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Targeting Beta-Catenin Signaling to Induce Apoptosis in Human Breast Cancer Cells by Z-Guggulsterone and Gugulipid Extract of Ayurvedic Medicine Plant Commiphora Mukul

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Targeting Beta-Catenin Signaling to Induce Apoptosis in Human Breast Cancer Cells by Z-Guggulsterone and Gugulipid Extract of Ayurvedic Medicine Plant Commiphora Mukul

Guoqin Jiang et al. BMC Complement Altern Med.

Abstract

Background: z-Guggulsterone (z-Gug) and Gugulipid (GL) have been used to treat a variety of ailments. We now report their anti-cancer effect and mechanism against human breast cancer.

Methods: Using the human estrogen receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cells as well as the normal human mammary epithelial cell line (HMEC), we evaluated the anti-breast-cancer efficacy and apoptosis inducing activity of GL. We determined the cellular and molecular mechanism of GL-inhibited breast cancer cell growth.

Results: GL significantly inhibited growth of MCF-7 and MDA-MB-231 cells with an IC50~2 μM at pharmacologically relevant concentrations standardized to its major active constituent z-Gug. The GL-induced growth inhibition correlated with apoptosis induction as evidenced by an increase in cytoplasmic histone-associated DNA fragmentation and caspase 3 activity. The GL-induced apoptosis was associated with down-regulation of the β-Catenin signaling pathway. The decreased expression of Wnt/β-Catenin targeting genes, such as cyclin D1, C-myc and survivin, and the inhibition of the activity of the transcription factor (T-cell factor 4, TCF-4) were observed in GL-treated breast cancer cells. The GL treatment resulted in a significant reduction of β-Catenin /TCF-4 complex in both of the cancer cells. The GL-induced apoptotic cell death was significantly enhanced by RNA Interference of β-Catenin and TCF-4. On the other hand, the normal human mammary epithelial cell HMEC, compared with the human breast cancer cells, is significantly more resistant to growth inhibition and apoptosis induction by GL.

Conclusion: The present study indicates that the β-Catenin signaling pathway is the target for GL-induced growth inhibition and apoptosis in human breast cancer.

Figures

Figure 1
Figure 1
A, Effect of GL (GL contains ~3.75% z-Gug and was standardized to z-Gug (μM)) on survival of MCF-7 and MDA-MB-231 (A) cells determined by the colonogenic survival assay. Cells were treated with different concentrations of GL for 24 h. Columns, mean of three determinations; bars, SE. *Significantly different (P<0.05) compared with DMSO-treated control by one-way ANOVA followed by Dunnett’s test. Similar results were observed in two independent experiments. Representative data from a single experiment are shown. Effect of GL (B and D) and z-Gug (C) on survival of MCF-7, MDA-MB-231 and HMEC cells determined by the trypan blue dye exclusion assay. Cells were treated with different concentrations of GL or z-Gug for 24 h. Columns, mean of three determinations; bars, SE. *Significantly different (P<0.05) compared with DMSO-treated control by one-way ANOVA followed by Dunnett’s test. Similar results were observed in two independent experiments. Representative data from a single experiment are shown.
Figure 2
Figure 2
GL induced apoptosis in MCF-7 and MDA-MB-231 cells, but not in HMEC, determined by (A and D) quantitation of cytoplasmic histone associated DNA fragmentation, and (B) flow cytomitry analysis of Caspase 3 activity. Cells were treated with the indicated concentrations of GL or z-Gug (C) or DMSO (control) for 24 hours. Results are expressed as enrichment factor relative to cells treated with DMSO (control). Results are mean±SE (n=3). *Significantly different (P<0.05) between the indicated groups by one-way ANOVA followed by by Dunnett’s test. Similar results were observed in at least two independent experiments. Representative data from a single experiment are shown. Effect of pretreatment with general caspase inhibitor Z-VAD on GL-induced cytoplasmic histone-associated DNA fragmentation (E) and caspase 3 activity (F) in MCF-7 and MDA-MB-231 cells. Columns, mean (n=3); bars, SE. a, p<0.05, significantly different compared with control; b, p<0.05, significantly different compared with GL alone treatment group (one-way ANOVA followed by Bonferroni’s test for multiple comparisons).
Figure 3
Figure 3
GL (A and C) and z-Gug (B) inhibited β-Catenin level in human breast cancer MCF-7 and MDA-MB-231 cells as well as HMEC. Cells were treated with DMSO (control) or 2.5 and 5 μmol/L GL standardized to z-Gug or 20 and 40 μmol/L z-Gug for 24h. β-Catenin level was determined by using the SurveyorTM IC Human total β-Catenin Immunoassay kit (R&D Systems) following the manufacturer’s instructions. Experiments were repeated twice with triplicate measurements in each experiment. Results are mean±SE (n=3). *Significantly different (P<0.05) between the indicated groups by one-way ANOVA followed by Dennett’s test. The results were consistent and representative data from a single experiment are shown.
Figure 4
Figure 4
Immunoblotting for β-Catenin, C-myc, Cyclin D1 and Survival proteins using lysates from MCF-7 (A) or MDA-MB-231 (B) cells treated with DMSO (control) or 5 and 10 μmol/L GL standardized to z-Gug or 10 and 40 μmol/L z-Gug for 24 h. The blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent change in protein levels relative to corresponding DMSO-treated control. Immunoblotting for β-Catenin protein using isolated cytosolic and nuclear fractions from MCF-7 (C) or MDA-MB-231 (D) cells following 24-h treatment with DMSO or 5 μmol/L GL or 40 μmol/L z-Gug. The blot was reprobed with anti-α-Tubulin or anti-PARP antibody to ensure purity of the nuclear fraction. The numbers on top of the immunoreactive bands represent change in levels relative to DMSO-treated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates.
Figure 5
Figure 5
Immunoblotting for β-Catenin using lysates from MCF-7 (A) or MDA-MB-231 (C) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated nonspecific control siRNA–transfected cells. Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (B) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean (n=3); bars, SE. *, P<0.05, significantly different between the indicated groups by paired t test.
Figure 6
Figure 6
Immunoblotting for TCF-4 using lysates from MCF-7 (A) or MDA-MB-231 (B) cells treated with DMSO (control) or 5 μmol/L GL for 24 h. Immunoblotting for TCF-4 using lysates from MCF-7 (C) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or TCF-4 -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated control cells (A-B) or relative to DMSO-treated nonspecific control siRNA–transfected cells (C-D). Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (E) or MDA-MB-231 (F) cells transiently transfected with a control nonspecific siRNA or TCF-4-targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean (n=3); bars, SE. *, P<0.05, significantly different between the indicated groups by paired t test. GL treatment inhibited β-Catenin binding to TCF in human breast cancer cells. Immunoblotting for TCF-4 using β-Catenin immunoprecipitates from MCF-7 (H) and MDA-MB-231 (G) cells treated for 24 h with DMSO (control) or GL 2.5 and 5 μM. The numbers on top of the immunoreactive bands represent change in levels relative to DMSO-treated control for each cell line. Each experiment was performed at least twice using independently prepared lysates.

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References

    1. Siegel R, DeSantis G, Virgo K, Stein K, Mariotto A, Smith T, Cooper D, Gansler T, Lerro C, Fedewa S, Lin C, Leach C, Cannady RS, Cho H, Scoppa S, Hachey M, Kirch R, Jemal A, Ward E. Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin. 2012;62:220–241. doi: 10.3322/caac.21149. - DOI - PubMed
    1. Badmaev V, Majeed M, Pacchetti B, Prakash L. Standardiation of Commiphora Mukul extract in dislipidemia and cardiovascular disease. NUTRA Foods. 2003;2:45–51.
    1. Shishodia S, Harikumar KB, Dass S, Ramawat KG, Aggarwal BB. The guggul for chronic disease: ancient medicine, modern targets. Anticancer Res. 2006;28:3647–3664. - PubMed
    1. Sinal CJ, Gonzalez FJ. Guggulsterone: an old approach to a new problem. Trends Endocrinol Metab. 2002;13:275–276. doi: 10.1016/S1043-2760(02)00640-9. - DOI - PubMed
    1. Urizar NL, Liverman AB, Dodds DT, Silv FV, Ordentlich P, Yan Y, Gonzaleg FJ, Heyman RA, Mangelsdorf DJ, Moore DD. A natural product that lowers cholesterol as an antagonist ligand for FXR. Science. 2002;296:1703–1706. doi: 10.1126/science.1072891. - DOI - PubMed

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