Limits...
Dietary compound isoliquiritigenin prevents mammary carcinogenesis by inhibiting breast cancer stem cells through WIF1 demethylation.

Wang N, Wang Z, Wang Y, Xie X, Shen J, Peng C, You J, Peng F, Tang H, Guan X, Chen J - Oncotarget (2015)

Bottom Line: In addition, WIF1 inhibition significantly relieved the CSC-limiting effects of ISL and methylation analysis further revealed that ISL enhanced WIF1 gene expression via promoting the demethylation of its promoter, which was closely correlated with the inhibition of DNMT1 methyltransferase.Molecular docking analysis finally revealed that ISL could stably dock into the catalytic domain of DNMT1.Taken together, our findings not only provide preclinical evidence to demonstrate the use of ISL as a dietary supplement to inhibit mammary carcinogenesis but also shed novel light on WIF1 as an epigenetic target for breast cancer prevention.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong.

ABSTRACT
Breast cancer stem cells (CSCs) are considered as the root of mammary tumorigenesis. Previous studies have demonstrated that ISL efficiently limited the activities of breast CSCs. However, the cancer prevention activities of ISL and its precise molecular mechanisms remain largely unknown. Here, we report a novel function of ISL as a natural demethylation agent targeting WIF1 to prevent breast cancer. ISL administration suppressed in vivo breast cancer initiation and progression, accompanied by reduced CSC-like populations. A global gene expression profile assay further identified WIF1 as the main response gene of ISL treatment, accompanied by the simultaneous downregulation of β-catenin signaling and G0/G1 phase arrest in breast CSCs. In addition, WIF1 inhibition significantly relieved the CSC-limiting effects of ISL and methylation analysis further revealed that ISL enhanced WIF1 gene expression via promoting the demethylation of its promoter, which was closely correlated with the inhibition of DNMT1 methyltransferase. Molecular docking analysis finally revealed that ISL could stably dock into the catalytic domain of DNMT1. Taken together, our findings not only provide preclinical evidence to demonstrate the use of ISL as a dietary supplement to inhibit mammary carcinogenesis but also shed novel light on WIF1 as an epigenetic target for breast cancer prevention.

No MeSH data available.


Related in: MedlinePlus

ISL inhibited breast CSCs in a WIF1-dependent mannerA. MDA-MB-231 cells treated with ISL at varying concentrations or time intervals were assayed by western blotting for β-catenin (cytoplasmic and nuclear) and ALDH1A3 antigen. β-actin and Lamin B were used as cytoplasmic and nuclear protein loading controls, respectively. The results indicated that ISL administration inhibits ALDH1A3 and β-catenin expression in a dose-and time-dependent manner; B. The relative β-catenin mRNA levels and the transcriptional activities of its downstream genes before and after ISL treatment in MDA-MB-231 were determined by real-time PCR analysis. (*P < 0.05, **P < 0.01, ***P < 0.001, values represented as the mean ± SD, n = 3); C. Breast CSCs of MDA-MB-231 were cultured in ISL-treated CM, and 2 or 4 μg/ml WIF1 neutralizing antibody was added to the culture system. Western blotting results revealed that WIF1 inhibition relieved the β-catenin-inhibitory effects of ISL, accompanied by the reactivation of Cyclin D1 and the phosphorylation of GSK-3β and AKT; D. WIF1 inhibition reversed the inhibitory effects of ISL on CSC populations in MDA-MB-231 cells; E. WIF1 inhibition abrogated the inhibitory effects of ISL on the mammosphere formation ability of CSCs. Breast CSCs were cultured in ISL-treated CM, and WIF1 neutralizing antibody was added to the culture system at 2 or 4 μg/ml. The number and size of the primary and secondary mammospheres were determined using fluorescence microscopy after 7 days (*P < 0.05, **P < 0.01 vs. negative control of the 1st passage spheres, *P < 0.05, **P < 0.01 vs. negative control of the 2nd passage spheres, values represented as the mean ± SD, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496402&req=5

Figure 7: ISL inhibited breast CSCs in a WIF1-dependent mannerA. MDA-MB-231 cells treated with ISL at varying concentrations or time intervals were assayed by western blotting for β-catenin (cytoplasmic and nuclear) and ALDH1A3 antigen. β-actin and Lamin B were used as cytoplasmic and nuclear protein loading controls, respectively. The results indicated that ISL administration inhibits ALDH1A3 and β-catenin expression in a dose-and time-dependent manner; B. The relative β-catenin mRNA levels and the transcriptional activities of its downstream genes before and after ISL treatment in MDA-MB-231 were determined by real-time PCR analysis. (*P < 0.05, **P < 0.01, ***P < 0.001, values represented as the mean ± SD, n = 3); C. Breast CSCs of MDA-MB-231 were cultured in ISL-treated CM, and 2 or 4 μg/ml WIF1 neutralizing antibody was added to the culture system. Western blotting results revealed that WIF1 inhibition relieved the β-catenin-inhibitory effects of ISL, accompanied by the reactivation of Cyclin D1 and the phosphorylation of GSK-3β and AKT; D. WIF1 inhibition reversed the inhibitory effects of ISL on CSC populations in MDA-MB-231 cells; E. WIF1 inhibition abrogated the inhibitory effects of ISL on the mammosphere formation ability of CSCs. Breast CSCs were cultured in ISL-treated CM, and WIF1 neutralizing antibody was added to the culture system at 2 or 4 μg/ml. The number and size of the primary and secondary mammospheres were determined using fluorescence microscopy after 7 days (*P < 0.05, **P < 0.01 vs. negative control of the 1st passage spheres, *P < 0.05, **P < 0.01 vs. negative control of the 2nd passage spheres, values represented as the mean ± SD, n = 3).

Mentions: Because Wnt/β-catenin signaling is closely correlated with the self-renewal ability of breast CSCs, we therefore determined the inhibitory effects of ISL on β-catenin expression in MDA-MB-231 cells. As shown in Figure 7A, ISL inhibited both cytosolic and nuclear β-catenin expression in a time- and dose-dependent manner but without great influences on its mRNA expression. Meanwhile, a series of genes downstream of β-catenin, including Cyclin D1, C-Myc, Survivin and Oct-4, were also simultaneously suppressed by ISL, indicating that the down-regulation of β-catenin might occur at the post-translational level (Figure 7B). To elucidate whether the enhanced WIF1 expression is critical for inducing the down-regulated β-catenin expression by ISL, we cultured breast CSCs in ISL-treated conditional medium (CM) of MDA-MB-231 cells, and a WIF1 neutralizing antibody was added to the culture system to see whether WIF1 inhibition would relieve the CSC-limiting effects of ISL. Western blotting results showed that when compared to the negative control, ISL CM significantly inhibited β-catenin expression via phosphorylating β-catenin and dephosphorylating GSK-3β. However, the expression of β-catenin was elevated by 24% and 65% after adding WIF1 neutralizing antibody to the culture system at 2 μg/ml and 4 μg/ml, respectively (Figure 7C). Meanwhile, flow cytometry results also demonstrated that WIF1 inhibition could reverse the down-regulation of breast CSCs induced by ISL in MDA-MB-231 breast cancer cells, but an isotype-matched IgG at 4 μg/ml did not (Figure 7D). On the other hand, mammosphere results showed that ISL CM could significantly limit the number and size of the mammospheres compared to the negative control, while the number and size of the primary mammospheres were increased to 1.3- and 2.4-fold, respectively, by adding anti-WIF1 antibody at 2 μg/ml, and to 2.1- and 3.6-fold, respectively, at 4 μg/ml, but not by an isotype-matched IgG (Figure 7E). These results indicated that ISL inhibited the Wnt/β-catenin signaling in a WIF1-dependent manner.


Dietary compound isoliquiritigenin prevents mammary carcinogenesis by inhibiting breast cancer stem cells through WIF1 demethylation.

Wang N, Wang Z, Wang Y, Xie X, Shen J, Peng C, You J, Peng F, Tang H, Guan X, Chen J - Oncotarget (2015)

ISL inhibited breast CSCs in a WIF1-dependent mannerA. MDA-MB-231 cells treated with ISL at varying concentrations or time intervals were assayed by western blotting for β-catenin (cytoplasmic and nuclear) and ALDH1A3 antigen. β-actin and Lamin B were used as cytoplasmic and nuclear protein loading controls, respectively. The results indicated that ISL administration inhibits ALDH1A3 and β-catenin expression in a dose-and time-dependent manner; B. The relative β-catenin mRNA levels and the transcriptional activities of its downstream genes before and after ISL treatment in MDA-MB-231 were determined by real-time PCR analysis. (*P < 0.05, **P < 0.01, ***P < 0.001, values represented as the mean ± SD, n = 3); C. Breast CSCs of MDA-MB-231 were cultured in ISL-treated CM, and 2 or 4 μg/ml WIF1 neutralizing antibody was added to the culture system. Western blotting results revealed that WIF1 inhibition relieved the β-catenin-inhibitory effects of ISL, accompanied by the reactivation of Cyclin D1 and the phosphorylation of GSK-3β and AKT; D. WIF1 inhibition reversed the inhibitory effects of ISL on CSC populations in MDA-MB-231 cells; E. WIF1 inhibition abrogated the inhibitory effects of ISL on the mammosphere formation ability of CSCs. Breast CSCs were cultured in ISL-treated CM, and WIF1 neutralizing antibody was added to the culture system at 2 or 4 μg/ml. The number and size of the primary and secondary mammospheres were determined using fluorescence microscopy after 7 days (*P < 0.05, **P < 0.01 vs. negative control of the 1st passage spheres, *P < 0.05, **P < 0.01 vs. negative control of the 2nd passage spheres, values represented as the mean ± SD, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496402&req=5

Figure 7: ISL inhibited breast CSCs in a WIF1-dependent mannerA. MDA-MB-231 cells treated with ISL at varying concentrations or time intervals were assayed by western blotting for β-catenin (cytoplasmic and nuclear) and ALDH1A3 antigen. β-actin and Lamin B were used as cytoplasmic and nuclear protein loading controls, respectively. The results indicated that ISL administration inhibits ALDH1A3 and β-catenin expression in a dose-and time-dependent manner; B. The relative β-catenin mRNA levels and the transcriptional activities of its downstream genes before and after ISL treatment in MDA-MB-231 were determined by real-time PCR analysis. (*P < 0.05, **P < 0.01, ***P < 0.001, values represented as the mean ± SD, n = 3); C. Breast CSCs of MDA-MB-231 were cultured in ISL-treated CM, and 2 or 4 μg/ml WIF1 neutralizing antibody was added to the culture system. Western blotting results revealed that WIF1 inhibition relieved the β-catenin-inhibitory effects of ISL, accompanied by the reactivation of Cyclin D1 and the phosphorylation of GSK-3β and AKT; D. WIF1 inhibition reversed the inhibitory effects of ISL on CSC populations in MDA-MB-231 cells; E. WIF1 inhibition abrogated the inhibitory effects of ISL on the mammosphere formation ability of CSCs. Breast CSCs were cultured in ISL-treated CM, and WIF1 neutralizing antibody was added to the culture system at 2 or 4 μg/ml. The number and size of the primary and secondary mammospheres were determined using fluorescence microscopy after 7 days (*P < 0.05, **P < 0.01 vs. negative control of the 1st passage spheres, *P < 0.05, **P < 0.01 vs. negative control of the 2nd passage spheres, values represented as the mean ± SD, n = 3).
Mentions: Because Wnt/β-catenin signaling is closely correlated with the self-renewal ability of breast CSCs, we therefore determined the inhibitory effects of ISL on β-catenin expression in MDA-MB-231 cells. As shown in Figure 7A, ISL inhibited both cytosolic and nuclear β-catenin expression in a time- and dose-dependent manner but without great influences on its mRNA expression. Meanwhile, a series of genes downstream of β-catenin, including Cyclin D1, C-Myc, Survivin and Oct-4, were also simultaneously suppressed by ISL, indicating that the down-regulation of β-catenin might occur at the post-translational level (Figure 7B). To elucidate whether the enhanced WIF1 expression is critical for inducing the down-regulated β-catenin expression by ISL, we cultured breast CSCs in ISL-treated conditional medium (CM) of MDA-MB-231 cells, and a WIF1 neutralizing antibody was added to the culture system to see whether WIF1 inhibition would relieve the CSC-limiting effects of ISL. Western blotting results showed that when compared to the negative control, ISL CM significantly inhibited β-catenin expression via phosphorylating β-catenin and dephosphorylating GSK-3β. However, the expression of β-catenin was elevated by 24% and 65% after adding WIF1 neutralizing antibody to the culture system at 2 μg/ml and 4 μg/ml, respectively (Figure 7C). Meanwhile, flow cytometry results also demonstrated that WIF1 inhibition could reverse the down-regulation of breast CSCs induced by ISL in MDA-MB-231 breast cancer cells, but an isotype-matched IgG at 4 μg/ml did not (Figure 7D). On the other hand, mammosphere results showed that ISL CM could significantly limit the number and size of the mammospheres compared to the negative control, while the number and size of the primary mammospheres were increased to 1.3- and 2.4-fold, respectively, by adding anti-WIF1 antibody at 2 μg/ml, and to 2.1- and 3.6-fold, respectively, at 4 μg/ml, but not by an isotype-matched IgG (Figure 7E). These results indicated that ISL inhibited the Wnt/β-catenin signaling in a WIF1-dependent manner.

Bottom Line: In addition, WIF1 inhibition significantly relieved the CSC-limiting effects of ISL and methylation analysis further revealed that ISL enhanced WIF1 gene expression via promoting the demethylation of its promoter, which was closely correlated with the inhibition of DNMT1 methyltransferase.Molecular docking analysis finally revealed that ISL could stably dock into the catalytic domain of DNMT1.Taken together, our findings not only provide preclinical evidence to demonstrate the use of ISL as a dietary supplement to inhibit mammary carcinogenesis but also shed novel light on WIF1 as an epigenetic target for breast cancer prevention.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong.

ABSTRACT
Breast cancer stem cells (CSCs) are considered as the root of mammary tumorigenesis. Previous studies have demonstrated that ISL efficiently limited the activities of breast CSCs. However, the cancer prevention activities of ISL and its precise molecular mechanisms remain largely unknown. Here, we report a novel function of ISL as a natural demethylation agent targeting WIF1 to prevent breast cancer. ISL administration suppressed in vivo breast cancer initiation and progression, accompanied by reduced CSC-like populations. A global gene expression profile assay further identified WIF1 as the main response gene of ISL treatment, accompanied by the simultaneous downregulation of β-catenin signaling and G0/G1 phase arrest in breast CSCs. In addition, WIF1 inhibition significantly relieved the CSC-limiting effects of ISL and methylation analysis further revealed that ISL enhanced WIF1 gene expression via promoting the demethylation of its promoter, which was closely correlated with the inhibition of DNMT1 methyltransferase. Molecular docking analysis finally revealed that ISL could stably dock into the catalytic domain of DNMT1. Taken together, our findings not only provide preclinical evidence to demonstrate the use of ISL as a dietary supplement to inhibit mammary carcinogenesis but also shed novel light on WIF1 as an epigenetic target for breast cancer prevention.

No MeSH data available.


Related in: MedlinePlus