Activated SIRT1 contributes to DPT-induced glioma cell parthanatos by upregulation of NOX2 and NAT10.

Parthanatos is a type of programmed cell death dependent on hyper-activation of poly (ADP-ribose) polymerase 1 (PARP-1). SIRT1 is a highly conserved nuclear deacetylase and often acts as an inhibitor of parthanatos by deacetylation of PARP1. Our previous study showed that deoxypodophyllotoxin (DPT), a natural compound isolated from the traditional herb Anthriscus sylvestris, triggered glioma cell death via parthanatos. In this study, we investigated the role of SIRT1 in DPT-induced human glioma cell parthanatos. We showed that DPT (450 nmol/L) activated both PARP1 and SIRT1, and induced parthanatos in U87 and U251 glioma cells. Activation of SIRT1 with SRT2183 (10 µmol/L) enhanced, while inhibition of SIRT1 with EX527 (200 µmol/L) or knockdown of SIRT1 attenuated DPT-induced PARP1 activation and glioma cell death. We demonstrated that DPT (450 nmol/L) significantly decreased intracellular NAD+ levels in U87 and U251 cells. Further decrease of NAD+ levels with FK866 (100 µmol/L) aggravated, but supplement of NAD+ (0.5, 2 mmol/L) attenuated DPT-induced PARP1 activation. We found that NAD+ depletion enhanced PARP1 activation via two ways: one was aggravating ROS-dependent DNA DSBs by upregulation of NADPH oxidase 2 (NOX2); the other was reinforcing PARP1 acetylation via increase of N-acetyltransferase 10 (NAT10) expression. We found that SIRT1 activity was improved when being phosphorylated by JNK at Ser27, the activated SIRT1 in reverse aggravated JNK activation via upregulating ROS-related ASK1 signaling, thus forming a positive feedback between JNK and SIRT1. Taken together, SIRT1 activated by JNK contributed to DPT-induced human glioma cell parthanatos via initiation of NAD+ depletion-dependent upregulation of NOX2 and NAT10.

TAX1BP1 contributes to deoxypodophyllotoxin-induced glioma cell parthanatos via inducing nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I.

Parthanatos is a type of programmed cell death initiated by over-activated poly (ADP-ribose) polymerase 1 (PARP1). Nuclear translocation of apoptosis inducing factor (AIF) is a prominent feature of parthanatos. But it remains unclear how activated nuclear PARP1 induces mitochondrial AIF translocation into nuclei. Evidence has shown that deoxypodophyllotoxin (DPT) induces parthanatos in glioma cells via induction of excessive ROS. In this study we explored the downstream signal of activated PARP1 to induce nuclear translocation of AIF in DPT-triggered glioma cell parthanatos. We showed that treatment with DPT (450 nM) induced PARP1 over-activation and Tax1 binding protein 1 (TAX1BP1) distribution to mitochondria in human U87, U251 and U118 glioma cells. PARP1 activation promoted TAX1BP1 distribution to mitochondria by depleting nicotinamide adenine dinucleotide (NAD+). Knockdown of TAX1BP1 with siRNA not only inhibited TAX1BP1 accumulation in mitochondria, but also alleviated nuclear translocation of AIF and glioma cell death. We demonstrated that TAX1BP1 enhanced the activity of respiratory chain complex I not only by upregulating the expression of ND1, ND2, NDUFS2 and NDUFS4, but also promoting their assemblies into complex I. The activated respiratory complex I generated more superoxide to cause mitochondrial depolarization and nuclear translocation of AIF, while the increased mitochondrial superoxide reversely reinforced PARP1 activation by inducing ROS-dependent DNA double strand breaks. In mice bearing human U87 tumor xenograft, administration of DPT (10 mg· kg-1 ·d-1, i.p., for 8 days) markedly inhibited the tumor growth accompanied by NAD+ depletion, TAX1BP1 distribution to mitochondria, AIF distribution to nuclei as well as DNA DSBs and PARP1 activation in tumor tissues. Taken together, these data suggest that TAX1BP1 acts as a downstream signal of activated PARP1 to trigger nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I.

ATF3 contributes to brucine-triggered glioma cell ferroptosis via promotion of hydrogen peroxide and iron.

Ferroptotic cell death is characterized by iron-dependent lipid peroxidation that is initiated by ferrous iron and H2O2 via Fenton reaction, in which the role of activating transcription factor 3 (ATF3) remains elusive. Brucine is a weak alkaline indole alkaloid extracted from the seeds of Strychnos nux-vomica, which has shown potent antitumor activity against various tumors, including glioma. In this study, we showed that brucine inhibited glioma cell growth in vitro and in vivo, which was paralleled by nuclear translocation of ATF3, lipid peroxidation, and increases of iron and H2O2. Furthermore, brucine-induced lipid peroxidation was inhibited or exacerbated when intracellular iron was chelated by deferoxamine (500 µM) or improved by ferric ammonium citrate (500 µM). Suppression of lipid peroxidation with lipophilic antioxidants ferrostatin-1 (50 µM) or liproxstatin-1 (30 µM) rescued brucine-induced glioma cell death. Moreover, knockdown of ATF3 prevented brucine-induced accumulation of iron and H2O2 and glioma cell death. We revealed that brucine induced ATF3 upregulation and translocation into nuclei via activation of ER stress. ATF3 promoted brucine-induced H2O2 accumulation via upregulating NOX4 and SOD1 to generate H2O2 on one hand, and downregulating catalase and xCT to prevent H2O2 degradation on the other hand. H2O2 then contributed to brucine-triggered iron increase and transferrin receptor upregulation, as well as lipid peroxidation. This was further verified by treating glioma cells with exogenous H2O2 alone. Moreover, H2O2 reversely exacerbated brucine-induced ER stress. Taken together, ATF3 contributes to brucine-induced glioma cell ferroptosis via increasing H2O2 and iron.

FOXO3a protects glioma cells against temozolomide-induced DNA double strand breaks via promotion of BNIP3-mediated mitophagy.

FOXO3a (forkhead box transcription factor 3a) is involved in regulating multiple biological processes in cancer cells. BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a receptor accounting for priming damaged mitochondria for autophagic removal. In this study we investigated the role of FOXO3a in regulating the sensitivity of glioma cells to temozolomide (TMZ) and its relationship with BNIP3-mediated mitophagy. We showed that TMZ dosage-dependently inhibited the viability of human U87, U251, T98G, LN18 and rat C6 glioma cells with IC50 values of 135.75, 128.26, 142.65, 155.73 and 111.60 µM, respectively. In U87 and U251 cells, TMZ (200 µM) induced DNA double strand breaks (DSBs) and nuclear translocation of apoptosis inducing factor (AIF), which was accompanied by BNIP3-mediated mitophagy and FOXO3a accumulation in nucleus. TMZ treatment induced intracellular ROS accumulation in U87 and U251 cells via enhancing mitochondrial superoxide, which not only contributed to DNA DSBs and exacerbated mitochondrial dysfunction, but also upregulated FOXO3a expression. Knockdown of FOXO3a aggravated TMZ-induced DNA DSBs and mitochondrial damage, as well as glioma cell death. TMZ treatment not only upregulated BNIP3 and activated autophagy, but also triggered mitophagy by prompting BNIP3 translocation to mitochondria and reinforcing BNIP3 interaction with LC3BII. Inhibition of mitophagy by knocking down BNIP3 with SiRNA or blocking autophagy with 3MA or bafilomycin A1 exacerbated mitochondrial superoxide and intracellular ROS accumulation. Moreover, FOXO3a knockdown inhibited TMZ-induced BNIP3 upregulation and autophagy activation. In addition, we showed that treatment with TMZ (100 mg·kg-1·d-1, ip) for 12 days in C6 cell xenograft mice markedly inhibited tumor growth accompanied by inducing FOXO3a upregulation, oxidative stress and BNIP3-mediated mitophagy in tumor tissues. These results demonstrate that FOXO3a attenuates temozolomide-induced DNA double strand breaks in human glioma cells via promoting BNIP3-mediated mitophagy.

Endoplasmic reticulum stress regulates oxygen-glucose deprivation-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS.

AIMS: Endoplasmic reticulum (ER) stress has been demonstrated to regulate neuronal death caused by ischemic insults via activation of apoptosis, but it still remains unclear whether ER stress participates in regulation of parthanatos, a new type of programmed cell death characterized by PARP-1 overactivation and intracellular accumulation of PAR polymer. METHODS: we used oxygen-glucose deprivation (OGD) and human SH-SY5Y cells to simulate neuronal damage caused by ischemia. RESULTS: Oxygen-glucose deprivation induced time-dependent death in SH-SY5Y cells, which was accompanied with upregulation of PARP-1, accumulation of PAR polymer, decline of mitochondrial membrane potentials and nuclear translocation of AIF. Pharmacological inhibition of PARP-1 with its specific inhibitor 3AB rescued OGD-induced cell death, as well as prevented PAR polymer accumulation, mitochondrial depolarization, and AIF translocation into nucleus. Similar results could be found when PARP-1 was genetically knocked down with SiRNA. These indicated that OGD triggered parthanatos in SH-SY5Y cells. Then, we found inhibition of overproduction of ROS with antioxidant NAC attenuated obviously OGD-induced parthanatos in SH-SY5Y cells, suggesting ROS regulated OGD-induced parthanatos. Additionally, OGD also induced upregulation of ER stress-related proteins. Mitigation of ER stress with chemical chaperone 4-PBA or trehalose suppressed significantly OGD-induced overproduction of ROS, PARP-1 upregulation, PAR polymer accumulation, and nuclear accumulation of AIF, and cell death in SH-SY5Y cells. CONCLUSION: Endoplasmic reticulum stress regulates OGD-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS.

Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1/RIP3 necrosome formation.

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 (RIP1) and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species (ROS), but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin (2-10 µmol/L) dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose-dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 (100 µmol/L) or the specific RIP3 inhibitor GSK-872 (5 µmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 µmol/L), which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.

Substance P reduces apoptotic cell death possibly by modulating the immune response at the early stage after spinal cord injury.

Previously, we have reported that substance P (SP) enhanced functional recovery from spinal cord injury (SCI) possibly by the anti-inflammatory modulation associated with the induction of M2-type macrophages at the injured lesion. In this study, we explored the cytokine expression profiles and apoptotic cell death in the lesion site of the SCI after an immediate intravenous injection of SP. SP injection increased the levels of interleukin-4 (IL-4), IL-6, and IL-10 at day 1 after the SCI approximately by 2-, 9-, and 10-folds when compared with the control SCI, respectively. On the basis of double immunofluorescence staining with IL-10 and CD11b, activated macrophages or microglia expressing IL-10 appeared in the margin of the lesion site at day 1 only after the SP injection. This SP-mediated alteration in the lesion microenvironment was shown to be associated with the lower cell death of neuronal cells at day 1 and oligodendrocytes at day 5 by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which was also accompanied by a decrease in caspase-3 activation. These findings suggest that SP may reduce the inflammation-induced secondary cell death, possibly through immune modulation at an early stage after the SCI.

Schwann-like cells from human melanocytes and their fate in sciatic nerve injury.

We induced human melanocyte dedifferentiation to Schwann cell-like cells in vitro by a combination of forskolin, neuregulin-ß1, neurotrophin-3, platelet-derived growth factor-aa, basic fibroblast growth factor, laminin, and heparin. Cultured human melanocytes constitutively expressed neural cell and melanocyte markers but melanocyte-specific marker, including microphthalmia-associated transcription factor and tyrosinase, expression was selectively lost after induction. In the sciatic nerve injury site, the induced cells were engrafted and closely aligned to axons and P0-expressing myelin sheaths, whereas uninduced cells were not colocalized with axons and myelin sheaths and reexpressed melanocyte-specific tyrosinase activity in vivo. Human melanocytes lose their melanocyte phenotype and transdifferentiate into Schwann cells under specific induction conditions and display their Schwann cell phenotype after transplantation to injured sciatic nerve tissue.

Schwann cells differentiated from spheroid-forming cells of rat subcutaneous fat tissue myelinate axons in the spinal cord injury.

In the present study, we found that nestin-expressing spheroid cells derived from multipotent adipose stem cells of subcutaneous fat tissue could efficiently differentiate into Schwann cells (SCs) in vitro based on expression of SC markers such as A2B5, GFAP, O4, p75, S100, Sox10, Krox-20, and L1. The induced SC is engrafted to spinal cord injury lesions and formed a peripheral nervous system (PNS)-type myelin sheath on central nervous system (CNS) axons. PNS-type myelin sheath formation in repaired tissue was confirmed by transplantation of both induced PKH26-labeled SC and induced EGFP-expressing SC generated from EGFP transgenic rats. In addition to direct participation as myelin sheath-forming SC in repaired tissue, the induced SC also expressed several neurotrophic factors, as did native SC, which may suggest an additional role for induced SC in stimulation of endogenous healing responses. Thus, spheroid-forming cells from subcutaneous fat tissue demonstrated rapid and efficient induction into SC, and such cells show therapeutic promise for repair of damage to the CNS and PNS.

Hyaline cartilage regeneration using mixed human chondrocytes and transforming growth factor-beta1- producing chondrocytes.

The purpose of this study was to investigate the efficacy of cartilage regeneration when using a mixture of transforming growth factor-beta1 (TGF-beta1)-producing human chondrocytes (hChon-TGF-beta1) and primary human chondrocytes (hChon) ("mixed cells"), compared with either hChon-TGF-beta1 or hChon cells alone. Specifically, mixed cells or hChon cells were first injected intradermally into the backs of immune-deficient nude mice to test the feasibility of cartilage formation in vivo. Both the mixed cells and the hChon-TGF-beta1 cells alone induced cartilage formation in nude mice, whereas hChon cells alone did not. To further test the efficacy of the cells in generating cartilage, an artificially induced partial thickness defect of the femoral condyle of a rabbit knee joint was loaded with hChon-TGF-beta1 cells with or without mixing additional untransduced hChon cells, and hyaline cartilage regeneration was observed at 4 or 6 weeks. The efficiency of complete filling of the defect and the quality of tissue generated after implanting were evaluated on the basis of a histological grading system modified from O'Driscoll et al. (J. Bone Joint Surg. 70A, 595, 1988). Significantly, mixed cells (14.2 +/- 0.9) produced significantly better results than hChon-TGF-beta1 (9.0 +/- 1.7) or hChon (8.0 +/- 1.8) cells alone. Histological and immunohistochemical staining of the newly repaired tissues produced after treatment with either mixed cells or hChon-TGF-beta1 cells alone showed hyaline cartilage- like characteristics. These results suggest that the implantation of mixed cells may be a clinically efficient method of regenerating hyaline articular cartilage.

Continuous transforming growth factor beta1 secretion by cell-mediated gene therapy maintains chondrocyte redifferentiation.

One of the most important factors in the production of cartilage is transforming growth factor beta1 (TGF-beta1). To obtain sustained release of TGF-beta1, a cell-mediated gene therapy technique was introduced. We infected chondrocytes with a retroviral vector carrying the TGF-beta1 gene. The single clone derivative showed sustained TGF-beta1 secretion. It also showed constitutive type II collagen expression. Whereas the TGF-beta1 protein itself is unable to induce formation of cartilage in vivo, human chondrocytes engineered to express a retroviral vector encoding TGF-beta1 showed cartilage formation in vivo when cells were injected into nude mice intradermally. These data suggest that cell-mediated gene therapy using TGF-beta1 as a transgene would be a promising treatment for osteoarthritis.