Figure 1.
HSE suppressed the growth of human breast cancer xenograft in mice.
Mice were treated daily with HSE at a dose ranging from 0 and 10 mg per kg body weight by intragastric administration. A, image of tumor-xenografted nude mice model at the end of the treatment. B, tumor size growth curves of MDA-MB 231 xenograft model during the treatment calculating the volume size of individual tumors. C, tumor size growth curves of MCF-7 xenograft model during the treatment calculating the volume size of individual tumors. D, H&E staining of MDA-MB 231 and MCF-7 xenografts. Dii, Image of necrosis in MDA-MD 231 breast tumor tissue sections at the end of the treatment. Arrow points the area of necrosis. Tumors were collected on day 30 after start of treatment and were formalin-fixed paraffin-embedded followed by staining with hematoxylin and eosin. Representative images are shown (C, magnification: ×200, scale bar = 200 µm). E, quantitative representation of tumor necrotic areas in comparison with vehicle. The area of necrosis in each section was quantified from six fields (magnification: ×200). The values are means ± S.E (n = 6) after normalization to vehicle (internal control). Asterisks indicate a statistically significant decrease (B) or increase (E) by t-test (*p<0.05, ***p<0.001).
Figure 2.
HSE down-regulated STAT5b/IGF-1R and STAT3/VEGF signal pathway, and inhibited HIF-1α related-protein expression in human breast cancer xenografts.
A and B, immunohistochemical (IHC) analysis of STAT5b/IGF-1R and STAT3/VEGF protein expression in both MDA-MB 231 and MCF-7 breast tumor tissues. The xenografts were sliced to 5 µm thickness and treated with primary antibodies specific for STAT3, STAT5b, VEGF and IGF-1R. Detection was done using secondary antibody, Alexa Fluor 488 (rabbit) and Alexa Fluor 594 (mouse). The IHC staining was performed thrice (magnification: ×400). C, studies confirmed the decrease in expression of STAT5b, IGF-1R, STAT3 and VEGF with no much alteration in the nucleus level. A, IHC specific for STAT5b/IGF-1R and B, IHC specific for STAT3/VEGF. C, the expression of p-STAT5, p-STAT3, p-IGF-1R, VEGF, VEGF-R2 and HIF-1α in breast cancer xenografts. The protein extracts (10 µg) were separated by 10% SDS-PAGE, and western blots were performed as described in experimental procedures. β-actin was used as a control for protein loading.
Figure 3.
HSE inhibited breast cancer cell growth, induce G1 cell cycle arrest, maintained the expression of tumor suppressor proteins and suppressed the expression of oncogenic proteins over other whole grain extracts.
A, breast cancer cells MDA-MB 231, MCF-7, and SKBR-3 were treated with HSE. After 24 h, cell viability was evaluated by the MTT assay. B, the expression of Bcl-2, Cyclin D1, Cyclin E, ppRb, pRb, p53, p21 and BRCA1 in MDA-MB 231 cells. C, the cell cycle arrest is confirmed through flow cytometry. Flow cytometry MDA-MB 231 cells using propidium iodide flow cytometry. D, the oncogenic protein expression of in MDA-MB 231 cells treated with whole grain extracts (10 µg/ml). The protein extracts were separated by 10% SDS-PAGE, and western blots were performed as described in experimental procedures. β-actin was used as a control for protein loading. Data represent the mean of at least three separate experiments, mean ± SEM. Asterisks indicate a IC50 value.
Figure 4.
HSE down-regulated the STAT5b/IGF-1R and STAT3/VEGF pathways in time and dose dependent manner.
A, human breast cancer cells MDA-MB 231, MCF-7, and SKBR-3 were exposed to different incubation time with HSE (10 µg/ml). B, MDA-MB 231, MCF-7, and SKBR-3 cells were exposed to various concentrations of HSE. The protein extracts (10 µg) were separated by 10% SDS-PAGE, and western blots were performed as described in experimental procedures. β-actin was used as a control for protein loading.
Figure 5.
HSE suppressed the binding activities of STAT5b to the IGF-1R and STAT3 to the VEGF promoter sites.
A, electrophoretic mobility shift assay (EMSA) showed, HSE suppressed the DNA-binding activity of STAT5b to the IGF-1R (i) and STAT3 to the VEGF (ii) binding sites in MDA-MB 231 cells treated with HSE for 24 h. B, nuclear protein extracts (NE) were separated and blotted onto nitrocellulose membrane showing decrease in the level of p-STAT5, p-STAT3, VEGF and IGF-1R. C and D, shows the activation of STAT5b/IGF-1R promoter and STAT3/VEGF promoter in COS-7 cells by HSE. COS-7 cells were transiently co-transfected with STAT5b and IGF-1R (C) and STAT3 and VEGF genes (D). Data represent means of at least three separate experiments. Asterisks indicate a statistically significant decrease by t-test (***p<0.001) in the crosstalks of STAT5b/IGF-1R and STAT3/VEGF respectively.
Figure 6.
Suppression of STAT3/DNA binding activity leads to the crackdown of target gene products.
A, western blotting analysis of whole cell lysates from MDA MB-231, MCF-7 and SKBR-3 cells treated with increasing concentration of HSE. B, RT-PCR analysis of MDA-MB 231, MCF-7, and SKBR-3 cells treated with different concentration of HSE showed transcriptional regulation of IGF-1R, Cyclin D1 and VEGF mRNA levels. 18S RNA was used as a control.
Figure 7.
HSE might block the metastasis of breast cancer to lungs in animal model.
A, analysis of the effect of HSE on the experimental lung metastases established by MDA-MB 231 and MCF-7 cells. Mice were injected with MDA-MB 231 cells (1×107 in 200 µL PBS per mouse) and MCF-7 cells (1×107 in 200 µL PBS per mouse). They are randomized into two groups for each cell model. One group of mice (n = 6) was injected with HSE for 30 days, and the other group of mice (n = 6) was injected with water using the same schedule. Lungs were collected on day 30 after the start of treatment and formalin-fixed paraffin-embedded lung tissues were stained with hematoxylin and eosin. Representative images are shown (A, magnification: ×200, scale bar = 200 µm). Arrow points the area of metastasis. B, quantitative analysis of tumor areas shown in A. The area of metastatic nodules in each section was quantified from six fields (magnification: ×200). The values are means ± S.E (n = 6) after normalization to vehicle (internal control). Asterisks indicate a statistically significant decrease by t-test (***p<0.001). C, HSE inhibited the migration of MDA-MB 231 cells treated with HSE (10 µg/ml for 24 h). MDA-MB 231 cells were plated into DMEM medium separately. After 24 hours, medium was replaced with and without HSE (10 µg/ml). Live cell images were acquired 24 hours after media changes (i and ii; pre-confluent), (iii and iv; confluent). Movie S1, MDA-MB 231 cells without HSE (control) and Movie S2, MDA-MB 231 cells treated by HSE.