The Effects And Mechanisms Of RIP-1on Shikonin Induced Necroptosis In Glioma Cells

Background：Glioma is the most common malignant primary brain tumors, accounting for35.26%~60.96%of primary intracranial tumors. Although surgery, radiation,chemical drugs, biological and other methods can be used for clinical treatment, it isvery difficult to eliminate malignant glioma cells, because surgical operation can notremove them out radically for their invasive growth and they are resistant topostoperative radiotherapy and chemotherapy. Median survival for patients treatedwith all the treatments above was14-16months, and recent studies show thatresistance to apoptosis is the major factor that makes malignant glioma cells tolerateclinical used medicines or radiotherapy. Thus, it is needed to find new medicines thatcould induce glioma cells death not via apoptosis pathway.Accumulating evidences have demonstrated that shikonin could induce apoptosisin various types of tumor cell lines such as breast cancer, hepatocellular carcinomaand glioma. However, shikonin has been found to cause necroptosis in leukemia celllines, which can circumvent cancer drug resistance, thus, whether shikonin couldinduce necroptosis in glioma cells is still needed to be examined as well. Clarifyingthis issue would help us to understand the mechanism underlying the anti-gliomaeffects of shikonin as well as provide powerful experimental and theoretical guidancefor overcoming tumor drug resistance clinically.Objectives:In this study, we use rat C6glioma cells and Human U87glioma cells toinvestigate whether shikonin could induce necroptosis in glioma cells as well as TheEffects and Mechanisms of RIP-1on Shikonin induced Necroptosis in Glioma Cells.Methods:1. MTT assay is used to detect the death of glioma cells induced by shikonin andto explore the role of Nec-1、z-VAD-fmk and NAC in the cell death. 2. Flow cytometry is used to further determine the death models of glioma cellsinduced by shikonin.3. Morphological changes in C6glioma cells induced by shikonin is observedthrough TEM (Transmission Electron Microscopy).4. Hoechst33342and PI staining are used to confirm the morphological changesin C6glioma cells induced by shikonin.5.The level of reactive oxygen species(ROS) was assessed by usingredox-sensitive dye DCFH-DA.6. The expressional level of Necroptosis associated protein RIP-1was analyzedby western blot.Results:1、MTT assay has proved that: Shikonin induced cell death in C6and U87glioma cells in a dose and time dependent manner. The cell death in C6and U87glioma cells could be inhibited by Necroptosis inhibitor necrotatin-1(Nec-1), not bypan-caspase inhibitor z-VAD-fmk, and anti-oxidant NAC can inhibit the cell deathsignificantly.2、Flow cytometry with Annexin V and PI double staining has showed that: after3hours incubation with different concentration of shikonin, the stage of AnnexinV-/PI+and Annexin V+/PI+rates of glioma cells increased respectively in a dose andtime dependent manner, which could be inhibited by Necroptosis inhibitor Nec-1, notby pan-caspase inhibitor z-VAD-fmk.3、Under transmission electron microscope, normal C6glioma cells displayedmicrovilli protruding from the entire surface, a smoothly outlined nucleus withchromatin in the form of heterochromatin and well-preserved cytoplasmic organelles.By contrast, shikonin-treated C6glioma cells presented electron-lucent cytoplasm,loss of plasma membrane integrity and intact nuclear membrane.4、The fluorescent microscope was used to observe the cells after3hoursincubation with different concentration of shikonin. Compared with normal C6gliomacells showing round nuclei with dark blue color, part of the C6cells treated byshikonin had nuclei in normal size but stained with dark blue and red color. Anotherpart of shikonin treated C6cells presented shrunken nuclei in light blue color, withbeing stained with red color. However, pretreatment with necroptosis inhibitor Nec-1 made significant reduction in the cells with nuclei in normal size and being stainedwith dark blue and red color, but the cells in light blue and red color remained.5、Redox-sensitive dye DCFH-DA staining showed that compared with controlgroup, the ROS level increased significantly when glioma cells incubated3h withshikonin, However, the increased ROS level caused by shikonin was attenuated byNec-1and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death inboth C6and U87glioma cells.6、Western blot results showed that：The expressional level of RIP-1wasup-regulated by shikonin in a dose and time dependent manner as well, moreover notonly Nec-1but NAC could suppressed RIP-1expression.Conclusions：1. The cell death caused by shikonin in C6and U87glioma cells was mainly viaNecroptosis.2. Shikonin killed glioma cells through Necroptosis mediated by RIP-1pathway.3. ROS participated in the activation of shikonin induced Necroptosis, and blockingit could attenuate the activation of Necroptosis obviously.