Herbal extract SH003 suppresses tumor growth and metastasis of MDA-MB-231 breast cancer cells by inhibiting STAT3-IL-6 signaling

Abstract

Cancer inflammation promotes cancer progression, resulting in a high risk of cancer. Here, we demonstrate that our new herbal extract, SH003, suppresses both tumor growth and metastasis of MDA-MB-231 breast cancer cells via inhibiting STAT3-IL-6 signaling path. Our new herbal formula, SH003, mixed extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz, suppressed MDA-MB-231 tumor growth and lung metastasis in vivo and reduced the viability and metastatic abilities of MDA-MB-231 cells in vitro. Furthermore, SH003 inhibited STAT3 activation, which resulted in a reduction of IL-6 production. Therefore, we conclude that SH003 suppresses highly metastatic breast cancer growth and metastasis by inhibiting STAT3-IL-6 signaling path.

Figure 1

HPLC profile of SH003. (a) Composition of SH003. (b) HPLC identification of components in SH003. Formononetin, decursin, and nodakenin were detected in Am and Ag. Three components in SH003 were detected at 3.6 min, 6.1 min, and 11.0 min.

Figure 2

SH003 suppresses tumor growth in vivo. (a) 1 × 10⁶ MDA-MB-231 cells were s.c. injected and nude mice (n = 5/group) were p.o. administrated with the indicatives until 34 days. Xenograft tumor volumes were measured three times a week by a caliper. *P < 0.05. (b) Body weights were measured three times a week. (c) Tumor tissues were stained with hematoxylin and eosin. Photo images were taken at 20x magnification. Tumor tissues were also stained with anti-CD31 antibody to detect tumor angiogenic vessels. The bar indicates 10 μm. (d) To measure tumor angiogenic vessels in tumor cohorts, CD31-positive vessels were counted. *P < 0.05. (e) Pulmonary metastases were determined by counting foci at lungs.

Figure 3

SH003 inhibits MDA-MB-231 growth and induces apoptosis. (a) Different breast cancer cells were seeded on 96-well plates and treated with each extract at different concentrations for 72 hours. Experiments were performed three times in sextuplicate. Representative data were presented as the means and standard deviations. Right triangles indicate the doses of each extract (0, 50, 100, 200, and 500 μg/mL), which was also marked with bars in different colors. (b) MDA-MB-231 cells were treated with 500 μg/mL of the each extract. Cells were stained with propidium iodide (PI, 50 μg/mL) at room temperature in the dark. PI-positive apoptotic cells were detected using FACSCalibur. *P < 0.05. (c) MDA-MB-231 cells were treated with the indicatives at 500 μg/mL for 24 hours and then subjected to western blots. Tubulin was used for the intimal control. (d) RIE cells were seeded on 96-well plates and treated with each extract at different concentrations for 72 hours. Experiments were performed three times in sextuplicate. Representative data were presented as the means and standard deviations.

Figure 4

SH003 inhibits metastatic abilities in vitro. (a) MDA-MB-231 cells were scratched and treated with the indicatives for 24 hours. Cell migration was determined by counting cell numbers migrated from the wounding region. *P < 0.05. (b) MDA-MB-231 cells were cultured on the upper chambers and treated with the indicatives for 24 hours. Invading cells were stained with crystal violet and then cell numbers were measured. *P < 0.05. (c) MDA-MB-231 cells were cultured in soft agars and treated with the indicatives for 15 days. Colonies were then stained with crystal violet. *P < 0.05.

Figure 5

SH003 selectively inhibits STAT3 phosphorylation and transcriptional activity. ((a) and (b)) MDA-MB-231 cells were treated with the indicatives at 50 or 500 μg/mL for 15 minutes and then subjected to western blots with the antibodies indicated. Tubulin was used for the internal control. (c) Cells were treated with the indicatives for 6 hours and then stained with anti-p-STAT3 antibody (green) and TOPRO-3 (blue). 20x objectives. A scale bar indicates 10 μm. (d) Representative data for the luciferase assays. 293T (left) and MDA-MB-231 (right) cells were transfected with the indicatives and then treated with each extract for 24 hours. Experiments were performed in triplicate. Bars indicate means and standard deviations. *P < 0.05.

Figure 6

SH003 inhibits STAT3 target gene expression. ((a) and (b)) MDA-MB-231 cells were treated with the indicatives at 50 or 500 μg/mL for 24 hours and then subjected to western blots with the antibodies indicated. Tubulin was detected as a loading control. (c) MDA-MB-231 cells were treated with the indicatives at 500 μg/mL for 24 hours and then subjected to real-time PCR for IL-6 mRNA expression levels. Experiments were performed in triplicate. Bars indicate means and standard deviations. *P < 0.05. (d) MDA-MB-231 cells were treated with the indicatives at 500 μg/mL for 24 hours and then harvested culture media. IL-6 levels were analyzed with ELISA assay. Experiments were performed in triplicate. Bars indicate means and standard deviations. *P < 0.05. (e) Cells were treated with SH003 for 6 hours and then subjected to chromatin immunoprecipitation assays to test STAT3 interaction with IL-6 promoter. (f) A schematic model for anti-TNBC roles of SH003. TNBC has highly metastatic characteristics with constitutively active STAT3. SH003 selectively targets STAT3-dependent IL-6 production, resulting in the inhibition of TNBC growth and metastasis.