Abstract LB-5: A proteomic approach to study molecular mechanisms of andrographolide-mediated degradation of Src oncoproteins.

Abstract

Abstract Andrographolide is a lactone diterpenoid compound isolated from Andrographis paniculata. Accumulating studies have demonstrated the anticancer activity of andrographolide. Andrographolide treatment could induce apoptosis of cancer cells and inhibit multiple malignant cancer phenotypes, such as aberrant proliferation, angiogenesis and metastasis. It is widely demonstrated that cancer cells require oncogene(s) for survival and maintenance of malignant phenotypes, a phenomenon called oncogene addiction. Thus, targeting oncoprotein(s) has been considered as an effective strategy for cancer therapy. Elevated protein expression and activity of Src oncoproteins have strong associations with carcinogenesis. Previous data from our laboratory have shown that andrographolide treatment led to degradation of Src proteins, suppression of Src-mediated cellular transformation and apoptosis of Src-transformed cells (J Biol Chem 2008; 283: 5023-33). To further reveal the molecular basis of andrographolide-mediated Src degradation, a proteomic approach was carried out to identify proteins specifically regulated by andrographolide. Proteomes from v-Src expressing RK3E cells treated with vehicle, andrographolide and NCTU-048 (an inactive andrographolide analog) were compared using two-dimensional electrophoresis and proteins with differential expression pattern in andrographolide-treated cells were further identified using mass spectrometry. Among the identified proteins, an N-terminal fragment of Hsp90 protein was selectively elevated (2.5-fold) in andrographolide-treated cells. Src oncoproteins are known client proteins of Hsp90 and their stability are modulated by Hsp90. We therefore investigated whether Hsp90 cleavage is a crucial event in andrographolide-mediated degradation of Src oncoproteins. Using Western blot analysis, we confirmed that Hsp90 cleavage was induced in various types of Src-transformed cells under andrographolide treatment in a concentration- and time-dependent manner. Notably, the Src-repressing activity of andrographolide and its derivatives correlated well with their ability to induce Hsp90 cleavage. Moreover, the amount of cleaved Hsp90 fragment induced by andrographolide and its derivatives positively correlated with the status of transformation suppression and apoptosis caused by these compounds. Notably, the andrographolide-induced Hsp90 cleavage, Src degradation, cellular transformation and induction of apoptosis were restored by ROS inhibitor, N-acetyl-cysteine (NAC). The molecular basis of andrographolide-induced biological effects via ROS is currently under investigation. Citation Format: Sheng-Hung Liu, Pei-Fen Chen, Chao-Hsiung Lin, Chung-Ming Sun, Chin-Wen Chi, Shu-Ling Fu. A proteomic approach to study molecular mechanisms of andrographolide-mediated degradation of Src oncoproteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-5. doi:10.1158/1538-7445.AM2013-LB-5