| Glioma is the most common malignant tumors of the nervous system. It is with high degree of malignancy, easy to relapse and spread locally. Currently, the main strategy of glioma treatment is still surgery. However, it locates in central nervous system, which makes it difficult to clear all the glioma cells. Thus, it is necessary to add other therapy methods such as radiation therapy, chemotherapy, immune therapy. The methods commonly used have achieved certain results for the treatment of glioma. However, due to some adverse reactions and drug resistance, it is necessary to seek drugs which are effective and with little toxicity. As a chemical plant, myricetin is widely found in plants. So, it is cheap and readily available. Meanwhile, it has been confirmed with multiple biological functions, such as anti-tumor, anti-oxidation, anti-inflammatory and so on. In recent years, research about biological function of myricetin mainly focused on its anti-tumor function. It has achieved some therapeutic effect on the treatment of non-small cell lung cancer, skin cancer, esophageal cancer and other tumors. Therefore, our study will apply myricetin to the treatment of glioma cells and explore its therapeutic effect on glioma.Our study mainly explores the inhibition effect of myricetin on glioma cells in vitro. The cells used in the experiments are mouse GL261 cell and human U251 cell. We conducted the study mainly from three aspects, namely, the effect of myricetin on the proliferation, apoptosis, migration and invasion of GL261 cell and U251 cell, the angiogenesis of HUVEC. On this basis, we aim to explore the therapeutic effect of myricetin on glioma. 1 The impact of myricetin on the proliferation and apoptosis of glioma cells 1.1 Myricetin inhibited the survival of glioma cellsWe detected the impact of myricetin on the survival of GL261 cells and U251 cell by CCK-8 assay. The result showed that myricetin can inhibit the survival of GL261 cells at a concentration of 20μM. Compared with the control group, the difference was significant(p<0.01). And, the inhibition is more obvious with the increase of myricetin concentration. What’s more, the inhibitory effect of 48 h showed stronger than 24 h. Myricetin could inhibited the survival of U251 cells at the concentration of 240μM(p<0.05). As the increase of myricetin concentration, the inhibitory effect was more obvious. And, the increase of time could also improve inhibitory effect. These assays proved that the inhibitory effect of myricetin is dose and time dependent. 1.2 Myricetin inhibited the proliferation of glioma cellsWe detected the influence of myricetin on both cells’ cycle by flow cytometry. The result showed that the proportion of cells which were in G1 phase increased when treated by myricetin. It proved that myricetin could arrest the cell cycle in G1 phase. Furthermore, we detected the cell cycle proteins by Western-blot assay. It showed that myricetin could inhibit the expression of Cycline D1 of U251 cells. These results suggested that myricetin could inhibit the proliferation of glioma cells. 1.3 Apoptosis of glioma cells induced by myricetinWe observed the apoptosis induced on myricetin GL261 cells and U251 cells by Hoechst 33342 staining. By fluorescence microscopy, we could found the proportion of cell apoptotic bodies which were induced by myricetin increased. Next, we examined the apoptosis effect of myricetin on U251 cells and GL261 cells by flow cytometry. The result showed that the percentage of early apoptotic and late apoptotic cells increased when treated by myricetin in both cells. And compared with the control group, the difference was of significance(p<0.05), which proved that myricetin could induce apoptosis of glioma cells. 2 Effect of myricetin on migration and invasion of glioma cells 2.1 Effect of myricetin on the migration of GL261 cells and U251 cellWe detected the effect of myricetin on the migration of GL261 cell by Transwell assay. The result showed that myricetin exhibited inhibitory effect at a concentration of 5μM on the migration of GL261 cells. Compared with the control group, the difference is significant(p<0.05). When the concentration of myricetin increased, the inhibition effect is more obvious. Transwell assay was also used to detected the effect of myricetin on the migration of U251 cells, and the result showed that both 100μM and 200μM concentration of myricetin exhibited inhibition effect on the migration of U251 cells compared with the parallel control(p<0.001). 2.2 Effect of myricetin on the invasion of GL261 cells and U251 cellsIn order to study the effect of myricetin on the invasion of glioma cells, we covered the bottom of the Transwell chamber with matrigel to mimic the extracellular matrix. The result showed that myricetin could reduce the number of GL261 cells and U251 cells which went through the chamber. Compared with the parallel control, the difference of cell number was statistically significant(p<0.05). RT-PCR was used to detect the level of MMPs m RNA in glioma cells which were treated by myricetin. The result showed that myricetin could reduce the m RNA levels of MMP-9 and MMP-2 in GL261 cells, and it could also reduce the m RNA levels of MMP-2, MMP-9 and MMP-14 in U251 cells. There were significant differences(p<0.05) compared with the corresponding control groups. 3 Effect of myricetin on HUVEC angiogenesisThe CCK-8 kit was used to detect the proliferation of HUVEC cell which was treated by myricetin. Through the wound-healing assay and tubule formation assay we detected the effect of myricetin on the ability of HUVEC angiogenesis induced by U251 cells. The result showed that myricetin could inhibit the proliferation of HUVEC cells in a dose and time dependent. Wound-healing assay showed that myricetin inhibited the migration of HUVEC cells, and compared with the control group, the inhibition was significant(p<0.001). In the tubule formation assay, we could find that myricetin could significantly reduce the length and formation of tubules induced by U251 cells. There was significant difference(p<0.05) compared with the corresponding control groups.Through these experiments, we have concluded that myricetin could inhibit the proliferation of GL261 cells and U251 cells and induce their apoptosis. Meanwhile, it could also inhibit the migration and invasion of these two kinds of cells. What’s more, myricetin may act on vascular endothelial cells and inhibit angiogenesis of HUVEC induced by U251 cells. |
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