Tongshu capsule down-regulates the expression of estrogen receptor α and suppresses human breast cancer cell proliferation

Abstract

The Tongshu Capsule (TSC) is a prevalent form of traditional Chinese medicine widely used for its purported effects in treating mammary gland hyperplasia and inflammation. Though successful in several clinical studies, there is no clear evidence as to why TSC has a positive treatment effect, and little known about underlying mechanism that may account for it. In this study, we examined the effects of TSC and found that it has a comparatively strong growth inhibition on ERα positive breast cancer cells. TSC seems to cause G1 cell cycle arrest instead of apoptosis. Interestingly, TSC also down-regulated the expression of ERα and Cyclin D1. Consistently, TSC suppressed E2 mediated ERα downstream gene expression and cell proliferation in ERα positive breast cancer cell lines MCF7 and T47D. Depletion of ERα partially abolished the effects of TSC on the decrease of Cyclin D1 and cell viability. Our findings suggest that TSC may have therapeutic effects on ERα positive breast cancers and moreover that TSC may suppress breast epithelial cell proliferation by inhibiting the estrogen pathway.

Figure 1. ERα-positive human breast cancer cells are more sensitive to TSC than ERα-negative breast cancer cells.

Two immortalized mammary epithelial cell lines (MCF10A and 184B5), two ERα-positive breast cancer cell lines (MCF7 and T47D), and two ERα-negative breast cancer cell lines (SW527 and HCC1937) were treated with different concentrations of TSC (0.125, 0.25, 0.5, 1, 2 mg/ml) for 48 hours. Cell viability was measured by the SRB assay. ERα-positive breast cancer cell lines were found to be more sensitive to TSC than ERα-negative breast cell lines.

Figure 2. TSC inhibits cell proliferation through inducing the G1 cell cycle arrest.

A. TSC significantly suppressed DNA synthesis in both MCF7 and T47D cell lines in a dosage dependent manner, as determined by the Edu incorporation assay. B. TSC caused G1 cell cycle arrest in both MCF7 and T47D cell lines in a dosage dependent manner as determined by flow cytometry. *: P<0.05; **: P<0.01.

Figure 3. TSC inhibits the expression of ERα and Cyclin D1.

A. TSC down-regulated the protein expression of ERα and Cyclin D1 in MCF7 in a dosage dependent manner. MCF7 cells were treated with different concentrations of TSC for 12 h. The protein expression levels of ERα and Cyclin D1 were examined using WB. B. The mRNA expression levels of ESR1 are decreased by TSC in a dosage dependent manner in both MCF7 and T47D cell lines, as measured by RT-PCR. C. TSC promoted the protein degradation of ERα in T47D but not in MCF7, as detected by the cycloheximide (CHX) chase assay. Quantitative results are show on the right. D. Knockdown of ERα decreased the expression of Cyclin D1 in MCF7 and partially abrogated the effect of TSC on Cyclin D1 expression down-regulation. E. Knockdown of ERα decreased the cell viability of MCF7 and partially abrogated the effect of TSC on inhibiting MCF7. F. TSC down-regulated the protein expression of ERβ in MCF7. MCF7 cells were treated with different concentrations of TSC for 12 h. The protein expression levels of ERα, ERβ, and Cyclin D1 were examined by WB.

Figure 4. TSC inhibits the estrogen signal pathway.

A. TSC inhibited the transcriptional activation of ERα in response to E2 in both MCF7 and T47D, as measured by the ERE luciferase reporter assay. **, P<0.01. B. TSC blocked the E2-induced CTSD and pS2 mRNA expression in both MCF7 and T47D, as measured by real-time PCR. **, P<0.01. C. TSC blocked the induction of Cyclin D1 by E2 in MCF7 and T47D cells. The E2 concentration was 10 nM.

Figure 5. TSC inhibits the E2-induced cell cycle progression and cell proliferation.

A. TSC blocked the E2-induced G1/S cell cycle progression in both MCF7 and T47D, as measured by flow cytometry. *, P<0.05; **, P<0.01. B. TSC significantly blocked the E2-induced cell growth in both MCF7 and T47D cell lines, as measured by the SRB assay.