Dihydroartemisinin Inhibits Proliferation And Induces Apoptosis Of U87Cells

Background Gliomas are the most common malignant tumor of all central nerve systerm, accounting for40%-60%of all brain tumors. Because of the biological characteristic of infiltrative growth, glioblastoma is the most aggressive glioma which difficult to eradicate by surgical therapy separated. With the high rate of recurrence, the averaged survival time of patients is only14months. Currently, the comprehensive treatment for glioblastoma includes neurosurgical techniques, radiation and chemotherapy. In recent decades, surgical techniques about neuroimaging and microsurgical technique have great development, but it is hard to completely remove lesions by neurosurgical treatment. Althought the therapeutic effect of patients with gliomas can be improved by radiotherapy after operation, most of the tumors are recrudescent because of the limited radiation dose and radiation insensitive in partial brain tumor. In addition, owing to drug resistance and low penetrability of blood brain barrier (BBB), the effect of chemotherapeutic drugs is also dissatisfactory.It is importance for oncogenesis, proliferation of cancer cell, metastasis of tumor and apoptosis, that genes and/or proteins altered in different signal pathways during gliomas generating process, such as the overexpression and amplification of epidermal growth factor receptor genes (EGFR/ErbB1), up-regulation of platelet-derived growth factor recetor (PDGFR), lose of INK4A-AR, p53genetic drop or Mutation and so on. Targeted therapies based on these are finding their way into clinical practice. Currently, it still is the hot spot that researches of chemotherapeutic drugs improve the survival rate of patients with gliomas.Artesunate is sesquiterpene lactones peroxides extracting from sweet wormwood Artemisia by scientists of China. Its derivants include dihydroartemisinin (DHA), artemether, and artesunate and so on. Artesunate and its derivants have been generally accepted by the world as high efficacy and low cytotoxicity. In the rcent decade, relevant researches indicated that Artesunate and its derivants can induce many kinds of intracorporal and extracorporal cells apptosis, especially tumor cells. So they were supposed to possess a broad spectrum of anticancer activities. Scientists research the anticancer mechanisms from different aspects, such as their directly cytotoxicity, the influence of proliferation cycle of tumor cells, promotment of apptosis and antiogenesis.(1) ROS mediate the toxic effect. The antimalarial mechanisms of Artesunate is that the peroxide bridge structure of sesquiterpene lactones in Artesunate react with Ferrous Ion. Then the peroxide bridge structure break and produce ROS, which can induce cells apptosis. Previous studies showed that the ion absorption of cells related to the numbers of transferrin receptors (STfR). Because of the DNA replication requires large quantities of ferrous ion during tumor cells division, the content of ferrous ion in tumor cells was far more than in normal cells. Therefor, the effect of Artesunate to tumor cells greater in contrast with to nomal cells, due to the hight ferrous ion concentration. This suggested that Artesunate increase the number of ROS in tumor cells and produce toxic effect for tumor cells, which maybe one of the antineoplastic mechanisms of Artesunate and its derivants. (2) Artesunate and its derivants can block the cell cycle process.The cell cycle, or cell-division cycle is a series of events that take place in a cell leading to its division and duplication (replication). It includes the time of cell division and growth, which is divided into five phases. Stationary phase, called "GO"phase, is the origin of cell division. The second phase is termed "Gl "phase. The next phase, called "S"phase, is DNA biosynthesis. The fourth phase is termed "G2"phase. The last phase is called "M"phase, in which one cell divides into two cells. There are two key check points in cell cycle, distributing at G2/M and G1/S respectively. Many experiments demonstrated that Artesunate and its derivants blocked the cell cycle, and the phases of some tumor cells in different organs distinctly. It maybe the selective of Artesunate and its derivants, but the reason is still unknown.(3) Artesunate and its derivants induce toumr cells apoptosis. Apoptosis, a kind of cell death, causes specific morphological changes and DNA fragmentation that are considered as the major cytopathologic hallmarks of the apoptotic process. There are so many apoptosis pathways.Two major apoptosis pathways have been identified: the death receptor pathway and the mitochondrial pathway. There are many different views about the apoptosis mechanisms of Artesunate and its derivants. For example, one study useing HL60and A549cell as models suggested that the mechanism of artesunate-induce apoptosis related to inhibiting the expression of survivin, as a member of IAPs family, with decrease of surviving mRNA and survivin protein. Another reseach, used hepatocarcinoma BEL-7402as a model, showed that the levels of expression of p53and p21protein had no change and bcl-2protein decreased. They concluded that the mechanism was p53-independent and related to down regulation of bcl-2. DHA induces Hela cells apoptosis by p53-independent in mitochondrial pathway. However, ART induces apoptosis by p53-independent and p53-dependent pathways in colon cancer cell line. In addition, it is considered that ROS result in cell death by activation of intracellular peroxide bridge. It maybe a mechanism that ART influence that topoisomerase (TOPO) mediate apoptosis in hepatocarcinoma. The other point of view is that increase of intracellular calcium ion result in blocking the signal transduction pathway which is mediated by CAMP. Scholars in China discover that DHA induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1cells, accompany with monosomic morphological changes and activation of caspase-3. Currently, it is unknown the mechanisms that Artesunate and its derivants induce toumr cells apoptosis.(4)Artesunate and its derivants can inhibt angiogenesis which is the main pathologic characteristics of tumor and an important foundation of turmor growth and metastasis. The exeriments showed thatangiogenesis is closely concerned with a wide variety of tumor formation, transfer, recurrence and prognosis. Angiogenesis is a complicated process which include several steps:proliferation and migration of vascular endothelial cell, formation of blood vessels solid cords, collagen deposition of blood vessel wall, formation of peripheral cells corresponding with these steps, we experimented that Artesunate and its derivants inhibted new vessel growth in gliomas. The experments showed that ART delayed the formation and reduced the number of new vessals. These suggested that ART inhibited the initial steps of new vessal growth, which related to the proliferation and migration of vascular endothelial cell. Another reseach show that ART prevented VEGF combination with receptor and down regulate the expression of VEGF and its receptor KDR flk-1on vascular endothelial cell. It means that ART has underlying value for antiangiogenesis, which maybe important for Artesunate and its derivants as antitumor drug.In addition, the toxic effects of ART and its derivants are not yet fully elucidated. There are more than ten thousands people who accepted the clinical test, which used ART as antimalarials, and were watchful waiting, but there were not any clinical evidences to prove the toxic effects of ART. The toxic effect take placed due to hight-does in animal testing. Study showsed that the toxic effect of ART and its derivants include several aspects, such as inhibition of function of hematopoietic system, Embryo Toxicity, neurotoxicity and so on. Neurotoxicity mainly occur on neurous rather than gliocyte. Otherwise, the toxic effect of ART impacted on various parts of brain is different. For example, it is the most sensitive to the toxic effect of artemether that are ventral nucleus trapezoid body located low brain stem, gigantocellular reticular nucleus and neurons of inferior cerebellar peduncle. Conversely, cerebral cortex neurons were influence slightly. The toxic effect is in dose-and time-dependent manner in vitro study. In addition, one study found that the toxic of ART by intramuscular injection was more than by oral, and the liposoluble one more toxic than the water-soluble one, which difference do not mean there are essential difference on drugaction among the derivants. As mentioned, one of the mechanisms displayed that ART has selectivity on tumor cells, and has mild effect on normal organs. In conclusion, we considered that ART and itsderivants are low toxic and no toxic effective in routine does.Therefore, in the present study we investigated the effects and the mechanisms of DHA on proliferation and Apoptosis of U87cells,Objective To investigate the effect of dihydroartemisinin(DHA) on apoptosis and proliferation of U87cells.Methods U87cells was cultured with DHA(10μmol/L、25μmol/L、50μmol/L、100μmol/L、125μmol/L) for24h,48h and72h respectively. Cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated U87cells. The induction of apoptosis was detected by Hoechst33258. Effect of DHA on cell cycle of U87cells was detected by flow cytometry analysis. Caspase-3activities measured with caspase-3Activity Assay Kit and ROS measured with Reactive Oxygen Species Assay Kit. The effect of U87cell proliferation by DHA was assessse with single factor analysis of variance (Univaraiate-ANOVA), in differet concentrations or time groups. If multiple comparisons are homogeneity of variance, we emloyed LSDmethod; on the contray, we used Dunnett’s T3method. Two independent sample t-test was used in statistical analysis of caspase-3protein expression in both groups.Results Our results indicated that DHA induced apoptotic cell death in a dose-and time-dependent manner, which was accompanied by the activation of caspase-3and ROS increased. The IC50at24h was97.04μmol/L,48h was59.76μmol/L and72h was33.20μmol/L respectively.Discussion This study showed that DHA can observably inhibits the proliferation of U87cells. With the increase of concentration of time of DHA treatment, the effect was enhanced. We found that U87cells were blocked in G0/G1phase and the number of them reduced observably. This finding illustrated that one of the mechanisms maybe DHA block tumor cell in G0/G1phase. So we considered that contral cell regulation is one of the mechanisms of the DHA inhibit the proliferation of U87cells.Apoptosis is the procedure of programmed cell death by multiple genes regulation, and relate with the activation of a series of intracelluar proteolytic enzymes. Caspase is an important protein family for the procedure of apoptosis. Activation of caspase is the crucial steps of apoptosis in which caspase-3is a key effector caspases of cell apoptosis cascade. This reseach illustrated that DHA-induce apoptosis is concerned with activation of caspase pathway, and DHA induce U87cell apoptosis by caspase-3pathway.In addition, our experiment founds that ROS conten in U87cells, which were cultured for24hours with DHA, incrasing obiously than that without DHA. It may be one of the anti-tumor mechanisms of DHA. Because of tumor cells are characterized by dividing rapidly in contrast with normal cells, and require large quantities of ferrous ion to replicate DNA during the phase of dividing rapidly, the conten of ferrous ion in tumor cell is more than that in normal cells. There is a peroxide bridge structure in ART and its derivants, which react with ferrous ion and produce ROS induceing cells apptosis. There are many TfR on the surfaces of tumor cells, so ART and its derivants have selective toxic effect on tumor cells.Conclusions The results showed that dihydroartemisinin exerts cytotoxic effect on U87cells by altering the cell cycle,activating caspase-3activities and increasing the ROS.