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Nucleoredoxin Knockdown in SH-SY5Y Cells Promotes Cell Renewal.

ABSTRACT: Nucleoredoxin (NXN) is a redox regulator of Disheveled and thereby of WNT signaling. Deficiency in mice leads to cranial dysmorphisms and defects of heart, brain, and bone, suggesting defects of cell fate determination. We used shRNA-mediated knockdown of NXN in SH-SY5Y neuroblastoma cells to study its impact on neuronal cells. We expected that shNXN cells would easily succumb to redox stress, but there were no differences in viability on stimulation with hydrogen peroxide. Instead, the proliferation of naïve shNXN cells was increased with a higher rate of mitotic cells in cell cycle analyses. In addition, basal respiratory rates were higher, whereas the relative change in oxygen consumption upon mitochondrial stressors was similar to control cells. shNXN cells had an increased expression of redox-sensitive heat shock proteins, Hsc70/HSPA8 and HSP90, and autophagy markers suggested an increase in autophagosome formation upon stimulation with bafilomycin and higher flux under low dose rapamycin. A high rate of self-renewal, autophagy, and upregulation of redox-sensitive chaperones appears to be an attractive anti-aging combination if it were to occur in neurons in vivo for which SH-SY5Y cells are a model.

SUBMITTER: Valek L

PROVIDER: S-EPMC7999887 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Nucleoredoxin Knockdown in SH-SY5Y Cells Promotes Cell Renewal.

Valek Lucie L Tegeder Irmgard I

Antioxidants (Basel, Switzerland) 20210313 3

Nucleoredoxin (NXN) is a redox regulator of Disheveled and thereby of WNT signaling. Deficiency in mice leads to cranial dysmorphisms and defects of heart, brain, and bone, suggesting defects of cell fate determination. We used shRNA-mediated knockdown of NXN in SH-SY5Y neuroblastoma cells to study its impact on neuronal cells. We expected that shNXN cells would easily succumb to redox stress, but there were no differences in viability on stimulation with hydrogen peroxide. Instead, the prolifer ...[more]

PMID: 33805811

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Project description:BackgroundSesamin is a well-known antioxidant extracted from sesame seeds that exhibits various curative effects. The present study investigated whether sesamin would protect neuroblastoma SH-SY5Y cells against mechanical stretch injury-induced increases in reactive oxygen species (ROS) and apoptosis. Additionally, the mechanisms underlying these actives were investigated. Following exposure to mechanical stretch injury, cells were incubated for further investigations. Lactate dehydrogenase and Cell Counting Kit-8 assays were used to assess cell viability, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometric analysis were performed to evaluate changes in mitochondrial membrane potential (ΔΨm). Furthermore, intracellular levels of ROS production were measured by 20, 70-dichlorofluorescein diacetate staining, the mRNA levels of matrix metallopeptidase 9 (MMP-9) were evaluated using real-time polymerase chain reaction analysis, and the determinations had also been made on related proteins by Western blot analysis.ResultsExposure to mechanical stretch injury significantly decreased cell viability but this decrease was attenuated by pretreatment with sesamin (50 μM). Sesamin also significantly inhibited mechanical stretch injury-induced increases in intracellular ROS production, attenuated declines in ΔΨm, diminished the expressions of pro-apoptotic proteins, and decreased cell apoptosis. Stretch injury increased Bax and cleaved caspase 3 levels, enhanced the gene expression of MMP-9, increased the phosphorylation levels of Akt, p38, and JNK and decreased Bcl-2 levels in the cells. However, pretreatment with sesamin reduced the mechanical stretch injury-induced overexpression of MMP-9.ConclusionsSesamin protected SH-SY5Y cells against stretch injury by attenuating increases in ROS levels and suppressing apoptosis. Accordingly, sesamin seems to be a potentially therapeutic agent in the treatment of traumatic brain injury.

Project description:BackgroundThe cancer caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection is one of the major causes of death in AIDS patients. Some patients have neurological symptoms, which appear to be associated with KSHV infection, based on the neurotropic tendency of this virus in recent years. The objectives of this study were to investigate the effects of KSHV infection on neuronal SH-SY5Y cells and to identify differentially expressed genes.MethodsKSHV was collected from islk.219 cells. Real-time PCR was used to quantify KSHV copy numbers. KSHV was used to infect SH-SY5Y cells. The KSHV copy number in the supernatants and mRNA levels of latency-associated nuclear antigen (LANA), ORF26, K8.1 A, and replication and transcriptional activator (RTA) were detected by real-time PCR. Proteins were detected by immunohistochemistry. The effect of KSHV infection on cell proliferation was detected by MTT and Ki-67 staining. Cell migration was evaluated by Transwell and wound healing assays. The cell cycle was analyzed by flow cytometry. The expression of CDK4, CDK5, CDK6, cyclin D1, and p27 were measured by western blotting. The levels of cell cycle proteins were re-examined in LANA-overexpressing SH-SY5Y cells. Transcriptome sequencing was used to identify differentially expressed genes in KSHV-infected cells. The levels of Notch signaling pathway proteins were measured by western blotting. RNA interference was used to silence Notch1 and proliferation were analyzed again.ResultsSH-SY5Y cells were successfully infected with KSHV, and they maintained the ability to produce virions. KSHV-infected SH-SY5Y expressed LANA, ORF26, K8.1 A, and RTA. After KSHV infection, cell proliferation was enhanced, but cell migration was suppressed. KSHV infection accelerated the G0/G1 phase. CDK4, CDK5, CDK6, and cyclin D1 expression was increased, whereas p27 expression was decreased. After LANA overexpression, CDK4, CDK6 and cyclin D1 expression was increased. Transcriptome sequencing showed that 11,258 genes were upregulated and 1,967 genes were downregulated in KSHV-infected SH-SY5Y. The Notch signaling pathway played a role in KSHV infection in SH-SY5Y, and western blots confirmed that Notch1, NICD, RBP-Jĸ and Hes1 expression was increased. After silencing of Notch1, the related proteins and cell proliferation ability were decreased.ConclusionsKSHV infected SH-SY5Y cells and promoted the cell proliferation. KSHV infection increased the expression of Notch signaling pathway proteins, which may have been associated with the enhanced cell proliferation.

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Nucleoredoxin Knockdown in SH-SY5Y Cells Promotes Cell Renewal.

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Nucleoredoxin Knockdown in SH-SY5Y Cells Promotes Cell Renewal.

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