Shikonin-induced Necroptosis In Nasopharyngeal Carcinoma Cells Via Regulation Of RIPK1/RIPK3/MLKL Expression

Background: Nasopharyngeal carcinoma(NPC)is a common head and neck cancer with high morbidity among patients in Southeast Asia.Chemotherapy is used as an effective method to treat advanced-stage disease.Nonetheless,treatment failure largely due to local recurrence and distant metastasis remains a challenge.In addition,drug resistance is a prominent problem that can compromise the efficacy of chemotherapy.There are several pathways through which cell death can occur,including apoptosis,necrosis,and autophagy.Necroptosis,a newly discovered type of cell death,is morphologically similar to necrosis.Necroptosis is a form of regulated necrosis that depends on receptor-interacting protein kinase 1(RIPK1)and receptor-interacting protein kinase 3(RIPK3).Accumulating evidence has demonstrated that activated RIPK1 interacts with RIPK3 to facilitate RIPK1/RIPK3 necrosome formation.Necroptosis signaling pathways can be targeted by therapeutic agents to eliminate apoptosis/drug-resistant cancer cells,which is critical for the success of cancer chemotherapy.Shikonin is a predominant type of naphthoquinone pigment that is extracted from the Chinese plant Lithospermum erythrorhizon.Previous research has shown that shikonin has been reported to induce necroptosis in 5-8FZ cell line.Nonetheless,the underlying molecular mechanisms of shikonin-induced necroptosis are not fully understood.we extended this investigation to another type of NPC cell line,5-8F cells,a 5-8F xenograft mouse model and biopsy specimens of patients with nasopharyngeal carcinoma.Clarifying the mechanisms of necroptosis by shikonin treatment may provide novel insight into the mechanism underlying the anticancer effects of shikonin.Objective: We used biopsy specimens of patients with nasopharyngeal carcinoma、nude mouse model of xenograft and human nasophrageal 5-8F cell in this study to investigate the roles of RIPK1、RIPK3 and MLKL in shikonin-induced necroptosis progress and the underlying mechanism.Methods: 1.Immunofluorescence staining was used to detect the expression of RIPK1 in biopsy specimens of patients with nasopharyngeal carcinoma.2.The mice model of 5-8F xenograft was used to investigate the effect of shikonin on nasopharyngeal carcinoma cell.Immunohistochemical staining was used to detect the expression of RIPK3 in xenograft tumor tissue.3.CCK8 assay was used to examine the viability of cells.4.Morphological changes in 5-8F cells induced by shikonin is observed through transmission electron microscopy.5.Flow cytometry(Annexin V-FITC and PI double staining)was used to investigate the cell death modes.6.DCFH-DA probe was used to evaluate the level of intracellular reactive oxygen species(ROS).7.Western-blotting was used to analyze the expressional level of related proteins in nasopharyngeal carcinoma cells.Results: 1.The results of immunofluorescence showed that RIPK1 was positively expressed in cytoplasm of nasopharyngeal carcinoma cell of biopsy specimens.2.The mice model of 5-8F xenograft revealed that the volume and the weight of xenograft became smaller in the shikonin group than that in the control.Immunohistochemistry staining demonstrated that the expression of RIPK3 was upregulated in the shikonin group.3.CCK8 assay showed that shikonin inhibited the viability of 5-8F cells in a range of concentration.4.As revealed by transmission electron microscopy.,the shikonin-treated 5-8F cells presented an electron-lucent cytoplasm,loss of plasma membrane integrity,and an intact nuclear membrane,indicating that shikonin induced necroptosis.Flow cytometry with Annexin V/PI double staining also indicated that shikonin induced necroptosis.5.Western blotting analysis showed that the expression of RIPK1,RIPK3,and MLKL was upregulated by shikonin in a dose-dependent manner.The upregulated expression of related proteins by shikonin was inhibited when 5-8F cells was pretreated with Nec-1.6.The ROS detected by DCFH-DA probe was overproduced in shikonin-treated 5-8F cells,which could be inhibited by Nec-1 and NAC.Conclusions: In conclusion,the results revealed that shikonin can induce necroptosis by ROS overproduction and regulating the expression of RIPK/RIPK3/MLKL.