Function And Mechanism Of RIP3 On Proliferation, Death And Chemosensitivity Of Glioblastoma Cell

BackgroundGlioblastoma or glioblastoma multiforme (glioblastoma multiforme, GBM) is the most encountered and malignant variants of gliomas in clinical practice. As with other tumors, the pathogenesis, development and its resistance to adjuvant treatments of GBM are associated with regulation of multiple genes and interaction of various factors within the tumor cells. Gene therapy is considered as a promising antitumor treatment to various malignancies. Because it has high efficiency of gene transfer, better controllability and it can integrate desired genes into non-dividing cells and dividing cell with stable expression of transgene, lentiviral vector-mediated gene transfer is a very useful way for gene therapy. Lentiviral vector-mediated RNA interference also is widely used to achieve a stable gene silencing in various cell lines. Apoptosis is programmed cell death, and there has been a major emphasis in the literature on the apoptotic form of cell death. Just recently, necrotic cell death is considered as a regulated and controlled process, like apoptosis, termed necroptosis or programmed necrosis. RIP3, a RIP family member, is a Ser/Thr protein kinase and shares homology with the kinase domain within RIP family. It is found that RIP3 is a key protein during the process of programmed cell necrosis. Dysfunction of apoptosis is believed to underlie GBM tumorigenesis and resistance to chemotherapy and radiotherapy. Although there are defects in apoptotic process, nearly all GBMs have different degree of intratumoral necrosis, which does not seem to be associated with the tumor size. In addition, some antitumor therapies have therapeutic effects on a variety of malignant tumors through the induction of tumor cells necrosis. It is a novel way to achieve a better therapeutic effect through the induction of tumor cells necrosis and this idea has gain increasing attention in recent years. Yet, there are limited literature shed light on inducing tumor cell necrosis to combat malignant tumors and no research has been reported to study the RIP3function on GBM. We construct recombinant lentiviral overexpression of human RIP3 vector and shRIP3 vector to modulate the expression of RIP3 in GBM cell lines to study the function and mechanism of RIP3 on proliferation, death and chemosensitivity of GBM cells. This would further reveal the function of RIP3 on GBM and provide basis for applications of RIP3 in GBM treatment.Methods and ResultsConstructing a lentiviral expression vector containing EF1 A-driving human RIP3 gene using Gateway technology and a lentiviral shRNA vector targeting human RIP3 gene using ligation and packaging into viral particles with high titers 1x108 TU/ml. Transducing human RIP3 gene and shRNA targeting human RIP3 into U251 respectively and establishing stable cell lines. U251-RIP3 and U251-shRIP3 by selection with antibiotic. Western blotting showed that the expression lever of RIP3 in U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. Some U251-RIP3 cells exhibited swelling-like death during culturing. This phenomenon was not inhibited by pan-caspase inhibitor zVAD. Annexin V/PI assay confirmed the death of U251-RIP3 cells was PI positive. Annexin V/PI assay was conducted to study the death rate. Death rate of U251-shRJP3 and U251-WT cells has significant differences with U251-RIP3 cell by Annexin V/PI assay. But there is no significant difference between U251-shRIP3 cells and U251-WT cells. ROS lever was measured by CellROXTM Deep Red Reagent to study the role of ROS in RIP3 induced cell death. ROS level of U251-RIP3 cells is significantly higher than that of U251-shRIP3 cells and U251-WT cells. And U251-shRIP3 did not show a significant difference with U251-WT cells. Death of U251-RIP3 cells is ROS dependent and BHA can reduce the death rate. Recombinant TNF-a do not enhance the death rate of U251-RIP3 cells. ELISA measured the secretion of TNF-a among U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. TNF-a level of U251-RIP3 cells is significantly higher than that of U251-shRIP3 cells and U251-WT cells. And U251-shRIP3 cells did not show any difference with U251-WT cells. MTT colorimetric assay showed the proliferation of U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. The proliferation of U251-RIP3 was lower than that of U251-shRIP3 cells and U251-WT cells. Western blotting analyzed the average activation of Akt in U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells. Our result showed that the Akt activation was suppressed in U251-RIP3 cells, whereas, both U251-shRIP3 cells and U251-WT cells had higher level of Akt activation and latter tow group has no significant differences. We analyzed the cell death in U251-RIP3 cells, U251-shRIP3 cells and U251-WT cells in the treatment of temozolomide. The average of cell death rate in the treatment of temozolomide measured by flow cytometry. Death rate of U251-RIP3 cells is much higher than that of U251-WT cells in treatment of temozolomide. MTT colorimetric assay showed that ectopic overexpression of RIP3 in GBM cell line U251 increase its chemosensitivity to temozolomide. U251-shRIP3 cells and U251-WT cells had the same low sensitivity to temozolomide. Western blotting analysis showed that the PARP-1 expression lever in U251-RIP3 cells was lower than that of U251-shRIP3 cells and U251-WT cells. This suggests RIP3 enhance the chemosensitivity of U251 to temozolomide at least partially via its impact on PARP-1.ConclusionRIP3 induces necrosis in GBM cell line U251. RIP3 activates the secretion of TNF-a, and then increases ROS production to cause necrosis. RIP3 inhibits proliferation of U251 at least partially by suppressing the Akt pathway. We propose RIP3 is a novel target to GBM gene therapy. RIP3 increases U251 cells sensitivity to temozolomide via the impact on PARP-1, RIP3 may be a targeted molecular to modulate the chemosensitivity of GBM.