Effect And Mechanism Of Oldenlandia Diffusa Extracts In Xiao’ai Jiedu Prescription Inhibits Colon Cancer

Objective:The superior curative effect of Xiao’ai Jiedu Prescription(XJP) by Professor Zhou Zhongying has be proved. The most commonly used traditional Chinese medicine in Xiao’ai Jiedu Prescription was Hedyotis diffusa, and the using rate of Hedyotis diffusa was up to 74% in Zhou’s prescription. Modernization of traditional Chinese medicine was focused on modernization of traditional Chinese medicine, but the key development and utilization of traditional Chinese medicine was to clear the anticancer mechanism of active ingredient. Hedyotis diffusa specifically inhibits the colon cancer, but its mechanism is still not clear.Methods:The HCT-116、Lovo、HT-29 and DLD-1 cells were purchased from Cell Bank of Chinese Academy of Medical Sciences (Shanghai, China). Cells were maintained in RPMI/DMEM medium (Sigma, St. Louis, MO), with a 10% FBS (Sigma, St. Louis, MO), penicillin/streptomycin (1:100, Sigma), and in a CO2 incubator at 37℃. Cells were treated with indicated Hedyotis diffusa (Ctr1、10、25、50、200μg/ml), and cultured for indicated time (0、 6、24、48、72、96h). Cell apoptosis was analyzed by Histone-DNA ELISA assay and Annexin V FACS assay. MTT assay and trypan blue staining were utilized to test cell survival. Activation of AMPK was reflected by increased expressions of phosphorylated AMPKa 1、ACC、 Caspase-3、Bc1-2、HIF-la、S6K、p53 activation and growth factor receptors expression were tested by western blots. AMPKα1 inhibitor compound C or target-shRNA lentiviral particles were applied to block AMPKα1 activation. Establishing CYP-D-shRNA, p53-shRNA, AMPKαl-shRNA and AMPK-αl dominant negative (DN) mutant (DN-AMPK-α1) cDNA stably transfected tumor cells, and using the above-described method further validated the changing of signaling pathway. Using the above-described method was to verify vitro activity of Hedyotis diffusa. Male nude mice were introduced to establish xenograft tumor model of HCT-116 cells, and to study the potential role of Hedyotis diffusa on HCT-116 cell growth in vivo. Mice survival rate and tumor size were recorded, meanwhile AMPKa expression was detected by immunohistochemistry. All chemicals and reagents mentioned in this study were purchased from Sigma.Results:Hedyotis diffusa significantly inhibited survival of colon cancer cell lines (HCT-116、Lovo、HT-29 and DLD-1 cell) survival. The effect of Hedyotis diffusa against these cancer cells was dose-and time-dependently. Cancer cell viability was examined through MTT assay, and results showed that Hedyotis diffusa alone only moderately inhibited HCT-116 colon cancer cell survival. Trypan blue staining assay and clonogenicity assay results showed that Hedyotis diffusa resulted in massive cell death. Histone DNA-ELISA assay and Annexin V FACS assay results failed to detect a significant cell apoptosis in Hedyotis diffusa-treated HCT-116 cells. Further, cell cycle progression, examined by FACS assay, was also not affected by HCT-116 in both cell lines. Western blot results demonstrated that, in HCT-116 cells, Hedyotis diffusa activated AMPK pathway (AMPK/ACC phosphorylations). Activation of mTORC1, detected by phosphorylation of S6K1 (Thr-389), and expressions of mTORC1-dependent genes, including Bcl-2 and HIF-la, were significantly downregulated by the co-administration. Inhibition of AMPKa1 activation, either by genetic silencing (shRNA knockdown) or by introducing a dominant negative (DN) mutation, restored S6K1 phosphorylation and Bcl-2/HIF-la expressions in co-stimulated cells. Importantly, Hedyotis diffusa-induced viability decease, cell apoptosis and cell necrosis were all inhibited by AMPK silencing or mutation in HCT-116 cells. On the other hand, AMPKal activator AICAR mimicked the actions on signalings by the co-administration, and promoted cancer HCT-116 cell apoptosis and necrosis. Thus, Hedyotis diffusa induced a profound AMPKa1 activation, causing mTORC1 in-activation, Bcl-2/HIF-la downregulation as well as cell apoptosis and necrosis. Inhibition of AMPKa1 by its inhibitor compound C or by shRNA-mediated silencing prevented subsequent cell death, indicating that AMPKa1 activation was required for cytotoxicity by Hedyotis diffusa. Further, we discovered that Hedyotis diffusa inhibited platelet-derived growth factor receptor (PDGFR)/epidermal growth factor (EGFR) and FSCN1 expressions and mTORCl activation in above cancer cells (HT-29 cells), which appeared also dependent on AMPKa1 activation. Inhibition of AMPK restored mTOR activation and PDGFR/EGFR/FSCN1 expressions in Hedyotis diffusa-treated cancer cells.Conclusion:Hedyotis diffusa significantly inhibited survival and elevated apoptosis of colon cancer cell lines (HCT-116、Lovo、HT-29 and DLD-1 cell). These results suggested that AMPKal and p53 activation by Hedyotis diffusa were required for growth factor receptors (PDGFR/EGFR/FSCN1) degradation.