIMBD compared to vehicle-treated controls. (*) indicates a P value <0.05 compared to respective controls.
Fig. 6
Fig. 6. TIMBD inhibits protein expression c-Myc in an ER-dependent fashion in breast cancer cells
a) MCF-10A, 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 TIMBD 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 TIMBD 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 TIMBD compared to vehicle-controls were calculated from four individual experiments and the mean values are given at the top of each blot. b) Binding of Resveratrol analog TIMBD into the active site of ERβ, as assessed by computer modeling studies. (*) indicates a P value <0.05 compared to respective controls.
Fig. 6
Fig. 6. TIMBD inhibits protein expression c-Myc in an ER-dependent fashion in breast cancer cells
a) MCF-10A, 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 TIMBD 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 TIMBD 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 TIMBD compared to vehicle-controls were calculated from four individual experiments and the mean values are given at the top of each blot. b) Binding of Resveratrol analog TIMBD into the active site of ERβ, as assessed by computer modeling studies. (*) indicates a P value <0.05 compared to respective controls.
Fig. 7
Fig. 7. TIMBD partially inhibits proliferation of MDA-MB-231-ERβ transfected cell line in an ERβ-dependent fashion
a) MDA-MB-231-ERβ cells were seeded in 6-well plates and were transfected with siRNA for ERβ followed by treatment with TIMBD or vehicle for 12 hours. After treatment, cells were incubated for an additional 8 days in fresh complete medium. The colonies obtained were fixed in methanol and stained with crystal violet solution. Three replicates were performed for each treatment group and representative pictures for each treatment group are shown. b) MDA-MB-231-ERβ transfected cells were seeded into 24-well plates and mammosphere assays were performed. Cells were grown in serum-free medium supplemented with growth factors as mentioned in Materials and Methods section. Cells were treated with TIMBD or vehicle control and the mammospheres formed were viewed under the microscope and photographed [25, 27, 30]. Three replicate wells from a 24-well plate were used for each experimental condition and representative pictures for each treatment group are shown. Western blot for ERβ after siRNA transfection is also presented to demonstrate that ERβ protein expression was decreased following siERβ transfection.
Fig. 8
Fig. 8. TIMBD is equally effective in inhibiting the proliferation of MDA-MB-231 and ERα-transfected MDA-MB-231 cells
a) MDA-MB-231 cells were transiently transfected with ERα and empty-vector plasmids using Lipofectamine 2000 transfection reagent for 48 hours in 6-well plates. Following transfection, cells were re-plated in 96-well plates and treated with 50 μM TIMBD or vehicle control for additional 48 hours. MTT assays were performed as described in Materials and Methods section. Three replicate wells were used for each experimental condition. Western blot for ERα expression in MDA-MB-231 cells following transfection is included to demonstrate that ERα protein expression was increased following ERα transfection. b) MDA-MB-231-ERα or vector-transfected cells were seeded in 6-well plates followed by treatment with TIMBD or vehicle for 48 hours. After treatment, cells were incubated for an additional 8 days in a complete medium. The colonies obtained were fixed in methanol and stained with crystal violet solution. Three replicates were performed for each treatment group and representative pictures are shown. Resulting colonies were counted and the bar graph represents the % colonies formed (Mean ± SEM) relative to respective vehicle-treated controls of the same cell type. (*) indicates a P value <0.05 compared to respective controls.
Fig. 8
Fig. 8. TIMBD is equally effective in inhibiting the proliferation of MDA-MB-231 and ERα-transfected MDA-MB-231 cells
a) MDA-MB-231 cells were transiently transfected with ERα and empty-vector plasmids using Lipofectamine 2000 transfection reagent for 48 hours in 6-well plates. Following transfection, cells were re-plated in 96-well plates and treated with 50 μM TIMBD or vehicle control for additional 48 hours. MTT assays were performed as described in Materials and Methods section. Three replicate wells were used for each experimental condition. Western blot for ERα expression in MDA-MB-231 cells following transfection is included to demonstrate that ERα protein expression was increased following ERα transfection. b) MDA-MB-231-ERα or vector-transfected cells were seeded in 6-well plates followed by treatment with TIMBD or vehicle for 48 hours. After treatment, cells were incubated for an additional 8 days in a complete medium. The colonies obtained were fixed in methanol and stained with crystal violet solution. Three replicates were performed for each treatment group and representative pictures are shown. Resulting colonies were counted and the bar graph represents the % colonies formed (Mean ± SEM) relative to respective vehicle-treated controls of the same cell type. (*) indicates a P value <0.05 compared to respective controls.
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