Mechanistic studies on suppression of invasive activity by timosaponin A-III, an active ingredient from Zhimu in triple negative breast cancer cell

• Main Authors: Chung-Hsin Tsai, 蔡崇鑫
• Other Authors: Jen-Hwey Chiu
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/34373696215015205252

博士 === 國立陽明大學 === 傳統醫藥研究所 === 102 === Triple-negative breast cancer (TNBC) is lack of estrogen receptor (ER), progesterone receptor (PR), and Human epidermal growth factor receptor 2 (HER2). There is no specific target to TNBC and the mainstay of treatment of TNBC is traditional systemic cytotoxic chemotherapy but with high recurrent and metastatic rate. The exact mechanisms of how TNBC recurs remain unclear. Since the treatment of breast cancer depends highly on the genomic subtypes such as ER/PR (+) or HER2 (+), further elucidation on TNBC subtypes and recurrence mechanism is very helpful for clinical judgment of therapeutic strategies in TNBC patients. Accordingly, the aims of this study were 1) to investigate the gene expression profiles between TNBC with subsequent recurrent and non-recurrent status; and 2) to investigate the mechanisms how Timosaponin AIII, inhibits HGF-induced invasion activity in triple negative breast cancer MDA-MB-231 cell line. This study used recurrent TNBC and non-recurrent TNBC samples to conduct microarray analyses suggested that 28 genes are differentially expressed between recurrent and non-recurrent TNBC tissues. The results showed that there was a marked different gene expression profiling in stage IIIc tumors compared to those with earlier stages. PAX/MET/COX-2 signaling pathways shows the key role in recurrent stage IIIc tumors. The other aim of the study was to investigate the mechanisms how Timosaponin A-III (TA-III), a pure compound derived from Anemarrhena aspodeloidea, inhibits HGF- induced invasion activity in triple negative breast cancer cell line. MDA-MB-231 breast cancer cell line [ER (-), PR (-), HER2 (-)] was used in this study. After pretreatment with different concentration (10-8~10-6M) of TA-III for 1 hr, cultured cells were treated with hepatocyte growth factor (HGF, 15 ng/mL). At different time intervals (5 min, 2 h, 24 h and 48 h) after co-incubation, parameters such as c-Met, ERK, COX-2, MMP-9 by Western blot, real-time PCR, invasion and wound healing assay were analyzed, respectively. Several signal transduction blockers were used to elucidate the possible mechanisms of TA-III effects on HGF-induced invasive activity. The results showed that HGF induced c-MET (5-min), ERK (2h) activation and increased COX-2 protein expression (24h) and subsequent invasive activity. TA-III concentration-dependently (10-8~10-6M) inhibited HGF-induced invasive activity and COX-2 gene expression despite increased phospho-form of c-Met and ERK at 5-min and 2 h after treatment were noticed, respectively. Besides, sustained cytoplasmic and nuclear ERK activation and hence a suppressed nuclear ATF2 activation were noticed, followed by down regulation of COX-2 and MMP-9 transcription. This study concludes that there is marked difference in gene expression profiling between early recurrent and late recurrent TNBC and that TA-III inhibited HGF-induced invasive activity on MDA-MB-231 cells through sustained ERK activation. These results provide important information in treatment of triple negative breast cancer.