Cancer chemopreventive effects of tussilagonone, igalan and chikusetsusaponin IVa methyl ester and their molecular mechanisms

Abstract

Cancer is widespread and growing worldwide. To combat it, people must avoid smoking and drinking alcohol, two major causes of cancer, and instead cultivate healthy lifestyle habits. In addition, the study of assistive drugs and foods that can supplement with cancer prevention and active cancer treatment must continue apace. Cancer is not onset suddenly. Its prognosis and symptoms appear at the cellular level, step by step, divided into four stages. An initiation step of changing from normal cells to initiated cells

a promotion step to preneoplastic cells

and a progression step of forming into neoplastic cells. These processes are referred to as carcinogenesis. Cancer chemopreventive activity, then, inhibits the development of cancer with suppressing and delaying the steps of carcinogenesis, and represses the progress of cancer. In this study, new natural substances that have the potential for chemopreventive activities and related mechanisms have been identified. In order to prevent the formation of cancer, tussilagonone, which is a compound isolated from Tussilago farfara L., and igalan, isolated from Inula helenium L., were identified by screening for natural products that can act in the initiation stage. The quinone reductase (QR) assay which measures chemopreventive activity through detoxification activity was used for screening. Tussilagonone showed greater than two-fold activity at a concentration of 2.5 μM, and igalan also started to show a two-fold activity at a concentration of 5 μM. Based on these assays, relevant molecular experiments were performed. And based on the results of the experiments, the Nrf2 activity was induced in HepG2 cells using two compounds, and an increase of the expression level of NQO1 and HO-1, target genes of Nrf2, was observed at the protein level. In order to investigate molecular mechanisms, a reporter assay and an oligo pull down assay were performed. The increase of binding to the ARE (Antioxidant Response Elements), on the promoter of the target genes, of Nrf2 was confirmed to induce the expression of target genes. To investigate which kinases are involved in the Nrf2 activation under each compound, various kinase inhibitors were used. Tussilagonone activated MEK and ERK1/2, and igalan inactivates GSK3β while also activating AKT by phosphorylation. In addition to detoxification, when strong reactive oxygen species was induced by t-BHP, ROS production was suppressed and cell death was reduced by tussilagonone. These results suggest that tussilagonone induces the activity of Nrf2 to detoxifying and antioxidative effects. Igalan has been shown to decrease the activity of NF-κB, a major marker of immune function, in addition to detoxify. Under exposure to TNFα, which induced an immune response, the inflammatory proteins of cells exposed to igalan was significantly lower than that of the control. ROS, xenobiotics, and immune responses are known to play a role in the development of cancer in the initial stage of normal cell transformation into initiated cells. Tussilagonone and igalan inhibit cancer development by suppressing carcinogenic activities. Next, the inhibitory effects of chikusetsusaponin IVa methyl ester (CME) on cell growth was evaluated by MTT assay. G0/G1 arrest was induced by this compound in colorectal cancer cell lines as a result of FACS analysis. In order to investigate molecular mechanisms to induce cell cycle arrest by CME, western blot analysis and other molecular approaches were performed. It is showed that Cyclin D1 expression, which is a representative target genes of Wnt/β-catenin signaling, was markedly decreased and reporter assays were performed using TOP flash. As a result, it was confirmed that the activity of β-catenin is inhibited. The inhibition of β-catenin nuclear translocation was confirmed by oligo pull down assay and protein expression in the nucleus. Cyclin D1, c-Myc and other CDK proteins, which promote cell division, are also decreased. It was confirmed that CME could inhibit the progression of cancer by inhibiting the nuclear translocation of β-catenin to promote cell proliferation. Taken together, it has been identified that novel functions of tussilagonone, igalan, and chikusetsusaponin IVa methyl ester, all of which are from natural products and have been shown to inhibit cancer development and progression. It means that those natural occurring compounds have chemopreventive activities and can be used to inhibit cancer development and progression.