Molecular Mechanism Study Of Recombinant Ganoderma Lucidum Immunoregulatory Protein Induced Apoptosis In HL60 Cells

The HL60 (Human promyelocytic leukemia cells) is a leukemic cell line. Leukemia, like other cancer, results from somatic mutations in the DNA. The mutations may activate oncogenes or deactivate tumor suppressor genes, which disrupt the regulation of cell death and differentiation. Only if the causes of leukemia are found, it would be a way to prevent the disease. Nowadays, many chemotherapeutics are effective in the treatment of leukemia,but more serious side effects come out in consequence. However, significant research into treatment of leukemia is being done.LZ-8,one of the fungal immunomodulatory proteins (Fips),was purified from the mycelium of Ganodermn lucidum,which have a wide range of pharmacological actions and immunomodulatory activities. In terms of anti-tumor activity, LZ-8 acts as a mitogen for mouse spleen cells, human peripheral lymphocytes and human peripheral blood mononuclear cells (hPBMCs), induces the production of cytokines and tumor necrosis factor (TNF) by activated macrophages and T lymphocytes. These are from the perspective of immunology to explore the indirect anti-tumor activity of LZ-8. In recent years, with the development of genetic engineering drugs, many anti-cancer drugs play a biological role through the apoptotic cell death. Our team have finished expressing recombinant LZ-8 (rLZ-8) and solved its crystal structure, most important among these work is that LZ-8 may be developed as an antitumor agent.Apoptosis is a regulatory mechanism for cleaning of unwanted cells, playing a crucial role in tumorigenesis and homeostasis. Apoptosis triggered by some stimuli, is characterized by a series of distinct morphological changes, including cell shrinkage, chromatin condensation and nucleosomal degradation. In addition, several signaling molecules are also important to apoptotic progress, and they need to be detected and analyzed by bioinformatics tools. At present, GeneChip testing technology has significant value in finding the causes and mechanism of the disease. Both of the changes of cell morphology and molecular action are regulated by gene expression. DNA microarray technology gives insights into the expression profiles of many thousands of genes. It is possible to map gene expression date into relevant pathway maps based on their functional annotation and known molecular interaction, thanks to the technologies for pathway analysis.In our previous study, we have finished expressing recombinant LZ-8 (rLZ-8) and solved its crystal structure.rLZ-8 was purified from fermentation. Purification was carried out by ultrafiltration, anion exchange chromatography (AIEC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC). Most important among these work is that LZ-8 may be developed as an antitumor agent. What we also found is that rLZ-8 could induce NB4 cells apoptosis, whose mechanism is still unknown. In the present study, we examined the effect of rLZ-8 on cellular proliferation in HL60 cells and the apoptosis rate by annxinV/PI assay, also investigated the relation between subcellular localization of rLZ-8 and its anti-tumor effect. Furthermore, we attempted to clarify how cell signaling pathways were regulated by rLZ-8 in the process of apoptotic cell death in HL60 cell.LZ-8, a fungal immunomodulatory protein found in Ganoderma lucidum, has been proposed to possess therapeutic effects on cancer and autoimmune diseases. However, few details are known about mechanism and process of cell death induced by LZ-8 in tumor cells. The HL-60 (Human promyelocytic leukemia cells) cell line is a leukemic cell line that has been used for anti-tumor research in laboratory, LZ-8 has shown promising results for some of types of tumor cell lines, Hl-60 cells remarkable especially. Under transmission electron microscope and flow cytometry, HL60 cells treated with LZ-8 exhibited several typical features of apoptotic cell death, these findings suggested that apoptosis may be the most important form of HL60 cell death induced by LZ-8. In the following researches, rLZ-8 accumulated on the nuclear, accumulating of rLZ-8 may trigger a stress to the nuclear for causing cell death. Using Fluo-3 -acetoxymethyl ester (AM) fluorescence imaging techniques, it was shown that rLZ-8 significantly elevated the intracellular calcium level ([Ca2+]i) in HL60 cells. Moreover, the nuclear may be an important site activated in the progress of calcium ion releasing. Moreover, in HL-60 cells treated with rLZ-8, a series of genes on MAPK signal pathway had expressed differently, which seemed to suggest MAPK signal pathway may play an important role in the process of cell death induced by rLZ-8.Comprehensive analysis of microarray, subcellular localization, intracellular calciumand mitochondrial pathway. mechanism of rLZ-8 induce HL60 cells apoptosis is the result of the interaction of different elements, which are closely linked. The mitochondrial pathway play a major role. Mitochondrial membrane potential, Cyt-c release and increased expression, activation of caspase-9 and caspase-3, then HL60 cells leading to apoptosis. Subcellular localization of rLZ-8 was in the nucleus.It maybe could trigger a nuclear calcium release and nuclear translocation factor TR3 expression. Nuclear calcium in apoptosis in the nuclear membrane and then on the closely related Bcl-2 family. Bcl-2/Bax expression decreased, with the trend to promote apoptosis, its role in mitochondrial outer membrane, can control the Cyt-c release in the regulation of apoptosis. The results show that p38 MAPK gene chip pathway also play a role.Therefore, the mechanism of rLZ-8 induced HL60 cells apoptosis may be the results by mitochondrial pathway and the MAPK pathway interaction.