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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 limited the self-renewal ability of breast CSCsA. Representative dot plots of CD44+CD24−/low cell surface markers in MDA-MB-231 and MCF-7 cells using the BD FACSAria SORP cell sorter. Breast cancer cells were incubated with 25 or 50 μM ISL for 24 h. CD44-FITC and CD24-PE antibodies were utilized to frame CSC-like subsets in the lower right quadrant (Q3). The isotype-matched antibody served as a negative control. The data analysis was performed using FlowJo software (*P < 0.05, **P < 0.01, values represented as the mean ± SD, n = 3). The results suggested that ISL administration significantly reduced the CSCs population in both cancer cells in a dose-dependent manner; B. The ALDEFLUOR assay was then used to determine the population of CSCs in MDA-MB-231 and MCF-7 cells after ISL administration. The ALDH-specific inhibitor DEAB was used as a negative control for minimizing background fluorescence. ALDHhi cells were shown as cells residing in the framed area analyzed by FlowJo software; time-dependent manner in both CSCs. C. Effects of ISL on the primary, secondary and tertiary mammospheres formed by the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without ISL (10, 25 or 50 μM) for 7 days. The number and size of the mammospheres were determined using fluorescence microscopy (*P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 1st passage spheres; *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 2nd passage spheres; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001 vs. negative control of the 3rd passage spheres, values represented as the mean ± SD, n = 3); the results showed that ISL administration could significantly limit the number and size of mammospheres formed by both CSCs populations; D. The effects of ISL on cell cycle distributions of the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without 25 μM ISL at 24 h and 48 h and then subjected to cell cycle investigation analyzed by Modifit LT software. The results indicated that ISL arrested cell cycle at G0/G1 phase in a time-dependent manner in both CSCs.
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Figure 5: ISL limited the self-renewal ability of breast CSCsA. Representative dot plots of CD44+CD24−/low cell surface markers in MDA-MB-231 and MCF-7 cells using the BD FACSAria SORP cell sorter. Breast cancer cells were incubated with 25 or 50 μM ISL for 24 h. CD44-FITC and CD24-PE antibodies were utilized to frame CSC-like subsets in the lower right quadrant (Q3). The isotype-matched antibody served as a negative control. The data analysis was performed using FlowJo software (*P < 0.05, **P < 0.01, values represented as the mean ± SD, n = 3). The results suggested that ISL administration significantly reduced the CSCs population in both cancer cells in a dose-dependent manner; B. The ALDEFLUOR assay was then used to determine the population of CSCs in MDA-MB-231 and MCF-7 cells after ISL administration. The ALDH-specific inhibitor DEAB was used as a negative control for minimizing background fluorescence. ALDHhi cells were shown as cells residing in the framed area analyzed by FlowJo software; time-dependent manner in both CSCs. C. Effects of ISL on the primary, secondary and tertiary mammospheres formed by the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without ISL (10, 25 or 50 μM) for 7 days. The number and size of the mammospheres were determined using fluorescence microscopy (*P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 1st passage spheres; *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 2nd passage spheres; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001 vs. negative control of the 3rd passage spheres, values represented as the mean ± SD, n = 3); the results showed that ISL administration could significantly limit the number and size of mammospheres formed by both CSCs populations; D. The effects of ISL on cell cycle distributions of the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without 25 μM ISL at 24 h and 48 h and then subjected to cell cycle investigation analyzed by Modifit LT software. The results indicated that ISL arrested cell cycle at G0/G1 phase in a time-dependent manner in both CSCs.

Mentions: After in vivo examination, the classic “stem-like” phenotype of CD44+CD24−/low was applied to determine the effects of ISL on the MDA-MB-231 triple-negative breast cancer cell line and the MCF-7 luminal cancer cell line. Flow cytometry results showed that ISL could significantly limit the population of breast CSCs in both cell lines (Figure 5A). In particular, 25 μM ISL eliminated the CD44+CD24−/low population of MDA-MB-231 cells by 23%, whereas 50 μM ISL resulted in over a 37% reduction of CSC-like cells. Similar findings were also obtained in MCF-7 cells. Compared to 6.64% of breast CSCs in the untreated MCF-7 cells, the proportion of CD44+CD24−/low cells dropped to 3.52% and 2.92% after treatment with 25 and 50 μM ISL for 24 h, respectively. ALDH was also applied for detection of stem-like cells with or without ISL plus the specific inhibitor DEAB. It was shown that ISL at 50 μM could reduce ALDH factions from 4.97% to 1.41% in MDA-MB-231 and from 1.24% to 0.298% in MCF-7, indicating that ISL could suppress the ALDHhi population in both cell lines (Figure 5B). The results suggested ISL could decrease CSC populations characterized with either ALDHhi or CD44+CD24−/low markers.


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 limited the self-renewal ability of breast CSCsA. Representative dot plots of CD44+CD24−/low cell surface markers in MDA-MB-231 and MCF-7 cells using the BD FACSAria SORP cell sorter. Breast cancer cells were incubated with 25 or 50 μM ISL for 24 h. CD44-FITC and CD24-PE antibodies were utilized to frame CSC-like subsets in the lower right quadrant (Q3). The isotype-matched antibody served as a negative control. The data analysis was performed using FlowJo software (*P < 0.05, **P < 0.01, values represented as the mean ± SD, n = 3). The results suggested that ISL administration significantly reduced the CSCs population in both cancer cells in a dose-dependent manner; B. The ALDEFLUOR assay was then used to determine the population of CSCs in MDA-MB-231 and MCF-7 cells after ISL administration. The ALDH-specific inhibitor DEAB was used as a negative control for minimizing background fluorescence. ALDHhi cells were shown as cells residing in the framed area analyzed by FlowJo software; time-dependent manner in both CSCs. C. Effects of ISL on the primary, secondary and tertiary mammospheres formed by the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without ISL (10, 25 or 50 μM) for 7 days. The number and size of the mammospheres were determined using fluorescence microscopy (*P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 1st passage spheres; *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 2nd passage spheres; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001 vs. negative control of the 3rd passage spheres, values represented as the mean ± SD, n = 3); the results showed that ISL administration could significantly limit the number and size of mammospheres formed by both CSCs populations; D. The effects of ISL on cell cycle distributions of the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without 25 μM ISL at 24 h and 48 h and then subjected to cell cycle investigation analyzed by Modifit LT software. The results indicated that ISL arrested cell cycle at G0/G1 phase in a time-dependent manner in both CSCs.
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Figure 5: ISL limited the self-renewal ability of breast CSCsA. Representative dot plots of CD44+CD24−/low cell surface markers in MDA-MB-231 and MCF-7 cells using the BD FACSAria SORP cell sorter. Breast cancer cells were incubated with 25 or 50 μM ISL for 24 h. CD44-FITC and CD24-PE antibodies were utilized to frame CSC-like subsets in the lower right quadrant (Q3). The isotype-matched antibody served as a negative control. The data analysis was performed using FlowJo software (*P < 0.05, **P < 0.01, values represented as the mean ± SD, n = 3). The results suggested that ISL administration significantly reduced the CSCs population in both cancer cells in a dose-dependent manner; B. The ALDEFLUOR assay was then used to determine the population of CSCs in MDA-MB-231 and MCF-7 cells after ISL administration. The ALDH-specific inhibitor DEAB was used as a negative control for minimizing background fluorescence. ALDHhi cells were shown as cells residing in the framed area analyzed by FlowJo software; time-dependent manner in both CSCs. C. Effects of ISL on the primary, secondary and tertiary mammospheres formed by the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without ISL (10, 25 or 50 μM) for 7 days. The number and size of the mammospheres were determined using fluorescence microscopy (*P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 1st passage spheres; *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control of the 2nd passage spheres; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001 vs. negative control of the 3rd passage spheres, values represented as the mean ± SD, n = 3); the results showed that ISL administration could significantly limit the number and size of mammospheres formed by both CSCs populations; D. The effects of ISL on cell cycle distributions of the sorted CSCs from MDA-MB-231 and MCF-7 cells. The CSCs were incubated with or without 25 μM ISL at 24 h and 48 h and then subjected to cell cycle investigation analyzed by Modifit LT software. The results indicated that ISL arrested cell cycle at G0/G1 phase in a time-dependent manner in both CSCs.
Mentions: After in vivo examination, the classic “stem-like” phenotype of CD44+CD24−/low was applied to determine the effects of ISL on the MDA-MB-231 triple-negative breast cancer cell line and the MCF-7 luminal cancer cell line. Flow cytometry results showed that ISL could significantly limit the population of breast CSCs in both cell lines (Figure 5A). In particular, 25 μM ISL eliminated the CD44+CD24−/low population of MDA-MB-231 cells by 23%, whereas 50 μM ISL resulted in over a 37% reduction of CSC-like cells. Similar findings were also obtained in MCF-7 cells. Compared to 6.64% of breast CSCs in the untreated MCF-7 cells, the proportion of CD44+CD24−/low cells dropped to 3.52% and 2.92% after treatment with 25 and 50 μM ISL for 24 h, respectively. ALDH was also applied for detection of stem-like cells with or without ISL plus the specific inhibitor DEAB. It was shown that ISL at 50 μM could reduce ALDH factions from 4.97% to 1.41% in MDA-MB-231 and from 1.24% to 0.298% in MCF-7, indicating that ISL could suppress the ALDHhi population in both cell lines (Figure 5B). The results suggested ISL could decrease CSC populations characterized with either ALDHhi or CD44+CD24−/low markers.

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