Multi-targeting chemoprevention of Chinese herb formula Yanghe Huayan decoction on experimentally induced mammary tumorigenesis

Abstract

Background: Development of safe and effective chemopreventive agents is a winning strategy in reducing the morbidity and mortality of breast cancer. The current study was to investigate the mechanism-based chemopreventive potential of a Chinese herb formula Yanghe Huayan (YHHY) Decoction on the classical 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary carcinogenesis model.

Methods: Female Sprague-Dawley rats at 42 days of age were orally administered with a human equivalent dose of YHHY Decoction at 0.02 ml/g (10 mg/ml) once daily, starting 1 wk. before and 4 wks following DMBA treatment. Mammary tumor occurrence was monitored every day. The length of time before palpable tumor is examined is defined as tumor-free survival time. High performance liquid chromatography (HPLC) analyses were adopted to identify major chemical compositions of the decoction. Following bioinformatics data mining and experimental analyses were performed to demonstrate the underlying mechanism of action.

Results: DMBA animals receiving YHHY Decoction exhibited a significant delay (P = 0.014) and in some animals prevention (P = 0.046) of tumor occurrence without obvious toxicity. Oncogenic myc activation was significantly suppressed in the DMBA-induced rats by the YHHY treatment. Eight major chemical compositions of the decoction were identified and were shown to interfere with multiple tumorigenic pathways simultaneously in the mammary tumors, including inducing tumor apoptosis and up-regulating pro-apoptotic protein Bax and down-regulating anti-apoptotic protein Bcl-2; suppressing abnormal cell proliferation and the MAPK/ERK, PI3K/AKT signalings; blocking neo-angiogenesis and the VEGF/KDR signaling, and inhibiting oxidative stress in the mammary tumors.

Conclusion: The multi-components and multi-targeting properties of the YHHY Decoction support its use as a potent chemopreventive drug in breast cancer.

Keywords: Chemoprevention; Chinese herb formula; Mammary tumorigenesis; Multi-components; Multi-targeting.

Fig. 1

Effects of YHHY Decoction on DMBA-induced tumorigenesis and oncogenic myc activation. a. Effect of YHHY Decoction treatment on body weight gain during DMBA-induced mammary tumorigenesis in rats. Each data point indicates mean ± SD. There were 10–18 animals in various groups. No significant difference in body weights was observed among various rat groups at any time point. b. Effect of YHHY Decoction treatment on the occurrence of palpable tumors in the DMBA-induced rats. The length of time before palpable tumor is examined is defined as tumor-free survival time. n = 18 of the YHHY+DMBA group, and n = 14 of the DMBA control group. P = 0.014, Log rank test. c. Histopathology of the tumors detected in the DMBA control and YHHY Decoction treatment groups. DMBA control tumors showed extensive epithelial proliferation (a) and nuclear pleomorphism (b). Tumors treated with YHHY Decoction showed significantly improved cell architecture (c) and almost normal ductal and alveolar structure (d). Scale bar: 50 μm d. Immunohistochemical staining of phospho-c-myc in the mammary glands. Rats’ tissue were harvested two weeks after DMBA induction. The YHHY decoction treatment was initiated 1 week before DMBA induction and lasted for two weeks. Extensive staining of phospho-c-myc was observed in all examined animals in the DMBA group, while none of the animals in the YHHY prevention group showed obviously positive staining (n = 3). Scale bar: 50 μm e. Expressions of phospho-c-myc and total c-myc in 3 individual tumors in the YHHY Decoction treated vs. DMBA control group

Fig. 2

YHHY Decoction induced tumor cell apoptosis. a. Representative TUNEL staining images showing the apoptotic cells in tumor sections in a: DMBA-induced rat and b: YHHY treated DMBA rat. Apoptotic cells were detected by TUNEL reaction with In Situ Cell Death Detection Kit. TUNEL-positive cells contain dark-brown nuclei. Scale bar: 50 μm. b. Quantitative analysis of the % of TUNEL positive cells in the tumor sections. P = 0.0023, student t test. n = 5 animals per group. c. Representative Western blot images of Bax and Bcl2 in one tumor sample from each group. d. Expressions of Bax and Bcl2 in 5 individual tumors in the YHHY Decoction treated vs. DMBA control group. e. Quantification of the western blot analysis for Bax and Bcl2 in normal mammary glands (normal and YHHY groups) and mammary tumors (DMBA and YHHY+DMBA groups). # p < 0.05 vs. normal; * p < 0.05 vs. DMBA; ^ p < 0.05 vs. DMBA. The up-right corner graph showing the ratio of Bax/Bcl2 in each group

Fig. 3

YHHY Decoction inhibited tumor cell proliferation. a. Representative images of Ki-67 immunohistochemistry staining showing the proliferative cells in tumor sections in a: DMBA-induced rat and b: YHHY treated DMBA rat. Tumor sections were stained with primary Ki-67 antibody and the DAB Detection kit for cell proliferation detection. Ki-67-positive cells contain dark-brown nuclei. Scale bar: 50 μm. b. Quantitative analysis of the % of Ki-67 positive cells in the tumor sections. P < 0.05, student t test. n = 5 animals per group. c. Representative Western blot images of ERK1/2, PI3K and AKT in one tumor sample from each group. d. Expressions of ERK1/2, PI3K and AKT in 5 individual tumors in the YHHY Decoction treated vs. DMBA control group. e. Quantification of the western blot analysis for ERK1/2, PI3K and AKT in normal mammary glands (normal and YHHY groups) and mammary tumors (DMBA and YHHY+DMBA groups). * p < 0.05 vs. normal; ^ p < 0.05 vs. DMBA

Fig. 4

YHHY Decoction inhibited tumor neo-angiogenesis. a. Representative images of CD-105/Endoglin immunohistochemistry staining showing the newly formed blood vessels in tumor sections in a: DMBA-induced rat and b: YHHY treated DMBA rat. Black arrows point to the CD-105 positive blood vessels. Scale bar: 50 μm. b. Quantitative analysis of the vascular density in the tumor sections. P < 0.01, student t test. n = 5 animals per group. c. Representative Western blot images of VEGFA, p-KDR(Tyr 951/996) and total KDR in one tumor sample from each group. d. Expressions of VEGFA, p-KDR(Tyr 951/996) and total KDR in 4 individual tumors in the YHHY Decoction treated vs. DMBA control group. e. Quantification of the western blot analysis for VEGFA, p-KDR(Tyr 951/996) and total KDR in normal mammary glands (normal and YHHY groups) and mammary tumors (DMBA and YHHY+DMBA groups). * p < 0.05 vs. normal; ^ p < 0.05 vs. DMBA