Inhibit NAD(P)H Oxidase Activity Induces Apoptosis Of U251 Glioma Cells

BackgroundGlioma is the most common primary intracranial tumor in humans, it origin from astrocyte or oligodendrocyte. Some studies showed that in recent twenty years the incidence rate of glioma (adult or children) is invreased. High grade astrocytoma is the most common glioma, recrudescence was take placed very often after postoperation, even though radiotherapy and chemotherapy was appled,80% patients were died in 1 year. Glioblastoma multiforme is the high malignant glioma, even though radiotherapy and chemotherapy was appled, meso life span was 1 year and the normal brain was damaged. Therefore, many oncologist thought that there was no progress in therapy of malignant glioma.It is thought that the malignant astrocytomas undergone mutisteps proceesing, which resulted in the deregulation of cell growth. These steps include:(a) the loss of function of the tumor suppressor p53; (b) the loss of function of cell cycle inhibitors, such as p16 and p19; (c) the amplification and mutation of genes encoding growth factor receptors; (d) activation of genes and proteins encoding growth-regulatory proteins, such as protein kinase C; Each of these proteins has been shown to play roles in tumor progression, cell proliferation, resistance to radiation treatment or chemotherapeutic agents, and cell migration.However, little is known about the signaling proteins that regulate survival of glioma cells.May be related to the mechanisms of anti-apotosis. Recent studies have found that 3 phosphoinositide-kinase (PI3K)/Akt channels, transcription factor NF-κB, mTOR could enhance the anti-apoptotic effect of glioma cells. Meanwhile, the study found that active oxygen species (ROS) acting on the (PI3K)/Akt and the transcription factor NF-κB and other targets,which play an important regulatory role on maintening the cell survival and proliferation. ROS include superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl ions (HO-), traditionally, it was viewed that ROS just were the toxins of cell metabolism.Exogenous H2O2 can promote apoptosis in many types of cells, or necrosis, therefore, it is agreed that ROS are the harmful factors which affect the cell survival and proliferation; But with in-depth study,it was found that ROS were important for intracellular signal transduction, not only the harmful factors of nucleotides, proteins and other cellular components.ROS can act on the PI3K/Akt, transcription factor NF-κB and other targets, which play an important role on the cell proliferation, apoptosis and the regulation of gene transcription.NF-κB is a multi-regulatory function of transcription factors,which widely participate in the transcriptional regulation of many genes, there was research indicated that NF-κB played a central regulatory role on tumor cell proliferation and apoptosis. NF-κB constructs by p50, p65. When cells are in resting state, p50, p65 through direct binding with the inhibitory protein IκB to form a non-active NF-κB/IκBs complex retained in the cytoplasm, but while a variety of stimulating extracellular signal transduction to the cytoplasm, a series of enzyme activation, resulting in IκB degradation, activation of NF-κB dimers into the nucleus, and then form a specific DNA-binding domain of target genes, thereby regulating the expression of the corresponding genes. However, the activation of transcription factor NF-κB and NF-κB-mediated gene transcription both require the involvement of ROS, when reducing the production of intracellular ROS can effectively block the ROS-dependent p38 and JNK signaling pathways to inhibit the activation of NF-κB. The intracellular ROS are mainly produced by NAD (P) H oxidase, Xanthine oxidase, lipoxygenase and cyclooxygenase, non-coupling nitric oxide synthase and mitochondrial respiratory chain, in which NAD (P) H oxidase is considered the main enzyme, NAD (P) H oxidase and its derived ROS involved in the control of glioma cell growth and apoptosis.However,the mechanism is still not very clear, intracellular ROS as intracellular signal transduction second messenger can regulate a variety of downstream factor activity,which involved in the control tumor cell growth and anti-apoptosis. Therefore, in this study, inhibited NAD (P) H oxidase activity by DPI, reducing the intracellular ROS generation, and using immunofluorescence detection of NAD (P) H oxidase inhibitor of nuclear factor-KB (NF-κB) activation, analyze the effect of NAD (P) H oxidase and intracellular ROS on glioma cell proliferation, survival, apoptosis.Objective:1.To evaluate the effect of NAD (P) H oxidase and intracellular ROS on glioma cell survival, proliferation and apoptosis.2. To evaluate the effect of NAD (P) H oxidase on nuclear factor-KB (NF-κB) activation.Methods:1. Cultured U251 glioma cells, packet processing U251 glioma cells:a normal control group; DPI treatment groups:1) 5μM group,2)15μM group,3) 25μM group; Tiron treatment group:10mM Tiron.2. Extracted total RNA from U251 glioma cells, the NOX genes express was detected though RT-PCR in the U251 glioma cell line.3. The cells were treated by DPI for 30 minutes, add carboxy-H2DCFDA probe,and then detected the intracellular ROS production by Flow cytometry. 4. The cell proliferation of U251 cells was tested by alamarBlue assay at 24h following adding 5μM,15μM,25μM NAD(P)H oxidase inhibitor DPI and lOmM antioxidant Tiron.5. the apoptotic U251 glioma cells were examined by flow cytometry at 24h following adding 5μM,15μM,25μM NAD(P)H oxidase inhibitor DPI and lOmM antioxidant Tiron.6. Immunofluorescence detected the effect of NAD (P) H oxidase inhibitor DPI on the nuclear factor-KB (NF-κB) activation.Results:1. The PCR result indicated that Nox4 mRNA was a major NOX homologue expressed in U251 glioma cells.2. DPI can inhibit the activity of NAD(P)H oxidase, which significantly reduces the generation of intracellular ROS in U251 glioma cells(P<0.01).3. Compared with the untreated control cells, the growth of U251 glioma cell was significantly inhibited by various concentrations of NAD(P)H oxidase inhibitor DPI(P<0.01) and 10mM antioxidant Tiron(P<0.01).4. when the U251 glioma cells were treated with various concentrations of DPI or 10mM Tiron, the amounts of apoptotic cells remarkably increased(P<0.01).Conclusion:1. NOX4 maybe is a major source which generates intracellular ROS in glioma cells.2. NOX4 semms to be involved in regulating cell proliferation, survival and apoptosis in glioma cells.3. NOX4 increase the intracellular ROS levels, which regulate the activation of NF-κB, to furthly regulate gene transcription within the cell, thereby affecting glioma cells proliferation and apoptosis.