Research On Signaling Transduction Mechanism Of Grifolin, A Low Molecular Probe Originated From Higher Fungi

Higher fungi belong to biological source of higher "creative index". In recent years, some low molecular weight secondary metabolite products isolated from fruiting body, mycelin and medium of higher fungi exhibit properties of anti-tumor lead compounds. The secondary metabolites of fungi possess novel structures, higher specificity and species-dependence after evolutionary pre-selection. It makes these bioactive substances good material and irreplaceable for biomedicine and natural innovative drug development. The unique chemical-framework and action mode of bioactive secondary metabolites enable them to gradually become ideal chemicals in R&D of natural antitumor drugs.Chemical low molecular probe is designated as chemicals that can highly selectively detect the structure, function of protein and the action mode. Its molecular weight is usually less than 700 Dalton. Chemical low-molecular probe may get involved in signal transduction, interfere, change or mimick the signal transduction progress and act as effective tool to elaborate the known biological signal transduction mechanism and explore the unknown transduction procedure.In previous study, for the first time in the world we screened out a novel bioactive low molecular probe, grifolin, from extractions of the mushroom Albatrellus confluens. Grifolin is a Farnesyl phenolic compound, the molecular formula of which is C22H32O2, and the molecular weight is 328. It has been shown to inhibit the growth of some cancer cell lines, induce cell cycle arrest and significant apoptosis. However, the molecular signaling mechanism underlying the anticancer effect of this compound is not completely understood yet. To investigate the signal transduction mechanism of biological function of grifolin, the research is conducted from below three parts.High-throughout scanning of apoptosis-related gene expression array provided a clue that the death-associated protein kinase 1 (DAPK1) was significantly up-regulated after grifolin treatment in CNE1 cells. DAPK1, as a stress-activated tumor suppressor protein, plays an important role in pro-apoptotic signal transduction pathways. Growing evidence of clinical sample detections and biological experiments suggest that loss of expression of DAPK1 might be an important initiator in tumorigenesis and development, which promotes us to conduct deeper research on molecular mechanism of DAPK1 protein expression regulated by grifolin in tumor cells.We took human nasopharyngeal carcinoma cell CNE1 as a model, and observed that protein as well as mRNA level of DAPK1 was up-regulated by grifolin in a dose-dependent manner. It was further proved in other tumor cell lines. Based on it, we revealed the molecular mechanism of DAPK1 expression induced by grifolin via means of siRNA, over-expression, EMSA and ChIP assays.By Western blot detection, we found that grifolin increased Ser392 and Ser20 phosphorylation level of transcription factor p53 protein, which could promote its transcription activity. While Ser15 and Thr81 phosphorylation levels of p53 had no detectable change after grifolin treatment. It suggests that grifolin may activate the transcription activity of p53 upon phosphorylation.Bioinformatics analysis manifests the promoter region of dapkl includes p53 binding site. It effectively depleted the expression of p53 that introducing p53 siRNA to CNE1 for 72 hours. Meanwhile, DAPK1 protein expression was down-regulated significantly. Vice versa, introducing p53 expression vector pEGFP-C3-p53 to H1299, a p53-/-cell line, p53 protein was re-expressed and DAPK1 was up-regulated as well. Therefore, the expression of DAPK1 protein was identified to be positive proportional to p53 protein expression. To evaluate whether grifolin promotes p53 binding to dapkl promoter region to up-regulate DAPK1 expression, we used EMS A and ChIP assays to detect in vitro and in vivo, respectively. We observed that nuclear protein treated with grifolin binding to dapkl DNA manifested higher than untreated CNE1 cells. Supershift-EMSA assay further demonstrated the transcript factor binding to dapkl DNA was p53 protein. The specificity of recruitment of p53 to dapkl promoter region was confirmed by ChIP assay. We found that compared with the untreated group,the recruitment of p53 to dapkl gene promoter increased after being treated with 30μM grifolin for 24 h.The results above indicate that grifolin up-regulate the expression of DAPK1 via targeting p53-DAPK1 pathway and provide sound basis for revealing the molecular mechanism of apoptosis in tumor cells induced by grifolin.On the basis of demonstrating that dapkl gene expression was upregulated by low molecular probe grifolin, we further investigated how it represents pro-apopotic effect by targeting DAPK1. Currently there are two known proteins interacting with death domain of DAPK1, including ERK kinase. It suggests that grifolin may promote the interaction of DAPK1 and ERK 1/2 to activate DAPK1 and inhibit the nuclear translocation of ERK 1/2, thus inducing tumor apoptosis.By means of LCFM, separation of cytoplasmic and nuclear protein and Western blot, we observed the total protein level of ERK 1/2 did not change in CNE1 when treated with grifolin. Grifolin induced the cytoplasmic arrest of ERK 1/2, meantime, it restricted the nuclear translocation of ERK 1/2 in a dose-dependent manner. LCFM, Co-IP and reverse IP assays were used to determine that grifolin enhanced the interaction of DAPK1 and ERK 1/2 proteins. From the above experiments, we deduce that grifolin induces apoptosis by promoting the interaction of DAPK1 and ERK1/2 to block the nuclear accumulation of ERK 1/2.To evaluate whether grifolin is involved in apoptotic effect induced by grifolin, DAPK1 siRNA was introduced into CNE1 cell. The results showed the suppression of DAPK1 expression effectively reversed the activation of Caspase-3,8,9 and apoptotic effect induced by grifolin.The data above indicate that grifolin, a low molecular probe, exhibits biological activity of inducing apoptosis in tumor cells via promoting the interaction of ERK1/2 and DAPK1 proteins to inhibit the nuclear location of ERK1/2.In our previous studies, we found grifolin had some selectivity for tumorous and non-tumorous cells and also relatively specifically targeted MAPK signal transduction pathway, moreover, down-regulated the phosphorylation level of ERK1/2 kinase in a dose-dependent manner. The above important findings suggest that grifolin has significant inhibitive effect on signal moleculars of MAPK pathway, which is frequently activated aberrantly in tumors.In order to estimate whether grifolin regulate the ERK1/2 pathway, we detected the phosphorylation level of ERK1/2 and its kinase activity in human Burkitts Lymphoma cell line Raji, human breast cancer cell line MCF7, human cervical cancer cell line HeLa and nasopharyngeal carcinoma cell CNE1, respectively. In Raji, MCF7 and CNE1 cells, grifolin significantly inhibited the phosphorylation level of ERK1/2 as well as its kinase activity, while had not much influence on ERK1/2 protein expression. In Hela, the phosphorylation level of ERK1/2 and its kinase activity as well as the total ERK1/2 protein were down-regulated by grifolin. In vitro kinase assay confirmed that grifolin can effectively down-regulate ERK2 kinase activity. Using grifolin-sepharose 4B pull down and fluorescence quench of ERK2 kinase assays, we demonstrated that grifolin interfered the ERK1/2 kinase activity via binding to it. When Ile 31, Val 39 and Leu 156 amino acids in ATP binding cavity of ERK2 protein are mutated simultaneously, grifolin can not bind to the ERK2 mutant, thus demonstrating that grifolin binds to the ATP cavity of ERK1/2 and inhibit the ERK2 kinase activity in ATP-competitive manner.In previous study, we observed that grifolin induced G0/G1 arrest in tumor cells. In view of cell cycle regulation, it is beneficial to clarify the molecular basis of cell proliferation, and define specific target proteins related to chemical low molecular probe. In vivo experiment demonstrated p21 protein is direct substrate of DAPK family member ZIP kinase. It is known that DAPK family members possess high-conservative catalytic domain. ERK and mitogen-activated protein kinase (MAPK) signaling cascades, have been shown to regulate DAPK1 activity. Taken together, it suggests that grifolin may induce G0/G1 arrest via targeting ERK1/2-DAPK1-p21 signaling pathway to activate p21.In this study, firstly we identified that grifolin effectively induced the dephosphorylation at Ser 308 of DAPK1 kinase to activate it. Secondly, we used Western blot and IP analysis to verify grifolin upregulated the phosphorylation level of p21 in a dose-dependent manner and activated it. Finally, the role of DAPK1 involved in G0/G1 arrest induced by grifolin was confirmed by introduing siRNA targeting DAPK1.We demonstrated from above results that grifolin effectively inhibit the kinase activity of ERK1/2 in ATP-competitive manner and further induce G0/G1 arrest in tumor cells via ERK1/2-DAPK1 - p21 pathway.In summary, we demonstrate that grifolin is an important chemical probe possessing the ability to induce apoptosis and cell cycle arrest in tumor cells. On one hand, it activates p53 to up-regulate the expression of DAPK1 and enhance the interaction of ERK1/2 and DAPK1 proteins to inhibit the nuclear accumulation of ERK1/2 and induce apoptosis in tumor cells. On the other hand, it activate p21 to induce G0/G1 arrest in tumor cells via targeting ERK1/2-DAPK1-p21. The purpose of this research is to reveal the new components of signal transduction networks related to cell apoptosis and cell cycle arrest, and further clarify the interaction between low molecular chemicals from higher fungi and multi-protein of signaling pathway. It will explore the knowledge of biological active essence of low chemical probe. Meanwhile, it brings out new evidence for grifolin as kinase inhibitor candidate.