Abstract
Background and PurposeShikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.MethodsShikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.ResultsShikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.ConclusionsWe demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.
Highlights
Malignant gliomas account for approximately 70% of the 22,500 new cases of malignant primary brain tumors that are diagnosed in adults in the United States each year [1]
Under light microscope, C6 glioma cells were found to present the morphological features of dying cells, we limited the incubation time of C6 glioma cells with shikonin to 3 hours
For studying the molecular mechanism underlying the inhibitory effects of shikonin on glioma cell viability, we examined the expressional level of RIP-1 that was thought to play a crucial role in initiating necroptosis [24]
Summary
Malignant gliomas account for approximately 70% of the 22,500 new cases of malignant primary brain tumors that are diagnosed in adults in the United States each year [1]. Recent studies show that resistance to apoptosis is the major factor that makes malignant glioma cells survive current clinically used medicines or radiotherapy [3]. It is needed to find new medicines that could induce glioma cell death not via apoptosis pathway [4]. Necroptosis (a type of programmed necrosis) is found to be a new form of programmed cell death that is different with apoptosis [5]. Necroptosis is able to overcome resistance to cancer drugs mediated by P-glycoprotein, Bcl-2, and Bcl-xL in cancer cell lines [14]. Necroptosis has become a new target to induce tumor cell death. Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. It is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms
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